Bioinformatics analysis revealed that many candidate genes were responsible for the two traits. Of these, FASN, ACACA, SCD, ME3, PDHB, ACSS1, ACSS2, and ACLY were identified, which functioned in regulating the level of hexadecanoic acid, hexadecenoic acid, octadecenoic acid, and monounsaturated fatty acid. And LPGAT1, PDK4, ACAA1, and ADIPOQ were associated with the content of stearic acid, octadecadienoic acid, and polyunsaturated fatty acid. Candidate genes, which were responsible for fatness trait, consisted of FGF2, PLAG1, ADIPOQ, IRX3, MIF, IL-34, ADAM8, HMOX1, Vav1, and TLR8. In addition, association analysis also revealed that 34 and 57 genes significantly correlated to the fatness and fatty acid composition trait, respectively. Working out the mechanism caused by these lncRNAs and candidate genes is proven to be complicated but is invaluable to our understanding of fatness and fatty acid composition traits.Tibetan pigs from the Tibetan Plateau are characterized with a significant phenotypic difference relative to lowland pigs. In this study, a significant difference of the fatness and fatty acid composition traits was observed between the Tibetan and Yorkshire pigs. To uncover the involved mechanism, the expression profile of long noncoding RNAs (lncRNAs) and genes was compared between them. After serial filtered steps, 1,964 lncRNAs were obtained through our computational pipeline. In total, 63 and 715 lncRNAs and genes were identified to be differentially expressed. Evidence from  Tibetan pigs mainly originated from the Tibetan Plateau, which had undergone the long-term natural selection in the harsh environment. It indicated that many candidate genes related to germplasm of the Tibetan pigs, such as energy metabolism, might contribute to the adaptability to hostile environment . HoweverSo we took another look on this scientific question that is due to thicker back fat and higher intramuscular fat in the Tibetan pigs, which could stably deliver the energy to the body and guarantee the inflammatory response in such a harsh environment. Additionally, phenotype difference of the fatness and fatty acid composition traits was also observed between the Tibetan and Yorkshire pigs in this study. Multiple elements, including coding genes , miRNA , and DNAAll phenotypes of the Tibetan and Yorkshire pig group, which included the fatness and fatty acid composition traits, were collected at days 180 . Each grSequencing data were assessed for quality using FastQC. High quality clean data was used in RNA-Seq analysis. Clean pair-end reads with FASTQ format were aligned to the Sscrofa 11.1 genome assembly, and novel splice junctions were determined using Hisat2 (v2.0.0-beta). The default parameters were used in the analysis, except for the “-G” option tighter with the GTF file of Ensembl gene annotation (Sscrofa 11.1.91). The alignment data with the BAM format were subjected to the StringTie (v1.3.0) to assemble transcripts. All transcripts from all samples were merged together using StringTie. Then, gffcompare (v0.10.1) was applied to match the transcript pool to Ensembl gene sets (Sscrofa 11.1.91). Transcripts, of which class code with “i,” “u,” “o,” and “x,” were subjected to lncRNA identification. To avoid incomplete assemble and false positive rate, the putative lncRNA transcripts were filtered with transcript length > 200 bp and exon number > 2. Remaining transcripts were scored with the CNCI tool . The traP value < 0.05). Enrichment analysis (GO and KEGG pathway) was performed with the Metascape database based on human homology gene list [R environment. The Bonferroni correction was used to determine the significance level of each association event.The number of reads that mapped to each gene was calculated with HTSeq . Then, Dene list . Associacis-regulating role of lncRNAs was defined as those exerting effects on neighboring genes [The ng genes . Differeng genes . The posμg of total RNA, following the protocol accompanied with the FastQuant RT Kit (Tiangen). Quantitative PCR (qPCR) amplification was conducted as described previously [Seven differentially expressed mRNAs and six differential lncRNAs were validated via qPCR. Total RNA was extracted from back-fat tissue using TRIzol reagent according to the manufacturer's instructions (Life Technologies). cDNA synthesis was performed with 1 eviously .Phenotypes of the fatness and fat composition traits from the Tibetan and Yorkshire pigs were collected and analyzed. Higher fat content was observed in the Tibetan group than the Yorkshire group, such as back-fat thickness at different positions and leaf fat percentage . EvidencClean reads were mapped to the Sscrofa 11.1 genome assembly, which mapping rates more than 83.1%. Of these, more than 84% reads were aligned on the CDS, 5′UTR, and 3′UTR region . A totalTo further understand the potential genes responsible for the phenotype difference, differentially expressed genes were subjected to functional enrichment analysis. Biological process and KEGG pathway terms significantly enriched by upregulated DEGs included small molecule biosynthetic process , many immune-related classes , and 4 KEGG pathway classes  .FASN, SCD, ME3, PDHB, ACACA, ACLY, ACSS1, and ACSS2. It had been demonstrated that the polymorphism of the FASN gene is associated with blood monounsaturated fatty acid and saturated fatty acid [FASN is a metabolic enzyme that catalyzed the synthesis of long-chain fatty acids through condensing acetyl-CoA and malonyl-CoA, and its main product is hexadecanoic acid [ACACA (acetyl-CoA carboxylase) [SCD (stearoyl-CoA desaturase) [SCD was demonstrated to correlate with the oleic acid, SFA, and MUFA content in pork [ME3 is an important paralog of ME1, together with ACACA and PDHB (Pyruvate Dehydrogenase E1 Beta Subunit) involved in the fatty acid synthesis process and pyruvate metabolism pathway  synthesis process [ACLY was demonstrated to be associated with the fatty acid content of milk in dairy cattle [FASN, ACACA, SCD, ME3, PDHB, ACSS1, ACSS2, and ACLY, were responsible for the higher monounsaturated fatty acid, octadecenoic acid, hexadecenoic acid, and hexadecanoic acid in the Tibetan group relative to the Yorkshire group.Many upregulated DEGs were enriched in fatty acid biosynthetic-related terms, which included tty acid . FASN isoic acid . Malonyloxylase) . Furtheraturase) . Variati in pork . ME3 is  pathway . ACSS1 a process . ACLY way cattle . It suggLPGAT1 (lysophosphatidylglycerol specific acyltransferase) recognized various acyl-CoAs and LPGs as substrates but preferred to long-chain saturated fatty acyl-CoAs and oleoyl-CoA as acyl donors [LPGAT1 gene was associated with stearic acid content of plasma [PDK4-specific DNA methylation was positively associated with polyunsaturated fatty acid, i.e., eicosapentaenoic and arachidonic acid [ACAA1 was reported to be involved in the fatty acid β-oxidation signal pathway [ADIPOQ level was associated with the polyunsaturated fatty acid content in animal models [ELVO6 is the enzyme that catalyzed the transformative reaction from hexadecanoic acid into stearic acid, but no significant difference was observed between groups in this study. These findings revealed the LPGAT1, PDK4, ACAA1, and ADIPOQ genes were associated with the higher level of stearic acid, octadecadienoic acid, and polyunsaturated fatty acid in the Yorkshire pigs.The biological process class of the “monocarboxylic acid metabolic process” was also enriched by downregulated DEGs , which cl donors . Genome-f plasma . PDK4-spnic acid . ACAA1 w pathway . The ADIl models . ELVO6 iMIF (macrophage migration inhibitory factor) deficiency ameliorated high-fat diet-induced insulin resistance in mice and reduced adipose inflammation [IL-34 is associated with obesity, chronic inflammation, and insulin resistance [ADAM8 was involved in chronic inflammation in fat tissue that is contributing to the development of obesity-related insulin resistance [HMOX1 (heme oxygenase 1) could rescue adipocyte dysfunction in mice, and this gene is important to the adaption of chronic hypoxemia [Vav1 in MSCs led to spontaneous adipogenic and displayed an increase in fat content [TLR8 gene was significantly upregulated in obesity as compared with lean individuals, the increase of which has consensus with inflammatory signatures [It has been surveyed that fat mass will be augmented in response to excess caloric intake leading to obesity and further to inflammatory signaling . As partammation . IL-34 isistance . ADAM8 wsistance . HMOX1  is secreted from adipose tissue into the bloodstream to regulate other organ and to be inversely correlated with body mass index [IRX3 could promote browning of white adipocyte and correlate with human obesity [Many interesting terms of biological process were also enriched by downregulated DEGs , includir organs –40 but wogenesis . Variantpulation . Adiponess index , 44. IRX obesity . It is w obesity , 47. So,LPXN, IL7R, GRPEL2, ACTG2, WISP2, ST14, ZNF277, SCEL, CAMK2A, and HACD1, were related to the RFT trait. Eight genes, including ND1, PLEK, RHOBTB3, HSDL2, GPR83, SCN3B, KIAA1211, and CBR1, were associated with the LFP trait. There were also 8 genes, including ENSSSCT00000014378, MXRA5, PENK, SGCG, ESPN, FERMT3, AARD, and ARMCX6, associated with the LFT trait. Five genes were correlated to the SFT trait, including SLC36A2, SHMT1, GSTM3, PFKFB1, and TIMP3. Four genes were significantly correlated to the AFT trait, such as DPP6, SLC36A2, PFKFB1, and TIMP3. Only 2 genes, S100B and XKR6, were involved in BFT. Furthermore, three genes, SLC36A2, TIMP3, and PFKFB1, were associated with both the SFT and AFT traits. Of these genes, some had been demonstrated to be involved in fat or adipocyte-related physiological process. TIMP3 overexpression in macrophages resulted in reduced activation of oxidative stress signals related to lipid peroxidation in fat tissue [IL-7R deletion ameliorated diet-induced obesity and insulin resistance in mice [SLC36A2 was identified as membrane surface marker of brown and beige adipocytes [XKR6 gene, was associated with body fat distribution [To better identify the potential function of differentially expressed gene, association analysis between gene expression level and phenotypes was investigated. After statistical testing, 34 DEGs were significantly associated with the fatness trait, which consisted of 14 positive and 20 negative correlation events . A totalt tissue . IL-7R d in mice . SLC36A2ipocytes . A signiribution .CYBB, CLU, CD84, ADGRE1, S100B, RGS17, C1QA, CFP, ENSSSCG00000028376, CST3, INPP5D, FAAH, NYAP2, and ALDH3A1. There were also 10 genes correlated to polyunsaturated fatty acid, which included CES1, CYBB, CD84, S100B, RGS17, C1QA, CFP, MYO1F, ENSSSCG00000028376, and CST3. Nine genes, such as ACACA, KDELC2, CAMKK1, PRUNE2, MUM1L1, SLIT2, SLC37A2, PARM1, and ATP1B1, were related to hexadecanoic acid level. Totally, 7 genes, LCP1, CRLF2, CD53, C1QC, SYCP2, ENSSSCG00000013144, and TIMELESS, were associated with the octadecenoic acid content. PENK, RGS2, SGCG, ESPN, AARD, and KIT were associated with the stearic acid content. SMPD3, ANO2, INPP5D, FAAH, NYAP2, and ALDH3A1 were related to the octadecadienoic acid content. For octadecatrienoic acid, ND1, LSAMP, LGALS12, VSTM4, EGR2, and ENSSSCG00000001252 were related. There were also 5 genes, SPHK1, NCS1, CTSB, SHROOM1, and SPOCK2, associated with the eicosadienoic acid level. Four genes, SMPD3, HCLS1, FABP7, and RUNX1, were correlated to monounsaturated fatty acid. There were only 3 genes, LEPR, TNFAIP6, and HSPA5, related to hexadecenoic acid. Some genes were associated with more than one trait. For example, CYBB, CD84, S100B, RGS17, C1QA, CFP, ENSSSCG00000028376, and CST3 were associated with both the eicosenoic acid and polyunsaturated fatty acid levels. However, no gene was observed to significantly associate with the other phenotypes of fatty acid. Most genes were first reported to be associated with the fatty acid content in this study. The reason behind is that most genes might indirectly involve in fatty acid composition, and less studies about fatty acid composition were reported. SNPs of LEPR were reported to be associated with SFA in subcutaneous fat. In this study, the LEPR expression level significantly correlated with the hexadecenoic acid content. To some extent, these findings are partly similar to published result and would be regarded as important foundational evidence for further study.There were also 57 DEGs significantly associating with the fat composition trait . A totalcis- and trans-regulating models were analyzed. For the cis-acting model, DEGs physically located within 100 kb near the differentially expressed lncRNAs were obtained. The 9 pairs of lncRNA-DEGs with cis-regulating effect were identified (cis-regulatory effect of MSTRG.8034 on ACACA is positive, but it is not significant. It was reported that ACACA catalyzed the first committed step of fatty acid synthesis, from carboxylation of acetyl-CoA to malonyl-CoA, leading to the biosynthesis of long-chain fatty acids [cis-regulatory effect of MSTRG.29167 on the RDH16 gene was also observed (RDH16 was correlated to fatness or fat composition trait, but it also showed differentially expressed in fat tissue between lean and obese pig breeds [To find the potentially biological function of differentially expressed lncRNAs, the entified . Of thesty acids . A signiobserved . No obvig breeds .Trans-acting correlations of expression between differential lncRNAs and DEGs were examined. A total of 49 significant targeting events between 21 lncRNAs and 42 DEGs were found (MSTRG.14097-SCD and MSTRG.42256-NPDC1, were identified by both the Trans- and Cis-acting models. SCD was discussed to be involved in the fatty acid composition trait in pigs as before. It was demonstrated that NPDC1 might participate in antiobesity effects of the sodium alginate in high-fat diet-induced obese mice [CBR1, CD74, and SCD were enriched in unsaturated fatty acid biosynthetic process term. The regulatory lncRNAs of CBR1 and CD74 were MSTRG.30986 and MSTRG.4275, respectively. The CD74 and CBR1 expression of adipocyte is involved in fatty acid synthesis process and metabolic complications of obesity [re found . Two paiese mice . Functio obesity , 56. It cis- and trans-targeting analysis demonstrated that some lncRNAs functioned as regulators of fatness and fatty acid composition. Functional analysis revealed that 12 candidate genes contributed to fat acid composition and 10 genes are involved in the pig fatness trait. Association analysis also identified 34 and 57 genes correlated to fatness and fatty acid composition trait, respectively. These findings will help us decipher the fatness and fatty acid composition trait of pig and deserve to be further explored.In this study, the lncRNA and gene profiles from back-fat tissue were investigated in the Tibetan and Yorkshire pigs. In total, 1,964 lncRNAs were identified through our computational pipeline. Evidence from lncRNAs


TP53, MDM2 and ZBTB4 were evaluated in an independent set of cases by qPCR. PC cases presented genomic complexity, while PIA samples had a small number of CNAs. Recurrent losses covering well-known tumor suppressor genes, such as ATM, BRCA1, CDH1, MEN1 and TP53, were found in PC. The in silico functional analysis showed several cancer-related genes associated with canonical pathways and interaction networks previously described in human PC. The MDM2, TP53, and ZBTB4 copy number alterations were translated into altered expression levels. A cross-validation analysis using The Cancer Genome Atlas (TCGA) database for human PC uncovered similarities between canine and human PCs. Androgen-receptor-negative canine PC is a complex disease characterized by high genomic instability, showing a set of genes with similar alterations to human cancer.Canine carcinomas have been considered natural models for human diseases; however, the genomic profile of canine prostate cancers (PCs) has not been explored. In this study, 14 PC androgen-receptor-negative cases, 4 proliferative inflammatory atrophies (PIA), and 5 normal prostate tissues were investigated by array-based comparative genomic hybridization (aCGH). Copy number alterations (CNAs) were assessed using the Canine Genome CGH Microarray 4 × 44K (Agilent Technologies). Genes covered by recurrent CNAs were submitted to enrichment and cross-validation analysis. In addition, the expression levels of  Prostate cancer (PC) is the second most common cancer in men worldwide and its incidence is especially high in developed countries . Among mDespite all the differences, there are also some similarities at pathological, clinical, and molecular levels between canine and human PC. The concomitant presence of prostatic intraepithelial neoplasia, proliferative inflammatory atrophy (PIA), and bone metastases as well as the deregulation of cancer-related transcripts/proteins, including MDM2, PTEN, TP53, CTNNB1, and CDH1, are common events in dog and human PCs ,15,16,17The first reports using comparative genomic hybridization (CGH) analysis to evaluate copy number alterations (CNAs) in dogs were performed in a glial tumor cell line and lymphomas ,19. The TP53, ZBTB4, and MDM2) involved in regions with losses or gains were evaluated at the gene expression level. We also compared the genomic alterations in canine and human PCs. To our knowledge, this is the first study that has evaluated genomic imbalances in a cohort of dogs with spontaneous PC.In canine PC, the cell line named Probasco, derived from a mixed breed dog with spontaneous tumors, presented genomic complexity, including large deletions, and duplications mapped in various chromosomes . In thisAZIN2, ADIPOQ, BCL6, CRYGS, MIR28, MIR8903, and SST genes) were exclusively detected in PIA cases. Furthermore, 17 genes  were covered by CNAs in both PIA, and more than 20% of PC cases (≥3 cases). The detailed data are presented in The genomic profiling of 4 PIAs revealed 45 CNAs , with a mean of 11.25 alterations per case A. TwentyA total of 370 CNAs  were found in 14 PC samples. Particularly, three cases  presented more than 60 CNAs. Almost all chromosomes had genomic alterations, except chromosome 19 B. LossesRecurrent alterations found in more than 20% of PC samples are depicted in The interspecies cross-validation analysis performed using human PC from The Cancer Genome Atlas (TCGA) database revealed 79 genes altered by genomic losses in both species (>20% of samples) . The synTP53 , and MDM2 (three cases): 1.874149; 0.471251; 0.333158. The qPCR analysis showed a significant down-expression of A comprehensive evaluation of canine cancer has the potential to uncover molecular mechanisms common to the human disease, including tumor progression, metastasis, and diagnostic, and prognostic markers. The presence of a high level of similarities opens avenues to identify combined or alternative therapies to better treat the disease in both species .ADIPOQ, and BCL6) were previously described in human PC [AK1 silvia.regina.rogatto@rsyd.dk (S.R.R.) silvia.regina.rogatto@rsyd.dk (S.R.R.) silvia.regina.rogatto@rsyd.dk (S.R.R.) AKT2, BRD2, CD4, FAM83H, GNB3, MIR221, MIR338, SCN2B, TIMP1, and TPI1) of 17 genes affected by CNAs in PIA, and also PC samples were associated with different aspects of human prostate carcinogenesis . A previous report using gene expression analysis showed that TIMP downregulation was implicated in the progression of prostatic intraepithelial neoplasia to PC [In a limited number of PIA cases studied, we detected a small number of CNAs, which revealed a low level of genomic instability. Among the seven genes exclusively identified in PIA, two  was characterized by CNAs . AlthougBRCA1, BRCA2, CDH1, and TP53, are common in human PC [ATM, BAX, BRCA1, CDH1, MEN1, and TP53. Four homozygous deletions harboring genes  were identified in three different PC cases. Among them, LOC403555 has an uncertain function, and TNNT2 is related to contraction of cardiac muscle cells [In human PC, chromosomal imbalances are prevalent and significantly more frequent than somatic mutations ,33. Delehuman PC ,35,36. Sle cells .QK1, BRD2, and EMR4P). Among them, BRD2 has been described as overexpressed in human castration-resistant PC [BRD2. In human PC cases from TCGA, CNAs mapped at BRD2 are rare , being 18 gains, and 23 losses.Gains and amplifications have been associated with the activation of proto-oncogenes, which promote gain of function and contribute to cancer development or progression. We detected 14 high copy number gains and only 3 covered known genes (stant PC . NeverthCLU), deleted in ~55% of TCGA cases, was associated with neoplastic transformation and tumor progression, while downregulation of MX1 was involved in proliferation, progression and metastasis of PC cells [ZBTB4 gene, a transcriptional repressor gene that binds to the GC-rich promoter regions of their targets, increases genomic instability (aneuploidy) and promotes tumorigenesis in human neoplasia, including PC [ZBTB4 overexpression is related to an increased survival time for PC-affected patients [Interestingly, the synteny map showed similarities between recurrent losses detected in canine PCs with chromosomal losses described in human PCs, including 2q, 3p, 8p, 12p, 16q, 17p, and 21q.36. An interspecies cross-validation analysis using TCGA database, which currently contains 492 human PC cases with CNA information, revealed additional similarities between human and canine PC. At least 79 genes were altered by gains/losses in more than 20% of our cases and those from the TCGA. The inactivation of the clusterin gene , and PTEN/NKX3-1 inactivation (point mutations or hypermethylation).In addition to the similarities, we also observed differences between canine, and human PCs. Highly prevalent alterations in human PC, such as gains of the one case ,34. Whil studies ,35. Usinpression ,35. A plAn interesting difference between canine and human PC relates to the AR. AR has been described as involved in the development, progression and survival of human PC cells ,51. In hAll canine PC AR-negative cases herein evaluated by CNAs presented a Gleason score of 6 and no metastasis. In addition, no information on AR protein expression in TCGA dataset was available, thus limiting a more detailed comparison. Although these factors are limitations of our study, we reported similar genomic alterations in both human and canine PC, addressing a gap in the literature. Further studies are needed to unravel genes and pathways involved in more aggressive canine PC, including in metastatic cases.This study was performed in accordance to the National and International Recommendations for the Care and Use of Animals. All procedures were performed under the approval of the Animal Ethics Committee from Faculty of Veterinary Medicine and Animal Science, UNESP, Botucatu, SP, Brazil (Protocol #10/2007).The total number of cases included in this study was 33 PCs, 4 PIAs, and 15 normal prostate tissues. Array-CGH assays were performed in 14 PC, 4 PIA, and 5 normal prostatic tissues. The necropsies were performed within an interval of 1 h from the animal’s death, with the consent of the owners. The mixed breed dogs were intact and had a mean age of 9 years. Although they had no clinical features of PC or PIA, lesions were found in the histopathological analysis. The prostatic gland was collected, fixed in 10% formalin, routinely processed, embedded in paraffin and sectioned. The areas of interest were identified and the cells were obtained from macrodissected samples using a 16-gauge needle, as previously described . Tumor aTP53, MDM2, and ZBTB4) by qPCR. The mean age was 10.2 years (8–15 years), and the mean survival time was 237.6 days (12–523 days). All 19 PC patients were evaluated by X-ray, and computerized tomography (CT) for screening of metastasis at the diagnosis. Thirteen of them (13/19) had metastatic disease at diagnosis in multiple sites including bone (7/13), lung (5/13), intestine (2/13), liver (1/13), and iliac lymph node (1/13). All PC cases were negative for androgen receptor immunostaining. In this cohort of 19 PC cases, 13 presented a Gleason score of 10, 6 a Gleason score of 6 and 3 a Gleason score of 8. The metastatic cases presented Gleason scores of 10 (10 of 13 cases) and 8 (3 of 13)  sample set composed by 19 PC (biopsy samples), and 10 normal prostate tissues (necropsy samples) were used to evaluate the gene expression levels (3 of 13) .Genomic DNA was extracted from FFPE samples using the Qiagen DNeasy Blood & Tissue Kit  according to the manufacturer’s recommendations. A pool of genomic DNA obtained from five FFPE canine normal prostate tissues was used as reference.http://visualization.ritchielab.psu.edu/phenograms/document).Copy number alterations were assessed using the Canine Genome CGH Microarray 4 × 44K G2519F , according to the manufacturer’s instructions. Data were extracted with Feature Extraction 10.1.1.1 software (Agilent), and the analyses were performed using Agilent Genomic Workbench Standard Edition 5.0.14 software . Nonredundant CNAs called by at least three consecutives probes and a log2 ratio > 0.3 for gains and <−0.3 for losses were considered as significant. The ideograms showing the CNAs identified in each case were constructed using the PhenoGram online software , considering the genes affected by CNAs in more than 20% of PC cases. In this analysis, the following parameters were used: core analysis, Ingenuity Knowledge Base (genes only) as a reference set, direct and indirect relationships, endogenous chemicals, 35 molecules per network and a maximum of 25 networks. Statistical significance was considered with p < 0.05 calculated using Fisher’s exact test. The activation (orange lines in the network) or inhibition (blue lines in the network) was used with z-score × 2.0 or ≤ −2.0, respectively. The CNA information of 492 human PCs from TCGA database, stored in the cBioPortal for Cancer Genomics , was used to explore the similarities between canine and human PCs.Enrichment analysis, based on human data, was performed with Ingenuity Pathway Analysis software  slide to guide the tissue dissection was demarcated and isolated using a 40-gauge needle in the FFEP block. Total mRNA was extracted using the RecoverAll™ Total Nucleic Acid Kit , according to the manufacturer’s instructions. cDNA synthesis was carried out as previously described . The quaTP53, MDM2, and ZBTB4) were selected and evaluated by qPCR based on their importance in carcinogenesis, network analysis and TCGA cross-validation analysis. TP53, and MDM2 are well-known cancer genes involved in the emergence of several human malignancies, including PC. TP53 has been implicated in several cancer-related canonical pathways, and MDM2 is a central node in the molecular network 2. In addition, ZBTB4 indirectly interacts with MDM2, and has also been related to prostate carcinogenesis. Moreover, TP53, and ZBTB4 deletions were cross-validated using TCGA database analysis.Three genes  were previously published [ZBTB4 was forward: 5′TTGGGATTTTGCCATTTCG 3′, and reverse: 5′GGGCAAGGAGGGCACAA 3′. qPCR for TP53, MDM2, ZBTB4, and the endogenous genes was conducted in a total volume of 10 μL containing Power SYBR Green PCR Master Mix , 1 μL of cDNA (1:10), and 0.3 μM of each primer. The reactions were performed in triplicate in 384-well plates using QuantStudio 12K Flex Thermal Cycler equipment . The PCR product specificity was determined by dissociation curve for all experiments. Relative gene expression was quantified using the 2−ΔΔCT method [The primer sequences of ublished ,60. The T method .TP53, MDM2, and ZBTB4 transcript levels between normal and PC samples. Statistical significance was set at p < 0.05.Statistical analysis was performed using GraphPad Prism 5 v.5.0 . The Kruskal–Wallis or Mann–Whitney U test was applied to compare TP53, and ZBTB4, and gains of MDM2 were translated in down- and overexpression, respectively, at the transcriptional level. Furthermore, TCGA cross-validation analysis pointed out similarities between human and canine prostate carcinogenesis. Canine PC is an interesting model to use in comparative prostate cancer research; however, it is important to note the differences, including those reported for the AR gene.To our knowledge, this is the first study to describe the genomic profile in a cohort of canine PIA, and PC samples. Our results revealed that PIA cases presented a small number of CNAs compared with PC. PC samples presented a large number of chromosomal imbalances, mainly deletions, which revealed a set of cancer-related genes involved in canonical pathways or networks previously described in human PC. Deletions of


Nanog, Oct3/4, Sox2, Klf4, and c-Myc, and two human cancer cell lines with similar stem cell gene expression. Deferasirox, an orally available iron chelator, suppressed expression of stemness markers and spherogenesis of cells with high stemness status in vitro. Combination therapy had a marked antitumor effect compared with deferasirox or cisplatin alone. Iron metabolism appears important for maintenance of stemness in CSCs. An iron chelator combined with chemotherapy may be a novel approach via suppressing stemness for CSC targeted therapy.Excess iron causes cancer and is thought to be related to carcinogenesis and cancer progression including stemness, but the details remain unclear. Here, we hypothesized that stemness in cancer is related to iron metabolism and that regulating iron metabolism in cancer stem cells (CSCs) may be a novel therapy. In this study, we used murine induced pluripotent stem cells that expressed specific stem cell genes such as  Iron is an essential element and plays crucial roles in our body, including roles in cell growth, proliferation, DNA synthesis, and energy metabolism. On the other hand, excess iron is associated with tumorigenesis in many types of human cancers ,2,3 and According to the cancer stem cell (CSC) hypothesis, CSCs exist in many types of cancer tissues and are considered resistant to conventional types of therapy such as chemotherapy and radiation therapy. CSCs are also related to recurrence and metastasis. CSCs have been reported in various types of cancer ,10,11,12CSCs possess the features of normal stem cells, including self-renewal and pluripotency, in addition to cancer cell features. CSCs are distinguished by the expression of stemness markers. From the viewpoint of stem cell hierarchy, embryonic stem cells (ES cells) possess the properties of pluripotency and self-renewal and are at the top of the stem cell hierarchy. Several transcription factors, including Nanog, Oct3/4, Sox2, Klf4, and c-Myc, regulate the stemness of ES cells and are also upregulated in various types of CSCs ,15,16,17To evaluate the effect of DFX on expression of stemness markers in miPS cells, the cells were cultured with several concentrations of DFX . DFX suppressed expression of stemness markers at concentrations over 50 μM A. To evaTo address the effect of DFX on tumorigenicity and expression of stemness markers in vivo, we employed a subcutaneous allograft model by using BALB/c nude mice. miPS cells were treated with 0.2% dimethyl sulfoxide (DMSO) for 48 h as the control group or with 50 μM DFX for 48 h as the DFX group and then injected into the right flank. Tumorigenesis was observed. Fourteen days after injection, tumors were harvested, and the tumor volume and immunohistochemistry of stemness markers were evaluated. The tumorigenesis of the DFX group was significantly suppressed compared to the control group A. The tuNext, to assess the effect of DFX and CDDP on cytotoxicity and expression of stemness markers in human cancer cell lines, we used HSC-2 cells and OE33 cells, which express similar stemness markers  as ES cells. The XTT assay showed that DFX suppressed proliferation and expression of stemness markers A,B in HSTo explore the effect of DFX on self-renewal, a sphere formation assay was performed. DFX suppressed the spherogenicity of HSC-2 cells and OE33 cells compared to the control group A. FurtheExpression of Oct3/4 and Klf4 in addition to Nanog was suppressed by si-Nanog C. HoweveTo evaluate the effect of combination therapy using DFX and CDDP on cytotoxicity, synergy, and stemness in HSC-2 cells and OE33 cells, the XTT assay, combination index, and western blot analysis were performed. The XTT assay showed that cytotoxicity of combination therapy against HSC-2 cells and OE33 cells increased in a dose-dependent manner A. CombinTo address the effect of combination therapy on tumorigenicity and expression of stemness markers in vivo, we employed the subcutaneous xenograft model of HSC-2 cells by using BALB/c nude mice. DFX was administered orally concerning clinical use. Tumor volumes of control group mice increased during the experimental period. Only the tumor growth of the combination group was significantly decreased compared to the control group A,C. In aIn our study, we employed miPS cells as a model of ES cells, which possess high stemness status. We also employed HSC-2 cells and OE33 cells as models of heterogeneous cancer tissue that includes CSCs. We focused on the point that some CSCs express similar stemness transcription factors  as ES cells. These transcription factors are important for the maintenance of pluripotency ,20,21. DOne problem in current cancer treatment is the existence of CSCs, which are resistant to conventional chemotherapy and radiation and are considered to be related to metastasis and recurrence ,23. SomeRaggi et al. reported that iron metabolism is related to stemness of cholangiocarcinoma stem-like cells . They shAs a therapeutic strategy for cancer and CSCs, attention is recently focused on iron metabolism ,36,37. WOur study has the following limitations. Although we confirmed that DFX suppressed the expression of stemness markers in CSCs, DFX did not recognized the CSCs from cell surface antigen. CD44 antibody includes both standard and variant isoforms. We did not check the ratio of CD44 variant isoform. We employed bulk cells as a model of heterogeneous cancer tissue and only focused on the effect of DFX against CSCs. In addition, we did not evaluate the relapse in vivo. Further studies are needed to evaluate the effect of DFX on other stemness markers.We also need to confirm the status of cells after treatment with DFX. DFX may induce specific cytotoxicity against cells with high stemness status or may lead to differentiation of such cells. Either way, the fact that DFX suppressed stemness in cancer tissue is important and suggests that DFX may be a useful treatment option. Thus, clarification of the mechanism is urgently needed.In conclusion, regulating iron metabolism may be a novel strategy via suppressing stemness for CSC targeted therapy.2.miPS cells were purchased from Riken Cell Bank . The human oral squamous carcinoma cell line (HSC-2) was obtained from Showa University. The human esophageal adenocarcinoma cell line (OE33) was purchased from The European Collection of Authenticated Cell Cultures . We also used the human fibroblast cell lines, FEF3 and WI38, as representative cells with a “normal” non-cancerous phenotype. FEF3 cells were isolated from human fetal esophagus as described previously . WI38 fel-glutamine, 0.1 mM 2-mercaptoethanol, 1000 U/mL leukemia inhibitory factor, 50 U/mL penicillin, and 50 U/mL streptomycin) on feeder layers of mitomycin C-treated mouse embryonic fibroblast cells . HSC-2, FEF3, and WI38 cells were maintained in DMEM containing 10% FCS, 50 U/mL penicillin, and 50 U/mL streptomycin. OE33 cells were maintained in RPMI containing 10% FCS, 50 U/mL penicillin, and 50 U/mL streptomycin.miPS cells were maintained in medium (Dulbecco’s modified eagle medium (DMEM) containing 15% fetal calf serum (FCS), 0.1 mM non-essential amino acids, 2 mM Deferasirox  was obtained from Novartis Pharma . For in vitro studies, DFX was dissolved in DMSO  at a stock concentration of 50 mM. For in vivo studies, DFX was dissolved in saline. Cisplatin  was purchased from Nippon Kayaku  and dissolved in phosphate-buffered saline (PBS).3/well), OE33 (3.0 × 103/well), FEF3 and WI38 (2.0 × 103/well).The XTT assay  was used to assess cell proliferation according to the manufacturer’s protocol. The cells were seeded in 96-well plates and treated with DFX and/or CDDP for 48 h at 37 °C. The combination index was calculated with CalcuSyn software . We seeded the cells as follows: HSC-2 (6.0 × 102 cells/well and maintained in DMEM/nutrient mixture F-12 (Sigma) containing B-27 supplement , 20 ng/mL epidermal growth factor (Sigma), 10 ng/mL fibroblast growth factor (Sigma), 0.4% bovine serum albumin (Sigma), and 5 μg/mL insulin (Invitrogen) for 7 days.Cells were seeded in 96-well ultra-low attachment plates  at a density of 5 × 10The Live/Dead viability assay was performed to assess cell viability of miPS cells after treatment with DFX. The miPS cells were treated with 0.2% DMSO or 50 μM DFX for 48 h, rinsed with PBS, and incubated with Calcein AM  and Ethidium homodimer-1 (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were visualized using a fluorescence microscope .To confirm the effect of Nanog on spherogenicity, HSC-2 cells were transfected with Silencer select siRNA against NANOG  or scrambled control  using Lipofectamine RNAiMAX (Invitrogen) at a final concentration of 20 nM and 10 nM, respectively. Sphere growth was initiated 48 h post-transfection.Protein was extracted from whole cells after 48 h of incubation in medium and reagents. The concentrations of extracted protein were measured using standard protocols. Cells were lysed using cell lysis buffer (50 mmol/L Tris-HCl (pH 7.4), 30 mmol/L NaCl, and 1% Triton X-100) containing protease inhibitors . Equal amounts of total cellular proteins (50 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred electrophoretically to polyvinylidene difluoride filter membranes  according to the manufacturer’s protocol. The following primary antibodies were used: anti-Nanog antibody , anti-Sox2 antibody , anti-Oct3/4 antibody , anti-KLF4 antibody , anti-c-Myc antibody , anti-transferrin receptor antibody , anti-DMT1 antibody , anti-ferroportin/SLC40A1 antibody , anti-ferritin heavy chain antibody , anti-β-actin antibody , anti-PARP antibody , anti-cleaved caspase-3 antibody , anti-caspase 3 antibody , anti-Stat3 antibody , and anti-phospho-Stat3 antibody . All primary antibodies were used at a 1:1000 dilution. The following secondary antibodies were used: anti-Mouse IgG, HRP-Linked Whole antibody Sheep , anti-Rabbit IgG, HRP-Linked Whole antibody Donkey , anti-Rat IgG, HRP-Linked Whole antibody Goat . All secondary antibodies (GE Life Sciences) were used at a 1:2500 dilution. The membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies. ECL prime Western Blotting Detection Reagent  was used to detect the peroxidase activity of secondary antibodies. Membranes were probed for β-actin as a loading control, and all sample data values were normalized to the corresponding control data values. Densitometric analysis was performed using Image J software (NIH).4 cells/mL in 6 well plates 24 h before treatment with different concentrations of DFX for 48h, after which cells and medium were recovered, centrifuged . Cells were suspended in PBS containing 2% fetal bovine serum and 0.1% sodium azide, and stained with the anti-mouse/human CD44 Antibody  and propidium iodide staining (Life Technologies Corporation) after 10 min of pre-incubation with human TruStain FcX . Cell fluorescence was detected with MACSQuant Analyzer  and analyzed with MACSQuantify Software.HSC-2 and OE-33 cells were seeded at 5 × 104 cells/mL in 6 well plates 24 h before treatment with different concentrations of DFX for 48 h. Total RNA was isolated from HSC-2 and OE-33 cells using Trizol Reagent  and High Pure RNA Isolation kit , respectively. First-strand cDNA was constructed from total RNA using the oligo (dT) primer. Real-time quantitative PCR analysis was performed using StepOne with Taqman PCR master mix . The primers used in this study were: GAPDH (Applied Biosystems) and Nanog . The quantification of the gene of interest was normalized to GAPDH and expressed as fold-increases relative to the negative control for each treatment at each time point as previously described.HSC-2 and OE-33 cells were seeded at 5 × 10To evaluate the supernatant VEGF secreted by HSC-2 and OE33 cells, we used a VEGF enzyme-linked immunosorbent assay (ELISA) kit . The cancer cells were plated in 6 well plates and were treated with different concentrations of DFX. After a 48-h treatment, the supernatant and cells were harvested and VEGF content was assayed by ELISA according to the protocol provided by the manufacturer.6) were injected subcutaneously into the right flank. When the tumor size reached 150~200 mm3, the mice were randomly divided into four groups . Each group was treated with oral gavage of saline or DFX (160 mg/kg) three times per week for 3 weeks and by intraperitoneal injection of saline or CDDP (6 mg/kg) once per week for 3 weeks. Tumor size and body weight were measured every 3 days. The tumor volume (mm3) was calculated with the formula d2 × D/2 where d and D are the shortest and longest diameters in mm, respectively. At the end of the experiment, the mice were sacrificed, and the tumors were excised, weighed, and processed for histological analysis.All animal experiments were performed according to the Japanese Welfare and Management of Animals Act and conducted in accordance with institutional guidelines at Shigei Medical Research Institute, Okayama, Japan. All animal experiments were approved by the Ethics Review Committee for Animal Experimentation of Shigei Medical Research Institute (#160401-1), Okayama, Japan. Female BALB/c (nu/nu) mice were purchased from CLEA Japan . Female BALB/c (nu/nu) mice were purchased from CLEA Japan. The experiment started when the mice were 9 weeks of age. HSC-2 cells in culture were harvested and resuspended in a 1:1 ratio of PBS and Matrigel (BD Biosciences). HSC-2 cells (3.0 × 10http://rsb.info.nih.gov/ij/).Harvested tumors were fixed in 10% paraformaldehyde and embedded in paraffin prior to immunostaining. The same anti-Nanog antibody, anti-Sox2 antibody, anti-Oct3/4 antibody, anti-KLF4 antibody, and anti-c-Myc antibody were used as described in the western blot analysis section. Evaluation of the Nanog, Sox2, Oct3/4, KLF4, and c-Myc area index was performed with Image J software . For two-group comparisons, Student’s Nanog, Oct3/4, Sox2, Klf4, and c-Myc was suppressed by iron chelator. By using iron chelator, regulating iron metabolism and combined with chemotherapy may be a novel strategy for CSC targeted therapy via suppressing stemness.In conclusion, to the best of our knowledge, iron metabolism appears important for maintenance of stemness in cell lines with high stemness status including CSCs. The expression of stemness markers such as


FLOWERING LOCUS T (FT). However, the function of FT(s) in alfalfa is largely unknown. Here, we identified MsFTa, an FT ortholog in alfalfa, and characterized its role in flowering regulation. MsFTa shares the conserved exon/intron structure of FTs, and the deduced MsFTa is 98% identical to MtFTa1 in Medicago trucatula. MsFTa was diurnally regulated with a peak before the dark period, and was preferentially expressed in leaves and floral buds. Transient expression of MsFTa-GFP fusion protein demonstrated its localization in the nucleus and cytoplasm. When ectopically expressed, MsFTa rescued the late-flowering phenotype of ft mutants from Arabidopsis and M. trucatula. MsFTa over-expression plants of both Arabidopsis and M. truncatula flowered significantly earlier than the non-transgenic controls under long day conditions, indicating that exogenous MsFTa strongly accelerated flowering. Hence, MsFTa functions positively in flowering promotion, suggesting that MsFTa may encode a florigen that acts as a key regulator in the flowering pathway. This study provides an effective candidate gene for optimizing alfalfa flowering time by genetically manipulating the expression of MsFTa.The production of hay and seeds of alfalfa, an important legume forage for the diary industry worldwide, is highly related to flowering time, which has been widely reported to be integrated by  FLOWERING LOCUS C (FLC)/MADS AFFECTING FLOWEIRNG (MAF), FLOWERING LOUCS T (FT), CONSTANS (CO) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), to determine the flowering time [Medicago, however, FLC/MAF clade flowering repressors are missing and CO-like genes do not seem to regulate flowering [FT, acting as a long-distance hormone signal between leaves and shoot apical meristems, is the most important flowering integrator of the legume species.Flowering time, the switch from vegetative to reproductive growth, is a key trait for the seasonal and geographical adaptation of flowering plants. The mechanism of flowering is best understood in the annual plant Arabidopsis, in which the timely onset of flowering is regulated by an intricate network with various genetic regulators responding to endogenous  or environmental  triggers, as reviewed in recent years ,2,3,4. Ting time ,6,7. In lowering ,9,10. HeFT-like genes have been identified in a wide range of annual plants, such as rice, soybean and barrel clover [M. truncatula five FT-like genes, namely MtFTa1, MtFTa2, MtFTb1, MtFTb2 and MtFTc, have been characterized. Among them, MtFTa1, MtFTb1 and MtFTc rescued the late flowering phenotype of Arabidopsis ft-1 mutant, while the other two genes failed to act as a florigen [FT paralogs derived from lineage-specific duplication has been observed in several species, such as soybean, potato and rice [FT paralogs is attributed to the expression pattern shift and the divergence in gene regulatory networks.Since the search for the flowering hormone florigen in the 1930s, an increasing number of l clover ,12,13. Fflorigen . Functioand rice ,15. The FT-integrated flowering regulation have focused on economically important perennial plants including grasses  [FT is expressed in both leaves and flowers of several perennial species such as sweet cherry, kiwifruit and leafy spurge [FT paralogs revealed the evolution of a complex perennial adaptive trait after genome duplication. For example, poplar FT1 determined the onset of reproductive stage in winter, whereas FT2 promoted vegetative growth and inhibited bud set in the growing season [FT perennial orthologs in Arabidopsis caused early flowering in most cases, suggesting a conserved function in perennial species [In recent years, studies on yegrass) ,17 and tyegrass) ,19,20. Iy spurge ,22,23. Fg season . Ectopic species ,19,20,22M. sativa), one of the most important forages globally, exhibits seasonal flowering. Different from perennial woody plants, which are not able to form flower buds during the first several years of their life cycle, herbaceous alfalfa grows vegetatively at early developmental stages and then switches to the reproductive stage in the same year. In Northern China, a main alfalfa planting region in the country, the first cutting coincides with the rain season, which severely limits hay production. Breeding alfalfa cultivars with optimal flowering time would facilitate the improvement of the quantity and quality of alfalfa. Our study here demonstrated that MsFTa, the first isolated FT from alfalfa, not only shared the conserved functional domains of its orthologs, but also acted ectopically in accelerating flowering. These findings would supply a potential candidate for the generation of alfalfa cultivars with an optimal flowering time for the main planting regions in Northern China via genetic modulation of MsFTa expression.Alfalfa (FLOWERING LOCUS T (FT) genes have been intensively documented in many plants including M. truncatula, a model annual legume species [MtFTs, namely MtFTa1, MtFTa2, MtFTb1, MtFTb2 & MtFTc, have been identified from M. truncatula. Using MtFTa1 as a query sequence, we did a BLAST search against the most updated M. truncatula genome database (http://plants.ensembl.org/Medicago_truncatula) and found that besides the four MtFT paralogs, one more gene (Mt6g033040) annotated as Flowering Locus Protein T (MtFT) was hit with a homology of 64–73% to its paralogous proteins  of 531 bp (GenBank: JF681135)  . The putF681135) . The putF681135) . Hence,  soybean . Sequenc soybean . The seq soybean . Multipl soybean  [24,25,2 soybean . Our pre soybean , indicatFT-like genes demonstrated that like most FT-like genes from a variety of species [MsFTa is composed of four exons separated by three introns (FT-like genes (Hv3Hr1g0871002 and Hv7Hr1g0246102)  at position 85  . The 2ndition 85 . TherefoMsFTa, 35S::MsFTa-GFP and 35S::GFP were transformed into onion epidermal cells separately. The transient expression of the GFP control was uniformly distributed throughout the cell, whereas the MsFTa-GFP fusion protein was observed in the nucleus and cytoplasm, especially the membrane   .MsFTa in modulating flowering, the construct of MsFTa driven by the cauliflower mosaic virus 35S promoter was introduced separately into wild type Arabidopsis and ft-10, a T-DNA insertion mutant of FT, by agrobacterium infiltration. The existence of a 35S::MsFTa construct in two independent transgenic Arabidopsis T2 lines (T2-1 & T2-4) with kanamycin resistance was confirmed by PCR (MsFTa transcript was abundantly expressed in both transgenic lines while absent in wild type Arabidopsis (p < 0.05) (p < 0.05)  d. Consis < 0.05) e. These ft-10 is a strong allele showing late-flowering phenotype. Under LD, ft-10 flowered when the mutant plant had 36.8 leaves, while wild type flowered with 15.4 leaves (p < 0.05)  f,g. For 5 leaves f,g, indiMsFTa to MtFTa1, both barrel clover wild type R108 and mtfta1-1, a late-flowering mutant (NF3307) caused by the insertion of a Tnt1 retrotransposon in the first exon of MtFTa1 [35S::MsFTa mentioned above. Flowering time of the transgenic plants was measured as days to produce the first flower under LD conditions. Compared with R108 which flowered at D56, the two independent T1 lines (T1-1 & T1-2) overexpressing 35S::MsFTa produced the first flower at D35 and D32, respectively. Over-expression of MsFTa in M. truncatula resulted in statistically significant early flowering (p < 0.05) , indicating that MsFTa successfully rescued the late flowering phenotype of fta1-1. These results suggested that similar to ectopic expression of MsFTa in Arabidopsis, introduction of MsFTa into M. truncatula caused strong flowering promotion.Given the close phylogenetic relationship and high sequence identity of f MtFTa1 , were tr < 0.05) a. For mtFLOWERING LOCUS T appears to be a universal regulator ultimately integrating flowering signaling in crops, vegetables, grass and trees [MsFTa, an FT homolog from alfalfa, functioned to accelerate flowering.Precise flowering time ensures that the onset of flowering occurs at an ideal time to optimize plant biomass and fitness in specific environmental conditions. Although diverse upstream components of flowering pathways have been identified from plants, nd trees ,18,26,30FT has a conserved exon/intron structure and highly identical protein sequence across species [MsFTa possessed the characteristic features of a classic FT gene. First, MsFTa, similar to FT-like genes, has four exons with conserved exon/intron boundaries [FT revealed that substitution of the fourth exon, especially the external loop caused late flowering, presumably the crystal structure was affected due to the sequence variation of the exon [FTs share a triad ending with residue asparagine (N) at site 152, followed by leucine  gene family,  species ,31. Indeundaries ,27,28. Othe exon . It has the exon ,32. Secothe exon . FTs sha leucine . The crylowering . Third, nal loop , both ofnal loop , implyinFT-like orthologs from a variety of plant species have been shown to promote flowering when ectopically expressed [M. truncatula, reverse genetics revealed that the loss-of-function mutant of MtFTa1, the closest homolog of MsFTa, caused late flowering and MtFTa1 fully complemented fta1-1 defects [MtFTa1 caused by retroelement insertions exhibited a dominant early flowering phenotype [FT, MsFTa from the perennial forage acts positively in flowering promotion. The notion is supported directly by the transgenic Arabidopsis and M. truncatula including both complementation and over-expression plants. Plants ectopically expressing MsFTa in either wild type or ft mutant of both plant species displayed a clear shift to early flowering (p < 0.05). Notably, compared with wild type, complementation plants flowered slightly late, suggesting that constitutive expression of MsFTa partially rescued the flowering time of ft mutants from Arabidopsis and barrel clover. These findings imply a minute functional distinction between MsFTa and its orthologs, FT and MtFTa1, in Arabidopsis and barrel clover, respectively. Application of the endogenous promoter of MsFTa to produce transgenic alfalfa is undergoing. Moreover, generation of a loss of function mutant of MsFTa using the CRISPR-Cas9 strategy would be necessary to elucidate the endogenous function of MsFTa in alfalfa flowering regulation.xpressed ,37,38,39 defects . Consisthenotype ,41, conflowering d–g. StatMsFTa in roots and stems, it is tempting to speculate that besides flowering promotion, MsFTa may participate in other development processes of alfalfa. FT orthologs have been documented to be involved in multiple functions like plant architecture [MsFTa resembled FT orthologs in perennial plants, such as sweet cherry, loquat, leafy spurge and tea-oil tree, in which FT-like genes are expressed in diverse tissues, including mature leaves, floral buds and reproductive organs (flower organs and fruits) [FTs in perennial plants, especially in species with multiple FT paralogs [M. truncatula, which has six identified FT-like genes, tetraploid alfalfa is more likely to have a similar number, or even more, MsFTa paralogs due to genome duplication. Long-term work of flowering regulation in alfalfa will be directed at paralogs of MsFTa.Given the detection of itecture , tuberizitecture , and doritecture . The exp fruits) ,22,27,44paralogs ,14,16,22Medicago sativa cv. ‘Zhongmu No.1’, bred by the Institute of Animal Science, the Chinese Academy of Agricultural Sciences, was used in this study. Seeds were germinated in regular soil in a 20 cm diameter pot. Medicago truncatula Tnt1 insertion mutant fta1-1 (NF3307) was obtained from the Noble Research Institute . Arabidopsis thaliana seeds of ft-10 (CS9869) were from Arabidopsis Biological Research Center (ABRC) and germinated in soil (vermiculite:soil = 1:3). All plants were grown in growth chambers at 21 °C under a 16/8 h (light/dark) photoperiod.FT-like genes in M. truncatula and sequenced. The obtained partial sequence of alfalfa FT-like gene was used to design primes for amplication the full-length MsFTa by 3′-RACE and 5′-RACE  with the specific primers according to the manufacturer’s instructions. PCR amplicons were purified after separated on 1% agarose gel and cloned into pMD19-T  and sequenced.Total RNA was extracted from 2-week-old alfalfa seedlings with TRIzol reagent . After DNase I  treatment, the first-strand cDNA was synthesized using Reverse Transcriptase III . A DNA fragment was amplified by using the two degenerate primers designed based on FT homologous genes, Arabidopsis FT (At1g65480) was used as query sequence to search against the database EnsemblPlants (http://plants.ensembl.org/index.html). Phylogenetic analysis was performed with bootstrap trials of 1000 using DNAMAN version 7.0 , and sequence alignment was analyzed using the same software with default parameters. Isoelectric point (pI) and molecular weight (MW) values were predicted using ExPaSy (https://prosite.expasy.org). The secondary structure of the protein was predicted using LYON-GERLAND PBIL (https://prabi.ibcp.fr); and the hydrophobic structure was predicted using ScanProsite (https://prosite.expasy.org/scanprosite).For MsActin serving as an internal control. The PCR product was analyzed in gel and images were captured using a Gel Image Analysis System FR-200A . For quantitative real-time PCR, SYBR Premix EX TaqTM qPCR Kit  was used according to the instruction with BIO-RAD CFX96TM Real-Time System . The Arabidopsis Actin gene was used as a loading control. The sequence of the primers used in this study is listed in For expression analysis in different tissues, roots, stems and leaves were harvested from 6-week-old alfalfa, while flowers were from 60-day-old plants. For the diurnal analysis, leaves were collected separately from 6-week-old alfalfa at zeitgeber time (ZT) 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 time points, respectively. After extraction of total RNA, cDNA was prepared as described above. Semi-quantitative RT-PCR was carried out using gene-specific primers with MsFTa ORF sequence was subcloned into the pA7-GFP (35S::GFP) expression vector [XhoI and SpeI restriction sites, respectively. The plasmid of the pA7-GFP and 35S::MsFTa-GFP fusion binary vector was then transformed into separate onion epidermal cells via particle bombardment . The subcellular localization of the transiently expressed MsFTa-GFP fusion protein was imaged using a confocal laser-scanning microscope .For the subcellular localization analysis of MsFTa-GFP fusion protein in onion epidermal cells, the construct of MsFTa-GFP was performed as follows: the n vector  using th35S::MsFTa, first, a binary vector harboring 35S::MsFTa was constructed by amplifying MsFTa ORF with primers named pBI-FTa-f and pBI-FTa-r, and the sequence-confirmed MsFTa was introduced into the pBI121 vector  using Xba I and Sma I restriction sites. Secondly, the 35S::MsFTa plasmid was transformed into Agrobacterium tumefaciens strain GV3101 by electroporation, and Arabidopsis transformation was performed via the floral-dip method. T1 Arabidopsis seeds were screened on a MS medium containing 40 mg·L−1 kanamycin, and T2 lines confirmed by semi-quantitative RT-PCR were used in this study for MsFTa expression and phenotypic analysis. For Medicago transformation, explants from 4-week-old plants (R108 and fta1-1) growing under LDs were cocultivated with the agrobacterium containing the construct of 35S::MsFTa according to the Medicago handbook [−1 was used to select transformant plants.For generation of the transgenic Arabidopsis plants expressing handbook . KanamycFT homolog from alfalfa, one of the most important perennial forages, was isolated and characterized. MsFTa has highly identical sequence composition, especially exon/intron boundary and functional domains, with FTs in a variety of plant species. The diurnally regulated MsFTa is expressed preferentially in leaves and floral buds. Functionally, MsFTa accelerated flowering in Arabidopsis and the annual legume M. truncatula, indicating that the novel alfalfa FT acts as a flowering promoter.In this study, the


SPL transcription factors. To date, SPL genes have been identified in many species of plants. Loquat, as a woody fruit tree of Rosaceae, is unique in flowering time as it blooms in winter. However, the study of its SPL homologous genes on the regulation mechanism of flowering time is still limited. In this study, four SPL homologs—EjSPL3, EjSPL4, EjSPL5, and EjSPL9—are cloned from loquat, and phylogenetic analysis showed that they share a high sequence similarity with the homologues from other plants, including a highly conserved SQUAMOSA promoter binding protein (SBP)-box domain. EjSPL3, EjSPL4, EjSPL5 are localized in the cytoplasm and nucleus, and EjSPL9 is localized only in the nucleus. EjSPL4, EjSPL5, and EjSPL9 can significantly activate the promoters of EjSOC1-1, EjLFY-1, and EjAP1-1; overexpression of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 in wild-type Arabidopsis thaliana can promote flowering obviously, and downstream flowering genes expression were upregulated. Our work indicated that the EjSPL3, EjSPL4, EjSPL5, and EjSPL9 transcription factors are speculated to likely participate in flower bud differentiation and other developmental processes in loquat. These findings are helpful to analyze the flowering regulation mechanism of loquat and provide reference for the study of the flowering mechanism of other woody fruit trees.The age pathway is important for regulating flower bud initiation in flowering plants. The major regulators in this pathway are miR156 and  FLOWERING LOCUS T (FT) is a crucial integrator in this network [SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), the floral meristem identity genes APETALA1 (AP1), LEAFY (LFY), and FRUITFULL (FUL) are located at downstream of the regulatory network [SQUAMOSA PROMOTER BINDING-LIKE (SPL) transcription factors [SPL transcription factors which were first discovered and confirmed in Antirrhinum majus, and were named SQUAMOSA PROMOTER BINDING PROTEIN (SBP) because they recognize and bind to the promoter region of MADS-box genes SQUAMOSA (SQUA) [SBP genes were also found in green plants and referred to as the SQUAMOSA PROMOTER BINDING-LIKE (SPL) genes [SBP gene families were characterized in model plants, woods, crops, and fruits, such as Arabidopsis thaliana [SPL genes contain a highly conserved DNA binding domain, which was called the SBP-box domain, and it consists of approximately 80 amino acid residues including two zinc finger structures and one nuclear localization signal [Flowering is regulated by extensive factors, such as plant hormones, photoperiod, vernalization, age, temperature, light quality, sucrose, etc. ,3,4,5,6. network , and the network ,12. The  factors ,14. Micr factors ,16. The A (SQUA) . SBP genL) genes . In recethaliana , barley thaliana , green athaliana , moss [2thaliana , tomato thaliana , salvia thaliana , rice [2thaliana , birch [thaliana , light bthaliana , cotton thaliana , grape [thaliana , apple [thaliana , corn [3thaliana , and so n signal .Arabidopsis thaliana, miR156 is in a higher expression level, which negatively regulates the target SPLs; as the plant grows, the expression level of miR156 decreases, and the target SPLs mRNA increases subsequently [SPLs, SPL9 and SPL15 positively regulate miR172, which inhibits the expression of transcription factor APETELA2 (AP2); AP2 inhibits the expression of FT and; therefore, delays flowering ultimately [AP1, LFY, and FUL, and SPL3, SPL4, and SPL5 can also recruit FT-FD complex and bind to the promoter regions of AP1, LFY and FUL and consequently initiate their transcription [SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), and then activate the expression of AP1, LFY, and FUL. SOC1 also negatively regulates SPL3, SPL4, and SPL5 to form a feedback loop [SPL genes in more and more species has been unearthed: Grape VpSBP11 can regulate floral transition and leaf development [BlSPLs in Betula luminifera interact with DELLA protein to participate gibberellin regulation [SPL genes are vital in also other physiological processes in plants, such as adventitious root development [The aging pathway regulates flowering under uninduced conditions, and also, it can be integrated with other pathways . In younequently . Among ttimately ,34. At tcription ,35. Furtack loop . At preselopment ; BlSPLs gulation . It is relopment ,37, fertelopment , fruit gelopment , floral elopment ,41, planelopment , morpholelopment , and strelopment .Eriobotrya japonica Lindl.) is a subtropical evergreen fruit that belongs to Maloideae subfamily of Rosaceae. Compared to its relative fruit trees like apple, pear, and peach, loquat has unique habits, in that it blooms in winter and fruits mature in late spring or early summer [EjTFL1 and EjLFY were identified earliest, and EjAP1 was characterized later which was found to promote flowering [EdFT, EdFD, EdCO, and EdGI were separated and were identified to be related to flowering regulation [EjSOC1s played a vital role in promoting flowering in cultivated loquat [Loquat . The spatiotemporal expression of the EjSPLs were analyzed, and their subcellular localization were observed in tobacco leaves. In addition, the interactions between EjSPLs and other flowering proteins or the downstream flowering gene promoters were detected. Finally, because of the transform system has not been built in loquat, the genetic functions of the EjSPLs were verified in Arabidopsis thaliana. This work discovered the roles of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 on flowering regulation, and provides further understanding of the unknown transcription factors attending reproductive growth in cultivated loquat.In this work, cultivated loquat was employed as research material. The EjSPL3, EjSPL4, and EjSPL5 from the cultivated loquat “JFZ” were annotated within the genome-wide scope of “JFZ” genome (from our unpublished data), and their coding sequences were isolated. EjSPL3, EjSPL4, and EjSPL5 had 303, 570, and 576 nucleotides encoding 100, 189, and 191 amino acids, respectively (Sequence S1). Clustering analysis between the predicted amino acid sequences of the above EjSPLs and their homologous genes in other plants was conducted, the phylogenetic tree showed that EjSPL3, EjSPL4, and EjSPL5 were clustered in different branches, and the closest homologs to them were from apple or pear: EjSPL3 and MdSBPB, EjSPL4 and MdSPL1 are the closest, and EjSPL5 is closest to PbrSPL1a , and flowers compared to other tissues. The expression level of EjSPL4, EjSPL5, and EjSPL9 in leaf buds was higher than that in flower buds, and EjSPL3 was expressed relatively higher in flower buds. However, EjSPL5 and EjSPL9 were also expressed in stem, and the transcription level of EjSPL9 was detectable in root and seed . Furthermore, the expression level of EjSPL4 and EjSPL5 gradually decreased to a low level by the end of the year, similar as that in the beginning of the year. The expression level of EjSPL9 came to a peak one month earlier than that of EjSPL4 and EjSPL5, and was kept in a relatively high level during the whole period of flower development; it just declined to a very low level after loquat finished flowering. The level of the EjSPL3 transcripts showed the first peak at the beginning of March, which was even earlier than that of EjSPL9, and also it showed another distinct peak during the early stage of flower bud development, and after that, decreased obviously to a low level by the end of the year  in loquat.In summary, these findings suggested that EjSPLs-encoded proteins in cells, we constructed an EjSPLs fusion expression vector carrying 35S promoter and a green fluorescent protein (GFP)-tagged protein, and introduced them into the epidermal cells of tobacco by Agrobacterium tumefaciens infection, and then their fluorescence signals were observed. The results showed that EjSPL3, EjSPL4, and EjSPL5 were localized in the cytoplasm and nucleus, and EjSPL9 was localized only in the nucleus . We used the PlaceCARE website to perform cis-acting element prediction on the promoter regions of these genes; some binding site sequences (GTAC) were found in their promoter regions (Sequence S2).In the previous experiment, the expression level of  is GTAC ,51. TherEjLFY-1pro, EjLFY-2pro, EjSOC1-1pro, EjSOC1-2pro, EjAP1-1pro, and EjAP1-2pro drive firefly luciferase (LUC). EjSPL3, EjSPL4, EjSPL5, and EjSPL9 were constructed into the effector vector driven by CAMV35S , and the  CAMV35S a. The tw control b.EjSPL3, EjSPL4, EjSPL5, and EjSPL9, overexpression vectors containing full length coding sequences of the four EjSPL genes were constructed and transformed into wild-type Arabidopsis thaliana (Col-0). The homozygous transgenic lines were screened out, and the flowering time and the expression of downstream flowering genes were analyzed in these transgenic lines. All transgenic lines with different genes showed early flowering phenotype compared to wild-type Col-0  [CiSPL5 removed the binding site of miR156 in the 3′UTR, to Arabidopsis wild-type, leading to early flowering and fewer small rosette leaves [PtSPL9 is expressed in various organs, with the highest expression in stems, and showed a nuclear localization [VpSBP11 (a homolog of AtSPL3/4/5) encoded 170 amino acids, contained a highly conserved SBP domain, and its protein was localized in the nucleus with the transcriptional activity; overexpressing it  into the Arabidopsis wild-type advanced flowering, and promoted the expression of FUL, AP1, LFY, and some other genes; and also, the phenotypic characteristics in transgenic plants were transformed [SPL genes are both functionally conserved and functionally differentiated during plant development.Functional verification of e leaves . MdSPL3 , AtFUL) . In citre leaves . PtSPL9 lization . In grapnsformed . CombineSPL is extremely complicated, and to date, the model plant Arabidopsis is the most thoroughly studied. Most of the SPL genes in Arabidopsis are targeted by miR156 and miR157 [SOC1, LFY, AP1, FUL, and so on [SPL binding sites (GTAC) in the promoter regions of EjLFY, EjSOC1, and EjAP1 (Sequence S2); and the dual-luciferase reporter assays verified that EjSPLs activate their expression, in which, EjSPL3, EjSPL4, EjSPL5, and EjSPL9 effectively activated EjLFY-1 and EjSOC1-1 expression; and EjSPL4, EjSPL5, and EjSPL9 significantly improve the expression of EjAP1-1  gene in the leaves and the AP1/MC in the shoot apex to promote flowering [Arabidopsis thaliana, SODIUM POTASSUIM ROOT DEFECTIVE1 (NaKR1) was found to respond to the changes in potassium, and it could regulate the expression of FT through the miR156-SPL3 module, which also affected the transport of FT protein in the phloem [CmNF-YB8 is a direct upstream gene of cmo-miR156, which can directly bind to its promoter region. Silencing this gene led to down-regulation of cmo-miR156 and up-regulation of CmSPL3, CmSPL5, and CmSPL9 [EjFT2 showed involvement in the process of flower bud differentiation in loquat [EjSPL genes in loquat, we need more in-depth research.There are also some novel regulatory models found in other plants. lowering . In the e phloem . The nucd CmSPL9 . Interesn loquat . In ordeEriobotrya japonica Lindl.) was used in this study. Adult loquat trees were grown under natural conditions in the loquat germplasm resource preservation garden, South China Agricultural University, Guangzhou, China. The wild-type of Arabidopsis, Arabidopsis thaliana Columbia-0 (Col-0), was used for gene transformation. Nicotiana benthamiana was cultivated for the assays of transient expression. The seeds of the Arabidopsis Col-0 and tobacco used in this study were provided by Xingliang Hou. Both of Arabidopsis and tobacco were grown under long-day conditions (16 h light/8 h dark) at 22 °C in a controlled environment room. Samples (loquat tissues and Arabidopsis plants) for qRT-PCR were immediately frozen in liquid nitrogen and stored in an ultra-low temperature refrigerator at −80 °C until use.Cultivated loquat  with specific primers (https://www.ncbi.nlm.nih.gov/). DNAMAN 6.0, ClustalX 2.0.12, GeneDoc 2.7, and MEGA 6 software were employed for the amino acid sequences alignment and the construction of the phylogenetic tree of SPL proteins, respectively.The coding region of  primers . The PCRArabidopsis was extracted by EASYspin Plus plant RNA extraction kit , and the cDNA was synthesized using PrimeScriptTM RT reagent Kit with gDNA Eraser . Quantitative real-time PCR (qPCR) was performed using iTaqTM universal SYBR Green Supermix  in the LightCycler 480 . Ejβ-actin [TUB2 (AT5G62690) were amplified as the internal control genes for normalization in loquat and Arabidopsis thaliana, respectively. Three technical and biological replicates were applied and data were analyzed with previous methods [Arabidopsis.The total RNA of loquat and jβ-actin  and TUB2 methods . Semi-quEjSPL3, EjSPL4, EjSPL5, and EjSPL9 without stop codon were separately cloned into the pGreen-35S-GFP vector [Agrobacterium tumefaciens strain GV3101::psoup by heat induction, and the transformed Agrobacterium cells were cultured and injected into epidermic cells of tobacco (Nicotiana benthamiana) leaves for transient expression following the previously described methods [The full length coding sequences of P vector . These p methods . After aEjSPL3, EjSPL4, EjSPL5, and EjSPL9 were alone cloned into pGreen-35S-6HA vector [EjSPLs. Then, these recombined vectors were transformed into Arabidopsis Col-0 by the floral dip through Agrobacterium-mediated method [The full length coding sequences of A vector  to generd method . FinallyEjLFY, EjAP1 and EjSOC1 were cloned from the DNA of “JFZ” using PrimeSTAR® Max DNA Polymerase (TaKaRa) with specific primers. Putative cis-acing elements in each promoter region were found in the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).The promoters of EjLFY, EjAP1, and EjSOC1, the above promoters were cloned into pGreenII 0800-LUC double-reporter vector, firefly luciferase (LUC), and Renilla luciferase (REN), and the full length coding sequences of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 without stop codon were cloned into pGreenII 62-SK effector vector [For the analysis of binding activity of EjSPL3, EjSPL4, EjSPL5, and EjSPL9 to the promoters of r vector .Agrobacterium-mediated protocol, as described above. After about 48 h of transformation, we quantified the activity of LUC and REN using the dual luciferase assay kit (Promega) with appropriate modification [The recombined effectors and reporters were co-transformed into tobacco leaves with different groups by fication  was quant test.All data analyses were performed by Excel and GraphPad Prism 6.01. Significance of differences between data was evaluated by Student’s Eriobotrya japonica transcription factors EjSPL3, EjSPL4, EjSPL5, and EjSPL9 are speculated to participate in flower bud differentiation and flower organ development by activating the expression of EjSOC1, EjLFY, or EjAP1.In summary, the


S-transferase, and monodehydroascorbate reductase. Combined effects of SA and SNP were observed to be more prominent in Cd-stress mitigation than the individual effects of SA followed by that of SNP, suggesting that SA and NO in combination more efficiently boosted physiological and biochemical responses to alleviate Cd-toxicity than either SA or NO alone. This finding signifies a cooperative action of SA and NO in mitigating Cd-induced adverse effects in rice, and perhaps in other crop plants.Cadmium (Cd) is one of the prominent environmental hazards, affecting plant productivity and posing human health risks worldwide. Although salicylic acid (SA) and nitric oxide (NO) are known to have stress mitigating roles, little was explored on how they work together against Cd-toxicity in rice. This study evaluated the individual and combined effects of SA and sodium nitroprusside (SNP), a precursor of NO, on Cd-stress tolerance in rice. Results revealed that Cd at toxic concentrations caused rice biomass reduction, which was linked to enhanced accumulation of Cd in roots and leaves, reduced photosynthetic pigment contents, and decreased leaf water status. Cd also potentiated its phytotoxicity by triggering reactive oxygen species (ROS) generation and depleting several non-enzymatic and enzymatic components in rice leaves. In contrast, SA and/or SNP supplementation with Cd resulted in growth recovery, as evidenced by greater biomass content, improved leaf water content, and protection of photosynthetic pigments. These signaling molecules were particularly effective in restricting Cd uptake and accumulation, with the highest effect being observed in “SA + SNP + Cd” plants. SA and/or SNP alleviated Cd-induced oxidative damage by reducing ROS accumulation and malondialdehyde production through the maintenance of ascorbate and glutathione levels, and redox status, as well as the better activities of antioxidant enzymes superoxide dismutase, catalase, glutathione  Modern-environment and human health are continuously challenged by a plethora of pollutants, which are originated by both anthropogenic and geological activities . Among t−1 dry weight) [S-transferase (GST), and non-enzymatic metabolites like ascorbate (AsA) and GSH [Although plants have several defense strategies against Cd-toxicity, most of the crop plants display severe vulnerability if they accumulate even a small amount of Cd in their leaf tissues (5–10 µg g weight) ,6. Cd ha weight) ,8. Addit weight) ,10. Desp weight) . To prot and GSH . Like RO and GSH . In addi and GSH . In resp and GSH ,14. StSABP2, StSOD, and StAPX to enhance potato (Solanum tuberosum) acclimatization under Cd-stress conditions [Oryza sativa), wheat (Triticum aestivum) and mustard (Brassica juncea) [The modulation of the endogenous SA levels by exogenous addition of SA enhances seed germination, photosynthesis, thermogenesis, transpiration, and membrane formation under various stress condition ,16,17. Snditions . Furthernditions ,20,21,22nditions ,14. The  juncea) ,13,23,24 juncea) ,26, sugg−1) have raised serious public health concerns [Crop plants cultivating in Cd-contaminated soils often show high Cd accumulation in edible parts in addition to poor growth performance ,28. Numeconcerns . Therefo2) for three (3) days triggered severe phenotypic disturbances, including stunted growth, leaf rolling, burning of leaf tips, chlorosis, and even yellowing of the entire plant , Chl b (61.00%), total Chls (34.16%), and carotenoids (18.82%) was observed in the leaves of “Cd” plants , Chl b , total Chls  and carotenoids  in the leaves of “SA + Cd,” “SNP + Cd,” and “SA + SNP + Cd” plants, respectively  ” plants . In contectively . Furtherectively .Rice plants exposed to Cd-stress displayed attenuation of leaf relative water content (RWC) by 27.23% in “Cd” plants, compared with that of control plants . Convers2•−) and H2O2, as evidenced by more scattered dark blue spots for O2•− , LOX activity , and MDA  in “SA + Cd,” “SNP + Cd,” and “SA + SNP + Cd” plants, respectively, in comparison with “Cd” plants , a significant accumulation of DHA (68.41%), and an attenuation in leaf AsA/DHA ratio (61.83%) when compared with control plants . On the In addition, Cd-stressed rice plants exhibited a decrease in the levels of GSH (37.08%) and GSSG (69.75%), and increase in GSH/GSSG ratio (107.22%) in comparison with control plants . RegardiIn comparison with control plants, “Cd” plants showed an increase of SOD activity by 45.96% and a decrease of CAT activity by 13.69% in leaves A,B. TreaIn comparison with “Cd” plants, the addition of SA, SNP, or SA + SNP reduced the APX activity  and GR activity , but increased the MDHAR activity by 49.24, 48.99, and 39.99% in the “SA + Cd,” “SNP + Cd,” and “SA + SNP + Cd” plants, respectively C,D,F. OnBrassica oleracea, and Pisum sativum) exposed to various levels of Cd for short or long periods [Zea mays) and mustard, respectively, demonstrating that application of these growth regulators could be a viable option in counteracting the toxic effects of Cd in important cash crops like maize and mustard. Similarly, co-application of SA and SNP promoted growth of wheat under osmotic stress conditions [Since cadmium (Cd) is not essential for plant growth and development, plants do not have specific mechanisms for up-taking Cd by their cells. Instead, Cd is accumulated in plant tissues through the transporters that are involved in the uptake of other essential elements like magnesium, calcium, manganese, iron, and zinc . Once ac periods ,31,32,33 periods  and Per  periods  observed periods  and SNP  periods  positivenditions , pointinHyperaccumulators, which are commonly used for phytoremediation can accumulate a large amount of toxic metals like Cd in their tissues without showing visible toxicity symptoms . Unlike Excessive Cd has negative effects on photosynthesis by damaging the photosynthetic pigments and electron transport processes, disrupting chloroplast structure and Chl-protein complexes, deactivating enzymes of Chl biosynthesis and Calvin cycle, and disturbing water balance ,8,37. InSolanum lycopersicum) [Plants’ typical physiological response against water deficiency is to accumulate various osmoprotectants, including Pro to overcoming water shortage-associated damage in tissues . In thisersicum) ,42. It sersicum) ,36,40.2 assimilation, and photorespiration [2•− and H2O2 in rice leaves (Brassica chinensis) [2•− and H2O2 [2O2 accumulation (•OH). The Cd-stressed plants seemed to compensate reduced CAT activity by stimulating the AsA-GSH system through the activation of APX, DHAR, and GR  ,44. In aand H2O2 , displaymulation B,C that , and GR B,C,E,F. d plants . Thus, dd plants A,C,E,F, d plants A–C. Mored plants  in additd plants B,H enfor2•−, H2O2, and MDA and the activity of LOX in rice leaves were significantly diminished by supplying exogenous SA and/or SNP to Cd-stressed seedlings .However, Cd-induced accumulation of Oeedlings A–E, whiceedlings , which teedlings . SA and/ and CAT A,B did n and CAT C,E,F. Ho and CAT . Thus, t and CAT . Dong etGSH-dependent GPX-GST system and Gly system are important in detoxification of lipid hydroperoxides and reactive aldehydes, thereby conferring tolerance to heavy metal toxicity ,12. In tOryza sativa cv. BRRI dhan52) were sprouted and hydroponically developed in a growth chamber under controlled conditions  [2). Fourteen-day-old healthy rice plants were grouped into five treatments, which were (1) control (C), (2) 500 µM CdCl2 (Cd), (3) 200 µM SA + 500 µM CdCl2 (SA + Cd), (4) 200 µM SNP + 500 µM CdCl2 (SNP + Cd), and (5) 200 µM SA + 200 µM SNP + 500 µM CdCl2 (SA + SNP + Cd). The plants exposed to the treatment solutions were further grown for a period of three days. The second leaves of the 17-day-old rice plants were harvested for determination of various physiological and biochemical parameters. Three independent replications of each treatment were used. Each replication consisted of 60 rice plants under the same experimental conditions.Healthy seeds of rice ( 65–70%) . Commerc−1. Cd contents in the oven-dried rice roots and leaves were quantified following the procedure described by Mostofa et al. [The biomass of rice plants was assessed by determining the dry weight (DW). To estimate the DW, whole seedlings (n = 10) were harvested from each treatment, and subjected to oven-drying for 48 h at 80 °C, and expressed as g seedlinga, b, and total Chls, and carotenoids in the freshly harvested second leaves were spectrophotometrically quantified according to the formula reported by Arnon [The levels of chlorophyll (Chl) by Arnon , and Licby Arnon , correspby Arnon . The conby Arnon , and modby Arnon .2O2) and lipid peroxidation product malondialdehyde (MDA) in the second leaves of rice plants were quantified following the methods of Mostofa and Fujita [Contents of hydrogen peroxide  and H2O2 in the second leaves of rice by using the solutions of 0.1% nitroblue tetrazolium (NBT) and 1% 3, 3’-diaminobenzidine (DAB), respectively. The technique of Mostofa and Fujita  was adopg for 15 min at 4 °C. The reduced and total ascorbic acid (AsA) contents were quantified following the method described in Mostofa et al. [Freshly harvested second leaves of rice plants (0.5 g/sample) were crushed in 3 mL of extraction buffer (1 mM ethylenediaminetetraacetic acid in 5% ice-cold metaphosphoric acid) and centrifuged at 11,500× a et al. . The conβ-mercaptoethanol and 10% (v/v) glycerol) was used for crushing the freshly harvested leaf samples (0.5 g/sample). After centrifugation, the resultant supernatants were collected and kept on ice bath for protein and enzyme analyses. The procedure of Doderer et al. [S-transferase , and glutathione peroxidase  activities. For the determination of glyoxalase (Gly) activities, the methodologies of Hossain et al. [Extraction buffer . The obtained data were subjected to a one-way analysis of variance (ANOVA) to test homogeneity of variance among the data using Statistix 10 software package. Least significant difference (LSD) post hoc test was carried out to identify the significant differences among the treatments at 5% level of significance (The present study revealed physiological and biochemical mechanisms associated with SA- and/or NO-regulated rice tolerance to Cd-stress. The Cd-induced toxic effects on rice growth was expressed in terms of increased Cd accumulations in roots and leaves, phenotypic changes, biomass reduction, leaf water imbalance, oxidative stress generation, and weakening of antioxidant defense and Gly systems. However, simultaneous application of SA and SNP with Cd resulted in substantial improvement of rice responses against Cd-toxicity, leading to improved performance of rice plants under Cd-stress conditions. SA and/or SNP restricted the Cd-uptake and accumulation, protected photosynthetic pigments, maintained leaf water balance, and reduced oxidative damage to membrane through increased activities of the antioxidant defense and Gly system. Although SA and SNP were effective in improving Cd tolerance, their co-operative actions proved to be the most effective in mitigating Cd-induced adverse effects on rice growth performance. Current findings, therefore, suggest that exogenous treatments of rice plants with SA and/or SNP, and perhaps genetic engineering to enhance SA and/or NO pathways in rice plants, could be a viable defense strategy in promoting their growth and development under Cd-stress conditions.


Moreover, flow cytometry experiments showed that the incidence of apoptosis was higher in the presence of T5224, RSPO2, or AZD5363 than among controls, and was increased by increasing the doses of the drugs. This suggests these drugs could be used as therapeutic agents to treat FPA. Finally, we found that cFos, WNT5A, NCAM1, JUP, AKT3, and ADCY1 are abnormally expressed in FPA cells compared to controls, which highlights these genes as potential prognostic and/or therapeutic targets.We tested whether the drugs T5224, RSPO2, and AZD5363 exert therapeutic effects against functioning pituitary adenoma (FPA). We analysed the gene expression profiles of four FPA mRNA microarray datasets  from the Gene Expression Omnibus database and identified genes differentially expressed in FPA vs control tissues. We then carried out Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction network analyses. We also measured the difference in expression of hub genes between human normal pituitary cells and FPA cells using qRT-PCR. Our  Functioning Pituitary Adenoma (FPA) accounts for 70% of all pituitary adenomas (PA) . EpidemiCurrently, early diagnosis and treatment have improved, owing to the widespread use of magnetic resonance imaging (MRI) . HoweverPrevious studies have demonstrated overexpression of high mobility group A (HMGA) in FPA, possibly due to downregulation of HMGA-targeting microRNAs (miRNAs) . For exaHere, we hypothesized that T5224, RSPO2, and AZD5363 should be effective treatments against FPA. To test this hypothesis, we looked for genes differentially expressed in FPA tissues compared to normal brain controls. We also performed bioinformatics analyses to investigate the molecular processes underlying FPA and used various biochemical and cell biology assays to test the effects of T5224, RSPO2, and AZD5363 treatments.We analyzed the gene expression profiles of four FPA mRNA microarray datasets  from the Gene Expression Omnibus database. There were 19,943 differently expressed genes (DEGs) picked up from GSE2175, of which 12,268 were upregulated while 7675 were downregulated. Altogether, 4635 DEGs were found out from GSE26966, among which 2159 were upregulated and 2476 were downregulated. Among 6472 DEGs were identified from GSE36314 with 2520 upregulated genes and 3952 downregulated genes. Lastly, 2020 DEGs were discovered from GSE37153, in which 2020 genes were upregulated and 1017 were downregulated. There were 178 mutual DEGs among the four datasets .The mutual DEGs were uploaded to DAVID for GO and KEGG pathway analyses , 1D. TheWe also conducted PPI network analyses of the previous 178 mutual DEGs. Genes with degrees ≥ 6 were screened as hub genes based on the STRING database. Altogether, 21 genes were identified as hub genes –1G, inclWe performed qRT-PCR in order to conform the expression of cFos, WNT5A, NCAM1, JUP, AKT3, and ADCY1 in normal pituitary cells and FPA cells . NCAM1, cFos, AKT3, and ADCY1 were consistently upregulated in FPA cells compared to normal pituitary cells (P < 0.05) while WNT5A and JUP were downregulated (P < 0.05)., with levels being slightly different across the tested cell lines . On the other hand, clonogenicities were approximately the same for the STO609 and Genipin groups, both of which were higher than those for the T5224, RSPO2, and AZD5363 groups (P < 0.05) . Higher Flow cytometry of FPA cells and controls treated with different doses of drugs for 48 h allowed us to measure the percentages of normal, necrotic, late apoptotic, and early apoptotic cells. For controls, the respective percentages were 57.35%, 16.04%, 21.33%, and 5.28%. On the other hand, for T-5224-treated cells they were 5.69%, 1.08%, 59.74%, and 33.49% in low-dose group (10 μmol/L); 0.36%, 0.55%, 70.23%, and 28.86% in the intermediate-dose group (20 μmol/L); and 0.16%, 0.91%, 83.13%, and 15.8% in the high-dose group (40 μmol/L) . For STOMany FPA patients present reduced quality of life, persistent morbidity, and slightly increased mortality in spite of receiving current therapies . In the GO analysis of abnormally expressed genes showed that upregulated genes were mainly associated with biological processes relevant to mitotic proliferation such as cell division, spindle formation, microtubule polymerization, protein binding, and ATP binding, which may explain the fast multiplication of cancer cells. Downregulated genes were primarily involved in biological processes underlying cell communication and signaling, including ion transport, cell junction and plasma membrane formation, calcium-dependent protein binding and calcium ion binding. Our results agree with a previous study showing that over-representation of genes can modify the course of the cell cycle, cell development, and cell differentiation/proliferation in pathologic adenoma cells . SeveralThe levels of some cell-cycle regulators  can predict the occurrence and proliferation of FPA . The P53c-Fos, one of the components of the activator protein-1 (AP-1) transcription factors, is hyper-activated in tumorigenesis and promotes cancer cell invasion and proliferation –19 for vWNT5A promotes malignant progression in tumor cells –23 and iNCAM1 is a neural cell adhesion molecule that promotes cell-cell and cell-matrix interactions during development and cellular differentiation . NCAM1 pThe junction plakoglobin gene (JUP) is a desmosomal anchor protein gene, whose normal functioning is necessary for having healthy inter-cellular junctions and microtubules –31. JUP Protein kinase AKT3 promotes progression, metastasis, and drug resistance in various types of cancer  36–39].–39.36–39ADCY1, adenylate cyclase 1, catalyzes the synthesis of cAMP –41 and wIn this study, cFos, NCAM1, JUP, AKT3 ADCY1, CCND2, PPP2R5A, CAMK2G, ATP2A2, and MAD2L1 as well as hub genes were shown to be dysregulated in FPA and may serve as therapeutic targets or prognostic and diagnostic makers.We used qRT-PCR to measure cFos, WNT5A, NCAM1, JUP, AKT3, and ADCY1 levels in normal pituitary cells (R1200) and FPA cell lines . We found that the expression of cFos, NCAM1, AKT3, and ADCY1 was lower in normal pituitary cells than in FPA cells (P < 0.05) while WNT5A and JUP levels were higher. This means that high expression of cFos, NCAM1, AKT3, and ADCY1 promotes tumorigenesis while high levels of AKT3 and ADCY1 inhibits it.We used MTT and colony-formation assays to evaluate the effects of T5224, RSPO2, and AZD5363 on FPA. For cell lines GT-1 and GH3, cell viability correlated negatively with T5224, RSPO2, AZD5363, STO-609, and Genipin treatments in a dose-dependent manner. Furthermore, the ratio of T5224 groups dropped more significantly than the rest of groups in GH1-1 cell lines, including RSPO2 and AZD5363 groups. That indicates that T5224, RSPO2, and AZD5363 have therapeutic effects on FPA cells and protective effects on normal pituitary cells. Similarly, colony-forming assays showed that the number and size of clonogenicities in the drug groups were remarkably smaller than in controls and correlating negatively with treatment in a dose-dependent manner, with T5224, RSPO2, and AZD5363 yielding the smallest clonogenicities, in agreement with the results of our MTT assays.Our flow cytometry experiments on FPA cells treated with different doses of T5224, RSPO2, AZD5363, STO-609, or Genipin for 48 h, showed that apoptosis correlated positively with treatment dose in all five drug groups compared to controls, suggesting beneficial effects from such drugs in the treatment of FPA.in vitro study here indicate that T5224 exerts anti- FPA effects specifically by inhibiting cFos pathways and that RSPO2 does so by inhibiting Wnt5A while AZD5363 inhibits Akt3. Further studies in vivo should be conducted to test the therapeutic effects we uncovered here in more clinically-relevant systems.Considering previous studies, the results of our https://www.ncbi.nlm.nih.gov/geo). The corresponding profiles were provided on platform GPL96 (GSE2175), GPL570 (GSE26966), GPL8300 (GSE36314), and GPL6480 (GSE37153) [The gene expression profiles of GSE2175, GSE26966, GSE36314, and GSE37153 were obtained from the GEO database (SE37153) –48. The th percentile were filtered out using the “filter probesets by expression” option. Then, the DEGs were identified using classical t test with P value cutoff of < 0.05 and a change ≥ two fold. We also computed Venn diagrams for each DEG (http://bioinformatics.psb.ugent.be/webtools/Venn/).Analyses of the raw data were carried out using GeneSpring software  for four groups of DEGs to fit four respective gene expression profiles. The category of each data set was derived from hierarchical clustering. Group FPA and normal tissues were identified. The probe quality control in GeneSpring was limited by virtue of principal component analysis (PCA), and probes with intensity values below the 20https://david.ncifcrf.gov/) provides a comprehensive annotation tools to understand the biological meaning underlying plenty of genes. GO (Gene Ontology) is a useful method for exposing biological process, molecular function, and cell component of genes. KEGG (Kyoto encyclopedia of Genes and Genomes) is a base for gene function analysis and genomic information linking. We performed GO and KEGG pathway enrichment analyses using DAVID for functional analyses of DEGs.The DAVID database  for PPI (Protein-Protein interaction) analysis. Then, we used Cytoscape software to screen hub genes and modules with MCODE (Molecular Complex Detection). Finally, we performed function and pathway enrichment analyses of DEGs in modules.We used the online database STRING  and FPA cells (GT1-1 and GH3) were received from the ATCC (American Type Culture Collection). Those cell lines were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum . The cell cultures were maintained at 5% CO-ΔΔCt method was used for qRT-PCR data analysis. The primers of genes were enumerated as followed: cFos sense, 5′-CCTCTCCATGCAGGAGTTAAGA-3′; cFos anti-sense, 5′-GGTCTCGGGTCCTTGATTTTCT-3′; WNT 5A sense, 5′-TACTGCGGTGGAGCAAGAAG-3′; WNT5A anti-sense, 5′-CATCTGCGCTTGACGGAGA G-3′; NCAM1 sense, 5′-AGCCCATCAATAAGGGAG GG-3′; NCAM1 anti-sense, 5′-ACCTGACACCCGTTT TAGCTG-3′; JUP sense, 5′-TACTGCGGTGGAGCAA GAAG-3′; JUP anti-sense, 5′-CATCTGCGCTTGACG GAGAG-3′; ADCY1 sense, 5′-TACTGCGGTGGAGC AAGAAG-3′; ADCY1 anti-sense, 5′-CATCTGCGCTT GACGGAGAG-3′.To verify the expression of cFos, WNT5A, NCAM1, JUP, AKT3, and ADCY1 in FPA cell lines and normal human pituitary cells, we used FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics) to perform qRT-PCR in a CFX96 Real-Time System (Bio-Red) according to the manufacturer’s instructions. Expression levels were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The 2The FPA cells  were plated into 96-well culture plate with a density of 500 cells/well, and were treated with different doses of T-5224, RSPO2, and AZD5363, respectively, as well as STO-609 (CaMKK inhibitor) and Genipin (aglycone derived from the iridoid glycoside), both of which protect against several types of tumors, including brain tumors. We used MTT  dissolved in PBS (5 mg/ml) to measure the viability of cells. On the day of measurement, the medium was replaced on fresh DMEM supplemented with 10% FBS and diluted MTT , and incubated for 3.5 h at 37 °C. Then, the incubation medium was removed and formazan crystals were dissolved in 200 μl solution of DMSO. We used an ELx800 absorbance microplate reader  to quantify the MTT reduction by measuring light absorbance at 570 nm. Each test was repeated four times.2. After 24 h in culture, the cells were treated with different doses of STO-609, Genipin, T-5224, RSPO2, and AZD5363, respectively. After 10 days of growth in vitro, colonies were counted and described according to Franken et al. Then, colonies were rinsed with PBS, fixed in 4% paraformaldehyde, stained with 5% crystal violet for 0.5 h, and rinsed twice with water.FPA cells  were seeded in Petri dishes with a density of 50 cells/cm5 cells/well and treated with different doses of STO-609, Genipin, T-5224, RSPO2 and AZD5363. After 48 h of culturing, the cells were harvested using accutase detachment solution . Annexin-V-FITC/PI labeling was conducted according to the manufactures’ instruction. a flow cytometer was used to count stained cells with the FACSDiva Version 6.2.The FPA cells (GT1-1) in the log growth phase were seeded into 6-well plates with a density of 2 × 10t tests for independent samples with P values < 0.05.All statistical data analyses were carried out using SPSS 18.0 , namely Supplementary Table