Toxocara canis infection. In this study, we conducted sequencing-based transcriptome profiling of miRNA expression in the spleen of Beagle puppies after T. canis infection to understand the potential biological roles of the altered miRNAs. Some differentially expressed miRNAs (DEmiRNAs) were identified after T. canis infection. The transcriptional profiling data suggested that the DEmiRNAs play significant roles in the pathogenesis of toxocariasis.As one of the neglected parasitic zoonoses, toxocariasis has gained increasing international attention. To date, however, little is known about the spleen microRNAs (miRNA) expression profiling in definitive or paratenic hosts after Toxocara canis is an unnoticed zoonotic helminth that causes severe disease in animals and humans. The spleen has a wide range of immunological functions in protecting the host against infection by many pathogens, but the function of the spleen in T. canis infection is still to be clarified, especially for the role of spleen microRNAs (miRNAs). In this study, deep sequencing of spleen RNA samples of 18 Beagle puppies was conducted to uncover the miRNAs expression profiling at 24 h post-infection (hpi), 96 hpi, and 36 days post infection (dpi). A total of 20, 34, and 19 differentially expressed miRNAs (DEmiRNAs) were identified at 24 hpi, 96 hpi, and 36 dpi, respectively. These DEmiRNAs  could play critical roles in Beagle puppies against T. canis infection, such as influencing inflammatory and immune-related cells and cytokines, by regulating target genes that are tightly associated with host immune function and enriched in immune response and immune pathways based on GO annotation and KEGG enrichment analysis. The current study discovered marked alterations of spleen miRNAs after T. canis infection, with potential effects on the pathogenesis of toxocariasis. Toxocara canis, the key member of the genus Toxocara, causes severe disease in humans and animals, which has led to increasing international attention [https://www.cdc.gov/parasites/npi/, accessed on 26 August 2022). T. canis does not develop into adults in intermediate hosts, such as humans, but causes various larval migrations, while, in the small intestine of the definitive host, T. canis develops into an adult, and the eggs are excreted into the environment in the feces [T. canis interactions are not fully clear, such as how T. canis evades the host’s immune surveillance. The understanding of pathogen–host interactions and disease ecology is changing with the advent of new molecular tools, such as high-throughput transcriptomics sequencing [ttention ,2. Accorhe feces . Howeverquencing . Large squencing . At the quencing . It is bquencing .T. canis infection [T. canis infection. Thus, to better understand the altered miRNAs, we undertook a sequencing-based transcriptomic analysis of miRNA expression in Beagle puppies’ spleen after T. canis infection at 24 h post-infection (hpi), 96 hpi, and 36 days post infection (dpi).A previous study revealed significant changes in miRNA expression profiles in Beagle puppies’ lives, as well as the roles of some miRNAs in a dynamic equilibrium between anti- and pro-inflammatory responses . The splnfection . NeverthAll animal experiments were approved by the Animal Ethics Committee of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences , and all experiments were performed in accordance with the Animal Ethics Procedures and Guidelines of the People′s Republic of China.Toxocara-naive, gender-neutral, specific pathogen-free (SPF) Beagle puppies were used and raised in the National Canine Laboratory Animal Resource Center without vaccination and feeding any medication in this study. The rearing environment has been described in a previous study [T. canis eggs and equal amounts of saline, respectively. Puppies were euthanized by injecting potassium chloride (KCl) solution under general anesthesia with Zoletil™ 50 . The collected splenic tissues were frozen in liquid nitrogen. None of the puppies, including infected puppies and control puppies, had any negative health effects before being euthanized.Eighteen 6–7-week-old us study . In brieus study . Brieflyg for 10 min at room temperature. Subsequently, the upper clear phase per sample was carefully absorbed and transferred to a new tube, followed by adding 0.5 mL isopropanol per each milliliter and was mixed vigorously by vortex, then the mixture was stood for 10 min and was centrifuged at 12,000× g for 10 min at 4 °C to collect the precipitated RNA. Then, the supernatant was carefully decanted, and the pellet was immediately resuspended in 1X SDS solubilization buffer with NaOAc to three M. The RNA was re-extracted with phenol and precipitated with 75% ethanol at 7500× g for five min at 4 °C to collect total RNA. The genomic DNA was removed by using DNase I . In brief, the total RNA was incubated in one μL 10X DNase I Buffer, one μL DNase I, and eight μL RNase-Free Water for 15 min at room temperature to remove genomic DNA. Then, 1 μL of 25 mM EDTA solution was added to the reaction and heated at 65 °C for 10 min to heat inactivation of the DNase I. RNA degradation and contamination were examined on 1% agarose gels for preliminary detection, and sample with distinct 18S and 28S bands and without significant stray bands was considered as high-quality RNA. RNA purity was examined using the NanoPhotometer® spectrophotometer , and sample with an OD260/280 value of around 2.0 was considered high-quality RNA. Utilizing the Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer , RNA concentration was measured. RNA integrity was evaluated using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system . High-quality total RNA samples with RNA integrity number (RIN) >8.0 were used to construct subsequent small RNA sequencing libraries.The total RNA of the 18 puppy spleen tissues was extracted using TRIZOL . In brief, 100 mg of frozen tissue was powdered in liquid nitrogen, and then 1 mL TRIzol was added to solubilize the tissue for five minutes at room temperature. Subsequently, 0.2 mL of chloroform was added and mixed vigorously by vortex for 15 s; then, the mixture was stood for 2–3 min and was centrifuged at 12,000× ®Multiplex Small RNA Library Prep Set for Illumina®  was used to generate the small RNA sequencing libraries, followed by adding index codes to attribute sequences to each sample. First-strand cDNA was synthesized by using M-MuLV Reverse Transcriptase (RNase H-), and then PCR amplification was carried out by utilizing the LongAmp Taq 2X Master Mix, SR Primer for Illumina, and index (X) primer. PCR products were purified using an 8% polyacrylamide gel . DNA fragments of 140–160 bp were dissolved in eight μL elution buffer. Finally, the quality of the libraries was evaluated on the Agilent Bioanalyzer 2100 system. The index-coded samples were clustered on a cBot Cluster Generation System using the TruSeq SR Cluster Kit v3-cBot-HS . Afterward, the small RNA libraries were sequenced on Illumina Hiseq 2000 platform, generating 50 bp single-end reads.Then, 3 μg total RNA was used as the input material, and NEBNextCanis lupus familiaris were downloaded from the Ensembl database (CanFam3.1) and mapped by Bowtie [p-value < 0.05 were considered differentially expressed.Clean data (clean reads) were generated by removing some of the low-quality reads from the raw data, such as containing ploy-N and 5′adapter contaminants, without 3′adapter or the insert tag. Moreover, GC-content, error rate, and Q30 of the raw data were calculated. To analyze the expression and distribution of the known miRNAs, the reference data of y Bowtie . Custom y Bowtie  were usey Bowtie  and miREy Bowtie . The expy Bowtie . The DEGp-value < 0.05.GOseq software and KOBAS software were used to implement the GO annotation and KEGG enrichment analysis for the underlying target genes of differentially expressed miRNAs (DEmiRNAs) ,18. The To validate the results of RNA-Seq, 15 DEmiRNAs (5 per infection stage) were randomly selected and their expression was analyzed by qRT-PCR on LightCycler 480 . The kit, amplification procedure, and melting curve analysis for qRT-PCR were conducted strictly according to the previous description . BrieflyIn total, 12.722 G data were generated from 18 spleen samples of Beagle puppies, obtaining 254,435,186 raw reads. Among them, an average of 0.678 G data was generated in the control group, and 0.736 G data were generated in the infected group. The GC-content, error rate, and Q30 of raw reads were 49.28%, 0.01%, and 97.63%, respectively. After screening, a total of 252,067,690 clean reads, with 96.07% mapping into the reference genome, were obtained from 18 samples. Compared to the reference sequences, 233,314,009 clean reads were identified as sRNAs, among which 163,137,451 clean reads were identified as known miRNAs that accounted for 69.99% of the total sRNAs, and 1,863,930 clean reads were identified as novel miRNAs that accounted for 0.81% of the total sRNAs. Most of the small RNA fragments are concentrated between 20 nt and 24 nt, and 182 novel miRNAs and 314 known miRNAs were identified A.Overall, 64 DEmiRNAs were identified in this study B. At 24 The validity of the RNA-seq outcomes was verified independently using the qRT-PCR method. The qRT-PCR validation results of the 15 randomly selected miRNAs showed the same trend as the sequencing results, demonstrating the reliability of the sequencing results D.irf1, tlr7, and masp1) of 18 DEmiRNAs were enriched in 490 GO terms; of those, 316 GO terms belonged to biological process , 64 GO terms belong to cellular component , and 110 GO terms belong to molecular function . At 96 hpi, 1499 potential target mRNAs of 33 DEmiRNAs  were enriched in 532 GO terms; of those, 368 GO terms belong to biological process , 47 GO terms belong to cellular component  and 117 GO terms belong to molecular function . At 36 dpi, 521 potential target mRNAs  of 19 DEmiRNAs of were enriched in 552 GO terms; of those, 414 GO terms belong to biological processes , 61 GO terms belong to cellular components , and 77 GO terms belong to molecular functions . The top 30 differential enriched GO terms in each infection stage are shown in Overall, 8242 potential target mRNAs of 470 miRNAs were predicted, including 676 potential target mRNAs of 18 DEmiRNAs at 24 hpi; 1738 potential target mRNAs of 33 DEmiRNAs at 96 hpi; and 545 potential target mRNAs of 19 DEmiRNAs at 36 dpi . The potpik3cd, pik3r5, and hmox1) of 10 DEmiRNAs were associated with six enriched KEGG pathways . At 96 hpi, 98 potential target mRNAs  of 17 DEmiRNAs were associated with seven enriched KEGG pathways . At 36 dpi, 19 potential target mRNAs  of six DEmiRNAs were associated with five enriched KEGG pathways . The top 20 KEGG pathways in each infection stage are shown in The potential target genes of DEmiRNAs were enriched by KEGG enrichment analysis . The resT. canis infection, which may have an impact on the pathology of toxocariasis. At the three different stages, overall, 496 miRNAs were identified, with 64 miRNAs being differentially expressed, including 20, 34, and 19 DEmiRNAs at 24 hpi, 96 hpi, and 36 dpi, respectively.The current study revealed the differential profile of splenic miRNA at three different stages after nfatc1), one of the predicted target genes of cfa-miR-320, was successfully assigned to two significantly enriched GO terms in this study, such as cytoplasm and cytoplasmic part. nfatc1 is integral for immune system development, which can induce the expression of cytokine genes in T cells [T. canis larvae by inducing an inflammatory response or inhibiting the T-cell function by targeting nfatc1 at the early infection stages. The downregulation of miR-193a clearly decreases the pro-inflammatory cytokines expression level [cd40, one of the predicted target genes of cfa-miR-193a, was successfully assigned to 33 significantly enriched GO terms, such as localization, tissue migration, and endothelial cell migration. cd40 ligation can induce monocytes to produce several cytokines, one of which is IL-10, an anti-inflammatory cytokine [T. canis infection by targeting cd40. cfa-miR-22 was downregulated 1.75 times at 24 hpi. Interferon regulatory factor 1 (irf1), one of the predicted target genes of cfa-miR-22, impacts myeloid lineages and lymphoid differentiation [irf1 in the spleen of puppies after T. canis infection.At 24 hpi, cfa-miR-320 was upregulated 1.43 times. A previous study showed that the miR-320 upregulation can boost the expression of inflammatory cytokines . The nucon level . At 24 hcytokine . The downtiation , the actnfatc1, myelin basic protein (mbp), semaphorin 7a (sema7a), TNF receptor superfamily member 1a (tnfrsf1a) and spleen associated tyrosine kinase (syk). Of these, at 96 hpi, GO annotation analysis showed that mbp was successfully assigned to two significantly enriched GO terms, such as neuron part and cellular component; sema7a was successfully assigned to 25 significantly enriched GO terms, such as cell growth, regulation of cell communication and positive regulation of cellular process; tnfrsf1a was successfully assigned to 15 significantly enriched GO terms, such as TNF-activated receptor activity, TNF binding, and oxoacid metabolic process; and syk was successfully assigned to 61 significantly enriched GO terms, such as kinase activity, biological adhesion, and protein kinase activity. syk was significantly enriched in Fc epsilon RI signaling pathway based on KEGG enrichment analyses. It was reported that mbp and its related transcripts are predominantly expressed in T cells [mbp-activated Th1 cells, increasing the expression of IFN-γ, TNF-α, -β, and IL-1β, while an anti-inflammatory response was started by mbp-activated Th2 cells, increasing the expression of IL-4, -10, and -13, which led to tissue damage and repair, respectively [sema7a, present on activated T cells, reduces inflammation by stimulating cytokine production by macrophages and monocytes via a1b1 integrin [tnfrsf1a is widely expressed in a variety of tissues, such as lymph nodes, spleen, thymus, lung, liver, and intestine. In parallel, in vitro, the tnfrsf1a gene suppresses the NF-κB signaling pathway while activating the TNF signaling pathway [syk expression is necessary for normal B-cell development and B cells activation [sema7a and tnfrsf1a, which are negatively associated with the inflammatory response.At 96 hpi, cfa-miR-331 is positively correlated with inflammatory response . It was  T cells . A pro-iectively . sema7a,integrin ,29. tnfr pathway ,31. Moretivation . At 96 hsyk and cd320) and was upregulated 1.47 times at 96 hpi. GO annotation analysis showed that cd320 was successfully assigned to three significantly enriched GO terms at 96 hpi, such as membrane part, membrane, and cellular component. cd320, FDC-signaling molecule 8D6, is a plasma membrane protein found on follicular dendritic cells (FDC) with functions as a co-stimulator to boost the stimulation of B-cell by antigen [cd320 enhances IL-10-directed B-cell to plasma cell differentiation in germinal centers [syk and cd320, reducing the B-cell response to antigenic stimulation at 96 hpi.cfa-miR-210 was predicted to regulate two potential target genes  was successfully assigned to 18 significantly enriched GO terms at 96 hpi, such as membrane part, enzyme regulator activity, and enzyme activator activity; and 25 GO terms at 36 dpi, such as intracellular, extracellular region and positive regulation of phosphorylation, based on GO annotation analysis. gprc5b regulated inflammatory response via NF-κB signaling, where overexpression of gprc5b boosted the NF-κB activation [gprc5b, was upregulated, suggesting that NF-κB activation promoted by gprc5b may be attenuated, and inflammatory response may also be attenuated.In the present study, cfa-miR-23b, which suppressed inflammatory responses , was uprtivation . At 96 hT. canis infection could be mediated by targeting the predicted target genes mbp, sema7a, tnfrsf1a, and IL12b. Among them, IL12b was successfully assigned to 64 significantly enriched GO terms at 36 dpi, such as defense response to protozoan, regulation of inflammatory response, and interleukin-12 alpha subunit binding. IL12b can serve as a growth factor for T cells and activated NK, increasing the lytic activity of NK/lymphokine-activated killer cells and inducing the production of IFN-γ [T. canis infection inhibits IL-12 production was confirmed in mice experimentally infected with T. canis [At 36 dpi, cfa-miR-339, which attenuated inflammation and apoptosis , was uprof IFN-γ . HoweverT. canis .β, -6) expression, whereas knockdown of miR-206 had the inverse effects [tcf7l2), one of the potential target genes of cfa-miR-206, was successfully assigned to 55 significantly enriched GO terms at 36 dpi, such as cell-cell signaling, regulation of response to stimulus and Wnt signaling pathway; and tcf7l2 was associated with four significantly enriched KEGG pathways, such as basal cell carcinoma, thyroid cancer, and melanogenesis, based on GO and KEGG analyses. Studies indicated that tcf7l2 is a pivotal effector in the composition of the tcf7l2/β-catenin complex [tcf7l2.At 36 dpi, cfa-miR-206 was downregulated 1.79 times, and overexpression of miR-206 increased the inflammatory cytokines  hallmark effector cytokine [IL17f is primarily involved in host defense by inducing neutrophilic inflammation [κB and MAP kinase pathways, ultimately leading to antimicrobial peptides, matrix metalloproteinases, cytokines, and chemokines transcriptional activation, with the potential for significant immunological inflammation [IL17f.At 36 dpi, cfa-miR-136 was downregulated 1.91 times. The upregulation of cfa-miR-136 alleviates inflammatory injury . IL17f, cytokine , particicytokine . Of whicammation  and actiammation . DownregT. canis infection. It can be shown that dynamic balances between pro-inflammatory and anti-inflammatory responses, and immuno-suppressive and modulatory effects induced by T. canis infection, and the innate and adaptive immune response of the puppies against T. canis infection. These findings supported earlier findings that the dynamic balance between pro-inflammatory and anti-inflammatory responses mediates the growth of T. canis in the definitive canine host [T. canis infection may be attenuated in puppies at this stage of infection, whereas at 96 hpi when T. canis appeared in the lungs of puppies, and at 36 dpi when T. canis settles in the intestinal tract after completion of tracheal migration, the host’s immune response against T. canis infection and mild parasite suppression essentially reached a dynamic balance, especially at 36 dpi.In this study, the up- and down-regulation of DEmiRNAs ultimately may impact the immune regulation of Beagle puppies against ine host ,12. HoweT. canis. As the infection progresses, the research showed transcriptional changes as well as notable differences in the expression and abundance patterns of miRNAs in the spleen between infected and uninfected puppies. Our findings indicate that the expression patterns of diverse populations of miRNAs in the spleen were particular to the advancement of T. canis infection. Investigating these miRNAs’ functional roles should provide additional knowledge for understanding the pathogenesis of toxocariasis.Using RNA-seq, the present study provided a quantized and holistic analysis of spleen miRNAs of Beagle puppies after infection by


Copper (Cu) is one of the essential microelements for all living systems. Studies have illustrated the biological significance of Cu homeostasis in human cancers, including breast cancer (BRCA). Nevertheless, the detailed roles of Cu homeostasis in BRCA need to be further explored. Here, we identified a downregulated Cu homeostasis-related gene FOXO1 and investigated the potential functions of FOXO1 in BRCA through several bioinformation databases. The BRCA patients with high level of FOXO1 displayed favorable prognostic values. Subsequently, enrichment analysis of FOXO1 coexpressed genes revealed that the top three enriched KEGG pathways were spliceosome, oxidative phosphorylation, and ribosome. Immunoinfiltration analysis indicated that aberrantly expressed FOXO1 showed positive correlations with the subcellular infiltration of macrophages and neutrophils in BRCA. Moreover, FOXO1 expression was positively associated with multiple immune checkpoints, such as sialic acid-binding immunoglobulin-like lectin 15 (SIGLEC15), indoleamine 2,3-dioxygenase 1 (IDO1), programmed cell death 1 ligand 1 (PD-L1/CD274), hepatitis A virus cellular receptor 2 (HAVCR2), programmed cell death 1 (PDCD1), cytotoxic T lymphocyte antigen 4 (CTLA4), and programmed cell death 1 ligand 2 (PDCD1LG2). Overall, these findings would deepen our understanding of FOXO1 in BRCA prognosis and immunotherapy response, representing a promising therapeutic strategy for BRCA patients. Breast cancer (BRCA) remains a major problem affecting women's health and is the second leading cause of cancer-related deaths among women worldwide. About 2.2 million people were diagnosed with BRCA each year, and a third of them die from this disease . In receCopper (Cu) is an essential micronutrient participating in various life processes. Precise regulation of Cu homeostasis is the key point to maintain fundamental biological functions and prevent the occurrence of related disease. Cu homeostasis has been reported to be involved in cell proliferation, angiogenesis, and metastasis . MeanwhiFOXO1, a winged-helix transcription factor, regulates a lot of physiological processes such as glucose homeostasis, apoptosis, autophagy, and cell cycle control. Additionally, FOXO1 promotes the expression of metal-containing antioxidant proteins including Cu-containing proteins and plays an important role in Cu homeostasis . FOXO1 iA comprehensive investigation was conducted to reveal the roles of Cu homeostasis-related genes in BRCA. This study mainly focused on the potential functions and mechanisms of Cu homeostasis-related gene, FOXO1, in BRCA, representing a promising prognostic and therapeutic target.p value was less than 0.01, and |log FC| was greater than 1. Next, a Venn diagram was exploited to identify the codifferentially expressed genes (co-DEGs) among two GEO datasets and the Cu homeostasis-related gene dataset.The data were obtained from the Gene Expression Omnibus (GEO) database according to the following conditions: (1) tumor type: BRCA, (2) species: Homo sapiens, and (3) analysis type: tumor vs. normal. We finally determined two GEO datasets (GSE10797 and GSE15852) . Then, gDRUGSURV, an open-access web resource, is devoted to providing the statistical evidence of the drugs affecting patients' outcomes by analyzing survival information of cancer patients . It was The following work focused on coexpression analysis of FOXO1, which was realized by the LinkedOmics algorithm . We obtaThe single-sample GSEA (ssGSEA) algorithm was performed to explore the associations between FOXO1 expression and tumor-infiltrating immune cells in BRCA. Subsequently, the Tumor IMmune Estimation Resource 2.0 (TIMER2.0) database  and Tumop value was less than 0.01 and |log FC| was greater than 1. We found 217 downregulated genes and 129 upregulated genes in GSE15852 and 396 downregulated genes and 29 upregulated in GSE10797, respectively. Subsequently, Venn plots between two GEO datasets and Cu homeostasis-related gene dataset presented two downregulated co-DEGs, FOXO1 and JUN  were exploited to screen differentially expressed genes between tumor tissues and normal tissues. The screen criteria were that  Figures . Unfortup < 0.05). However, there was no significant correlation between the JUN expression and patients' OS (p > 0.05). These findings supported the underlying roles of FOXO1 in BRCA patients' prognosis. Hence, FOXO1 was the main focus for further research.We utilized the DRUGSURV platform to obtain overall survival (OS) data of BRCA patients from GSE11121 , GSE3144Firstly, we found the lower FOXO1 expression in tumor groups compared with normal controls in GSE15852 and GSE10797 Figures . Then, ip = 0.004), race (p < 0.001), histological type (p < 0.001), PR status (p = 0.004), estrogen receptor (ER) status (p = 0.024), prediction analysis of microarray 50 (PAM50) (p < 0.001), menopause status (p = 0.013), and median age (p = 0.017). In contrast, N stage, M stage, pathologic stage, epidermal growth factor receptor 2 (HER2) status, and anatomic neoplasm subdivisions were not statistically significant. These results might provide a novel view for predicting the clinical progression of patients with BRCA based on FOXO1 expression.The associations between FOXO1 expression and clinical characteristics of patients with BRCA were investigated in TCGA-BRCA patients. As shown in The coexpression model of FOXO1 in TCGA-BRCA was confirmed by the LinkedOmics database, which was conducive to exploring the underlying biological functions. Firstly, 9171 genes positively and 5976 genes negatively associated with FOXO1 are displayed in Studies on the potential biological functions of FOXO1 were carried out though GSEA. The results of the KEGG pathway are shown in We used the ssGSEA algorithm to investigate the correlations between FOXO1 and immune-infiltrating cells in TCGA-BRCA cohort. The results suggested that the top 10 immune-infiltrating cells having positive correlations with FOXO1 expression were central memory T cells (Tcm), mast cells, immature dendritic cells (iDC), T helper cells, macrophages, effector memory T cells (Tem), natural killers (NK) cells, neutrophils, eosinophils, and those having negative correlations with FOXO1 expression were Th2 cells . We thenr = 0.414, p < 2.22e − 16), PDCD1LG2 , cell surface marker cluster of differentiation 96 (CD96) , and B and T Lymphocyte Attenuator (BTLA)  were the top four positively correlated molecules. Supplementary Figure r = 0.622, p < 2.22e − 16), 5′-Nucleotidase Ecto (NT5E) , ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1) , and CD40 ligand (CD40LG) . We then analyzed the relationships of FOXO1 expression and chemokines. The results indicated that the four significant molecules with the largest correlation coefficients were chemokine ligand12 (CXCL12) , C-C motif chemokine ligand 14 (CCL14) , C-C motif chemokine ligand 21 (CCL21) , and C-C motif chemokine ligand 22 (CCL22)  , C-C chemokine receptor type 4 (CCR4) , C-C chemokine receptor type 6 (CCR6) , and C-C chemokine receptor type 5 (CCR5)  were the most significant chemokine receptors associated with FOXO1. Taken together, these findings suggested the roles of FOXO1 in the immune regulation and immunotherapy response in BRCA patients.Next, other immune signatures related to the expression level of FOXO1 were investigated. Immunoinhibitors, immunostimulators, chemokines, and chemokine receptors were mainly included. The associations between FOXO1 expression and immunoinhibitors in patients with BRCA are displayed in Supplementary Figure In this research, we revealed the crucial role of Cu homeostasis-related gene FOXO1 in BRCA using multiple bioinformation databases. Firstly, two GEO datasets and the Cu homeostasis-related dataset were analyzed to screen the co-DEGs by Venn diagram. Then, the Kaplan-Meier plotter was used to evaluate the prognostic values of co-DEGs in BRCA patients. Moreover, the underlying roles of FOXO1 in the immune infiltration were also investigated. The results suggested the possibility of FOXO1 affecting BRCA progression though regulating the immune cell infiltration. Taken together, these findings could supply a new perspective for FOXO1 in the prognosis and treatment of BRCA patients.Cu is a kind of necessary trace element to participate in the regulation of numerous physiological activities. Cu metabolism needs to be strictly controlled to prevent various diseases, including metabolic syndrome . AbsolutFOXO1, as a member of the forkhead box O (FOXO) family, is involved in the transcriptional regulation of genes, affecting biological and physiological processes . It is wFor the past few years, a growing number of studies have focused on tumor immune microenvironment (TIME), which has shown a significance in tumor immunosuppression, metastasis, and drug resistance . More imThere were several deficiencies that need to be addressed. In this report, we mainly used comprehensive bioinformatics to demonstrate the downregulation of FOXO1 expression in BRCA. Moreover, we also demonstrated the potential roles of FOXO1 in the prognosis and immune response of BRCA patients. In the future, more in vivo and in vitro experiments will be conducted to clarify the biological function and underlying mechanisms of FOXO1 in BRCA pathogenesis, immune regulation, and therapeutic response.In this research, we revealed that FOXO1, a Cu homeostasis-related gene, was significantly downregulated in BRCA. Highly expressed FOXO1 indicated favorable prognosis values in patients with BRCA. In addition, the expression level of FOXO1 was linked to immune infiltrating regulation and might affect patients' therapeutic response, such as immunotherapy. Consequently, these findings would shed light on FOXO1 as a novel promising prognostic and therapeutic target for BRCA patients.


Brassica napus R2R3-MYB genes are still mainly unknown. In this study, 35 Brassica napus MYB (BnaMYB) genes were screened in the genome of Brassica napus, and details about their physical and chemical characteristics, evolutionary relationships, chromosome locations, gene structures, three-dimensional protein structures, cis-acting promoter elements, and gene duplications were uncovered. The BnaMYB genes have undergone segmental duplications and positive selection pressure, according to evolutionary studies. The same subfamilies have similar intron–exon patterns and motifs, according to the genes’ structure and conserved motifs. Additionally, through cis-element analysis, many drought-responsive and other stress-responsive cis-elements have been found in the promoter regions of the BnaMYB genes. The expression of the BnaMYB gene displays a variety of tissue-specific patterns. Ten lignin-related genes were chosen for drought treatment. Our research screened four genes that showed significant upregulation under drought stress, and thus may be important drought-responsive genes. The findings lay a new foundation for understanding the complex mechanisms of BnaMYB in multiple developmental stages and pathways related to drought stress in rapeseed.The R2R3-MYB sub-family proteins are composed of most members of MYB  protein, a plant-specific transcription factor (TF) that is classified into four classes depending on the number of MYB repeats. R2R3-MYB TFs are involved in physiological and biochemical processes. However, the functions of the  B. napus (Brassica napus L.). A method of accumulating secondary metabolites are formed through accumulating lignin [Populus), overexpression of the transcription factor PdNF-YB21 promotes root growth, along with drought tolerance [VlbZIP30 in grapevine (Vitis vinifera) [poCCoAOMT in tobacco (Nicotiana benthamiana) [IbLEA14 in sweet potato (Ipomoea batatas) [osTF1L in rice [PeLAC10, CmCAD2 and CmCAD2 in Arabidopsis  has been found [Zea mays) that are more lignified than drought-sensitive lines imply that lignification is a key mechanism for drought stress adaptation [In land plants, drought is often a major stress that can reduce crop productivity severely . Rapeseeg lignin ,6,7,8,9.hamiana) , IbLEA14batatas) , osTF1L  in rice , and PeLen found ,13. All MYB gene family that act as TFs for plants. There is a conserved DBD (DNA-binding domain) in the MYB transcription factor, which can be composed of up to four imperfect repeats, known as R repeats [There are several genes within the  repeats ,16. Each repeats ,17. In a repeats . Sequenc repeats . Members repeats .R2R3-MYB genes were identified in six brassica plants, including 130 in Arabidopsis, 236 in B. rapa, 247 in B. oleracea, 248 in B. nigra, 425 in B. napus, 422 in B. juncea and 412 in B. carinata. However, the number of R2R3-MYBs in Arabidopsis, B. rapa and B. napus were different from those found in previous studies, namely, 126 in Arabidopsis [B. rapa [B. napus [In a recent study , 2120 R2bidopsis , 256 in Pyrus bretschneideri) significantly activated the promoters of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2 and LAC18 by binding with the AC element in the promoter, thus positively regulating the lignification in the cell matrix of pear fruit [CAD and CCR genes involved in the lignin biosynthesis pathway, which has a negative regulatory effect on the lignin biosynthesis in wheat (Triticum aestivum L.) [COMT genes of maize (Zea mays L.) and Arabidopsis, and overexpression results in a reduction of lignin content in transgenic plants [Musa acuminata) [R2R3-MYB is responsive to drought stress while regulating lignin synthesis.R2R3-MYB TFs have been shown to synthesize hormones, participate in signal transduction, and be involved in physiological and biochemical processes, particularly in reaction to a variety of biotic and abiotic stimuli ,24,25,26ar fruit . The conar fruit ,29. The ivum L.) . ZmMYB31uminata) . AccordiArabidopsis, there are about 126 members of the R2R3-MYB subfamily, which is roughly divided into 25 subgroups and distributed across 11 branches [AtMYB26 can control the deposition of secondary walls in the anthers [AtMYB46 is a positive regulator of lignin biosynthesis in fibers and vessels [In branches . The fun anthers . AtMYB46 vessels . These tBnMYB genes of rapeseed were discovered by comparing the genomic sequences of the S13 and S16 subfamilies of the known R2R3-MYB from Arabidopsis thaliana. Additionally, we used the method of bioinformatic analysis to effectively process, analyze, and visualize a large amount of data from the field of life science. The physical and chemical characteristics of the BnMYB genes’ cis-acting promoter and their gene duplication were clarified, as well as their evolutionary relationships, genomic positions, gene structures, and three-dimensional membrane proteins. We also looked at how the BnaMYB gene family was expressed in various tissues and how they responded to drought treatment. Further research into the functional characterization of BnMYBs may benefit from our findings.In this work, 35 R2R3-MYB family genes in rapeseed, 12 AtTCP protein sequences were used as queries for a BlastP search against the rapeseed genome. As a result, 35 BnMYB genes were consistent with the R2R3-MYB domain  to 370 amino acids ; the MW ranged from 77,433.37 kDa (BnaMYB8) to 195,053.44 kDa (BnaMYB23); and the PI ranged from 4.89 (BnaMYB23) to 5.11 (BnaMYB8). The predicted subcellular location of each BnaMYB protein was in the nucleus. To identify B domain . HereaftBnaMYB genes were found on the chromosomes , B. rapa (A genome), and B. oleracea (C genome)  . The colWe compared the CDS sequences , which sBnaMYB promoters in order to determine the potential function of the BnaMYB genes, excluding components with uncertain functions and general transcriptional regulatory elements  (BioProject ID: PRJCA001495), the histo-specific expression profiles of BnMYB genes were detected in nine different tissues and organs . The expression patterns of all BnaMYB genes varied in the different tissues, as shown in BnaMYB3, BnaMYB5, BnaMYB7, BnaMYB11, BnaMYB14, BnaMYB15, BnaMYB16, BnaMYB17, BnaMYB18, BnaMYB19, BnaMYB23, BnaMYB25, BnaMYB26, BnaMYB27, BnaMYB28, BnaMYB30, BnaMYB31, BnaMYB33, BnaMYB34 and BnaMYB35. In the cotyledons, a few genes showed higher expression levels, including BnaMYB2, BnaMYB4, BnaMYB6, BnaMYB7, BnaMYB8, BnaMYB10, BnaMYB14, BnaMYB20, BnaMYB22, BnaMYB24, BnaMYB26, BnaMYB29, BnaMYB31 and BnaMYB32. In the roots, BnaMYB4, BnaMYB11, BnaMYB14, BnaMYB15, BnaMYB16, BnaMYB17, BnaMYB18, BnaMYB27, BnaMYB31 and BnaMYB33 displayed higher expression patterns. In the sepals, BnaMYB1, BnaMYB9, BnaMYB21 and BnaMYB28 exhibited higher expression patterns. In the siliques, BnaMYB5, BnaMYB15 and BnaMYB33 displayed higher expression patterns. Likewise, in the seeds, many genes, including BnaMYB12 and BnaMYB27, showed higher expression patterns. These findings led us to the conclusion that the BnaMYB gene family was essential for rapeseed’s development at all stages, and that other members with similar expression traits might serve related purposes.Through the use of RNA-seq data from BnaMYB genes had drought-responsive elements, which suggests that they may be involved in the response to drought, according to an analysis of the cis-acting elements in the upstream sequence of the BnaMYB promoters we conducted before. We chose 10 lignin-related genes  and submitted the 10 genes to drought treatment for 7 days to further discover their potential function  showed significantly upregulated expression under drought stress.The majority of R2R3-MYB gene family of B. napus and related species was biologically analyzed in this work, including their geographic position, phylogenetic analysis, gene structure, homologous protein domains, and predicted 3D protein structure. The findings demonstrated that the 19 rapeseed chromosomes contained an unequal distribution of 35 BnaMYB family members. These individuals were divided into three groups through phylogenetic analysis, and the majority of the genes within each subgroup shared similar gene structures, conserved protein domains, and three-dimensional protein structures, which is indicative of the BnaMYB gene family’s evolutionary stability.The Allotetraploid rapeseed is a crop that has been engaged in extensive genome replication and combination activities . One of MYB family has a significant function. The function of MYBs has been systematically researched in Arabidopsis [Setaria italica , Vitis vinifera [Zea mays [Populus trichocarpa [Gossypium raimondii [Arabidopsis and Oryza sativa [MYB gene subfamily in plants is the R2R3-MYB subfamily. Genetic methods have been used to characterize a large number of R2R3-MYB proteins, and it has been discovered that these proteins are involved in the regulation of a variety of plant-specific processes, such as (i) primary and secondary metabolism, (ii) cell fate and identity, (iii) developmental processes, and (iv) responses to biotic and abiotic stresses [R2R3-MYB gene family. The availability of rapeseed’s genome sequences makes it possible to identify rapeseed genes across the entire genome [BnaMYB genes in the genome of rapeseed.In plants, the bidopsis ,37, Setavinifera , Zea mayZea mays , Populuschocarpa , Gossypiaimondii , and otha sativa . The larstresses . To the e genome . Here, wBnaMYB gene family may have developed in the rapeseed genome through segmental events and many tandem duplications. A key strategy for gene expansion is tandem duplication. Genes that have been enlarged by tandem duplication are always dispersed throughout chromosomes as a group [BnaMYBs and members of the same group were studied, it was shown that MYB genes contributed equally to exon/intron allocation in terms of exon and intron numbers, while BnaMYBs showed similar motif patterns. These findings suggest that these R2R3-MYB members could play similar roles in a range of abiotic stressors.The  a group ,42. Exon a group ,44,45. WBnaMYB genes in diverse organs using publicly accessible data. The results showed that they varied in their patterns of expression in different tissues and that they were expressed in different tissues. In stems and cotyledons, 85.7% of BnaMYB genes were primarily expressed, while the primary expression of 10 genes was detected in the roots. BnaMYB12 was only strongly expressed in the seeds and less so in other tissues. BnaMYB1, BnaMYB9, BnaMYB21 and BnaMYB28 displayed higher transcription levels in the sepals. These findings suggest that the BnaMYB gene family might well be important for rapeseed’s development and expansion at all stages. Twenty of the BnaMYB genes were mostly expressed in the stems; therefore, they may play a crucial role in how the stems react to environmental influences. Little research has been conducted on Brassica napus, despite the fact that several R2R3-MYB genes have been linked to drought adaptations and endurance in some species, particularly in rice and Arabidopsis [BnaMYBs is helpful to analyze the response of R2R3-MYB to drought stress in Brassica napus.We looked at the expression patterns of 35 bidopsis ,47,48,49R2R3-MYB family have been identified as transcription factors that are triggered by abiotic stress to date [Arabidopsis overexpressing the R2R3-MYB gene GaMYB85 [Arabidopsis overexpressing LpMYB1 is more tolerant to salt and drought because of this R2R3-MYB factor [TaMYBsdu1 increases noticeably in the roots and leaves of wheat [TaMYB33 improves tolerance to salt and drought, partly by restoring the osmotic equilibrium and eliminating superoxide radicals [TaMYB30-B and TaMYB19-B can increase the ability of transgenic Arabidopsis plants to withstand drought stress [R2R3-MYB has the function of dealing with drought stress in wheat, cotton, and Arabidopsis and their function during drought stress is affected by the ABA synthesis and signal transduction pathway. For instance, the accumulation of 35S:GaMYB85 resulted in the transcription of RD22, RD29A, ADH1, AB15, and P5CS. The AB15 gene, which codes for a bZIP TF, is known to be an ABA-responsive gene that is important for seed germination under drought stress [Brassica napus is affected by the lignin content. MYB46 controls not only the signaling molecules but also the genes that produce all three of the primary components of the secondary walls: grain, hemicellulose, and lignin [MYB85 controls the production of lignin by regulating the lignin-synthesizing genes and, when overexpressed, results in ectopic lignin deposition [BnaMYB gene family may affect lignin synthesis, and lignin synthesis responds to drought stress. We will pay attention to this in future research to deeply understand the relationship between the BnaMYB gene family and lignin.Numerous members of the plant  to date ,50,51. C GaMYB85 . ArabidoB factor . Under pof wheat . TaMYB33radicals ,55. TaMYt stress ,57. Thest stress ,59. In ld lignin . Additioposition . This inBnaMYB gene had significant changes under drought conditions, which proved that 10 genes were responsive to drought stress. The expression of BnaMYB5 decreased significantly under drought stress, indicating that this gene could not cope with drought; BnaMYB3, BnaMYB19, BnaMYB23, BnaMYB25 and BnaMYB35 showed U-shaped curves, indicating that these five genes did not show drought resistance under mild drought stress but showed drought resistance under severe drought. Four genes  were significantly upregulated under drought stress, indicating that they are probably drought-resistant genes. According to our understanding, this is the first evidence indicating that R2R3-MYB genes in B. napus are implicated in the response to drought stress. This work is helpful for understanding the characteristics and functions of R2R3-MYB genes in different species.The tissue-specific expression patterns reflected which tissues of the plant the gene mainly acts on. We found that 20 genes were highly expressed mainly in the stem, indicating that these genes were probably involved in lignin synthesis. Ten of these genes were selected for drought treatment, and their expression profiles were detected under drought stress. Our RT-qPCR data showed that the http://cbi.hzau.edu.cn/bnapus/, accessed on 14 February 2022) [R2R3-MYB  was downloaded from the Pfam database . The cut-off E-value was chosen to 1e-10 when using the Markov model on HMMER software to preliminary screen the protein sequences of rapeseed. In order to exclude possible R2R3-MYB proteins without two conserved MYB DNA-binding domains, all candidate proteins were then uploaded to three online sources, SMART , NCBICDD , and PFAM . For the MW and pI prediction analyses, ExPASy  received the identified R2R3-MYB candidate genes. Additionally, the subcellular localization was predicted by WoLF PSORT .On the BnPIR website (ry 2022) , the genhttp://cbi.hzau.edu.cn/bnapus, accessed on 5 March 2022) provided information on the chromosome location of BnaMYBs. After that, the distribution status of the BnaMYBs discovered on the chromosomes was shown using MapChart software. With the aid of Adobe Illustrator, the outcomes were improved.The BnPIR website . The sequences of the protein in Oryza sativa and Zea mays were collected from the Ensembl website . The protein sequences of Capsella rubella and Raphanus sativus were collected from the NCBI website . OrthoMCL software (v2.0.3) [Arabidopsis thaliana, Brassica oleracea, Brassica rapa, Capsella rubella, Oryza sativa, Raphanus sativus and Zea mays using entire R2R3-MYB protein sequences. The E-value cut-off of an all-against-all BLASTP alignment process was set at 1 × 10−10, and the alignments with a match cut-off value lower than 50 were eliminated.We downloaded the protein sequences of the MYB family members of (v2.0.3)  was usedMYB family members from B. napus, Arabidopsis, B. oleracea, B. rapa, Capsella rubella, Oryza sativa, Raphanus sativus and Zea mays were retrieved. Additionally, MEGA 11.0 software was used to create the evolutionary tree using the neighbor joining method (NJ) once the results of sequence alignment were obtained [http://www.omicsclass.com/article/671, accessed on 13 March 2022) to visualize the evolutionary tree.These plants’ MYB protein sequences were analyzed using Clustal W after the protein sequences of obtained ,64. FinaR2R3-MYB genes contains intron and exon information [https://meme-suite.org/meme/, accessed on 30 March 2022) was used to examine the homologous domains of the genes using the BnaMYB protein sequences, increasing the maximum motif count to 10, the maximum motif amino acid count to 20, and the minimum motif width to 6, and leaving all other variables at their normal values. Finally, the similar motifs of BnaMYB proteins were viewed using TBtools software.The gene structure of the ormation . MEME Suhttp://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 25 April 2022).Thirty-five BnaMYB protein sequences were entered into the DNAMAN8.0 program for triple sequence comparison. We next predicted the 3D structure of the proteins using the internet tool Phyre2 (BnaMYB genes’ intra-species collinearity in B. napus and Circos software (v0.69-8) was used to visualize the associations. Additionally, the McScanX software (Python version) was used to visualize the collinearity.McScanx analysis was used to analyze the BnaMYB genes were input into DNAMAN8.0 for sequence alignment. We selected the CDS sequences of homologous gene pairs, uploaded them to the ALTER website , and outputted the results in ALN format. We converted ALN format to AXT format with AXT Converter and used KaKs Calculator 2.0 software to calculate the value of Ka/KS.The CDS sequences of 35 B. napus  provided 1500 bp of the upstream sequences of the BnaMYB genes. The cis-acting components were extracted using the online tool Plant CARE  and then visualized using the online tool DSGS.The publicly available online whole-genome information on Brassica napus genomic resource is a website  [http://www.heatmapper.ca, accessed on 2 May 2022).The BnTIR: ay 2022) . The steIn this study, the rapeseed genotype “ZS11”, a typical cultivated variety, was used for the stress treatments. The seeds of the ZS11 genotype were furnished by OCRI, CAAS, China. Before the stress treatments, some seeds were randomly selected from the same batch of seeds to determine the germination rate. The seeds with a 100% germination rate were considered to be vigorous seeds. The vigorous seeds were carefully chosen and sterilized with a 10% hypochlorous acid solution for 5 min. The seeds were grown on water-saturated filter paper in a chamber  until the plants grew to the five-leaf stage. When the plants had grown to the five-leaf stage, we stopped watering to impose drought stress. Samples from the stem were collected 0, 1, 2, 3, 4, 5, 6 and 7 days after the initiation of the drought treatment ,68. More2O. Specific primers were created using the online qPCR Primer Database  based on the coding sequences of the BnaMYB genes. The specific primers were amplified, and the primers with a ct value of no more than 30, a normal amplification curve, and no obvious impurity peak in the dissolution curve were selected for subsequent experiments. The quantitative experiment was conducted in real-time using SYBR Premix Ex TaqTM (TaKaRa). Three separate replicates were collected for this experiment, and the samples that had not been subjected to drought were used as standards. The 2−ΔΔCT technique is often used in investigations of the relative expression of genes [Using a carbohydrate phenolic total RNA extraction kit, the total RNA was isolated from leaves that had undergone various drought treatments . An ultramicroscopic spectrophotometer was used to measure the quantity and quality of the RNA . As samples for subsequent RT-qPCR experiments, we synthesized cDNA using a reverse transcription kit and diluted it 100 times with ddHof genes . The graBnaMYB genes were found in B. napus, dispersed unevenly across 19 chromosomes. We analyzed how the MYB proteins are related, their gene structure, their conserved motifs, and their three-dimensional structures. These genes were divided into three subfamilies, each of which had reasonably traditional gene architectures and patterns. The BnaMYBs promoter regions also contained cis-acting hormones and abiotic stress response components, along with drought-responsive genes. There were 48 pairs of large-segment repetitive genes discovered in B. napus through the analysis of collinearity. The R2R3-MYB genes of B. napus had undergone polyploidization and various degrees of loss and expansion, according to comparative genomic research. We also looked at the gene expression patterns of BnaMYB in various rapeseed tissues, and the results showed that the members of the BnaMYB gene family were crucial at different phases of the development of B. napus. In addition, we examined the expression patterns of 10 BnaMYB genes under drought. Overall, by carefully examining the conservation and divergence of the BnaMYB gene family’s activities, our work lays the biological groundwork for the subsequent functional discovery of R2R3-MYB genes in cruciferous plants.In total, 35


Dilated cardiomyopathy (DCM) is a clinical diagnosis characterized by left ventricular or biventricular dilation and systolic dysfunction. In most cases, DCM is progressive, leading to heart failure (HF) and death. This cardiomyopathy has been considered a common and final phenotype of several entities. DCM occurs when cellular pathways fail to maintain the pumping function. The etiology of this disease encompasses several factors, such as ischemia, infection, autoimmunity, drugs or genetic susceptibility. Although the prognosis has improved in the last few years due to red flag clinical follow-up, early familial diagnosis and ongoing optimization of treatment, due to its heterogeneity, there are no targeted therapies available for DCM based on each etiology. Therefore, a better understanding of the mechanisms underlying the pathophysiology of DCM will provide novel therapeutic strategies against this cardiac disease and their different triggers. MicroRNAs (miRNAs) are a group of small noncoding RNAs that play key roles in post-transcriptional gene silencing by targeting mRNAs for translational repression or, to a lesser extent, degradation. A growing number of studies have demonstrated critical functions of miRNAs in cardiovascular diseases (CVDs), including DCM, by regulating mechanisms that contribute to the progression of the disease. Herein, we summarize the role of miRNAs in inflammation, endoplasmic reticulum (ER) stress, oxidative stress, mitochondrial dysfunction, autophagy, cardiomyocyte apoptosis and fibrosis, exclusively in the context of DCM. Dilated cardiomyopathy (DCM) is a heart muscle disease characterized by left ventricle (LV) or biventricular dilatation and systolic dysfunction in the absence of coronary artery disease, hypertension, valvular disease or congenital heart disease . DCM is TTN) and lamin A/C (LMNA) genes, as they are responsible for nearly 10–15% and up to 35% of genetic DCM . Mo. MomiR-2ammasome . Furtherammasome . Finallyammasome . Finally (TRAF6)  28]..miR-221/+ T cells, was identified as a regulator of cell activation and proliferation . Mo. MoPak2 R-194-5p . This wo16 cells ,46  and cytochrome c oxidase subunit 1 (COX1), which are involved in the coding of complex I and complex IV of the electron transport chain. The intravenous injection of agomiR-1 increased the left ventricular ejection fraction and the expression of Nd1 and Cox1 in a mouse model of isoproterenol-induced HF ..ARL2) , xanthine oxidase, and uncoupled nitric oxide synthase . MultiplNOX2) expression, one of the major NOX isoforms expressed in the heart, and NOX2-derived ROS production are involved in the pathophysiology of Duchenne muscular dystrophy, a muscle-wasting disease accompanied by DCM . Th. ThATG13 complex . In addimulation .Becn1 in an in vitro model of doxorubicin-induced cardiotoxicity in rat primary cardiomyocytes , is activated, triggering the formation of the autophagosomes. In normal conditions, the Bcl2 family anti-apoptotic proteins interact with Beclin-1 and inhibit autophagy ,92. Howemyocytes  93], wh, whBecn1C from mitochondria in response to an apoptotic stimulus. Released cytochrome C, in the presence of dATP, forms an activation complex with apoptotic protein-activating factor-1 (APAF1) and caspase-9 (CASP9) that activates downstream caspases  . A. ACamk1d (Pa2g4)  112]. T. TCamk1d miR-675 .XIAP  XIAP  114]. M. MXIAP  [Col3a1) [JNK1 , the receptors TGFβR1 and TGFβR2, and ETS-1 (ETS proto-oncogene 1) [Diverse studies have shown that miRNAs play a central role in regulating the pathogenesis of myocardial remodeling by modulating cardiac fibrosis, angiogenesis, and inflammatory response through multiple mechanisms . In the trations ,131. In fibrosis . Bernards [Mst1) ,134. Mor[Col3a1) . In heal[Col3a1) . Accordi[Col3a1) ,137. Nis[Col3a1) . Signifiogene 1) . It has ogene 1) . In addiogene 1) , an miRNogene 1) . Wang etogene 1) . More reogene 1) . Finallyogene 1) .The main miRNAs involved in the regulation of cardiac fibrosis in the pathogenesis of DCM are reported in The functional validation of miRNAs has enabled a better understanding of the cellular and developmental biology of various diseases at the molecular level . As outlTo date, clinical trials (phase 1 and 2) of antimiRs against miR-122-5p   have proOn the other hand, numerous pharmacological challenges remain to be solved. Although the development of synthetic miRNA mimics and chemically modified antimiRs has helped us to overcome major hurdles, the delivery of these molecules, as well as the establishment of effective concentrations in target tissue, still pose considerable challenges. Their hydrophilic nature that hinders their membrane penetration, degradation in the bloodstream of systemically delivered oligonucleotides, as well as the poor delivery to the target site, in particular for cardiovascular tissue, of systemically delivered oligonucleotides are barriers that need to be overcome. Different approaches have been developed to circumvent these obstacles, of which many hold promise for cardiovascular applications, e.g., LNA antimiRs, as they penetrate membranes as “naked” molecules . On the Finally, further work is necessary to address some aspects that make data interpretation very challenging. When working with human samples, there can be conflicting observations regarding the miRNA expression profile in DCM. Since a large proportion of the current evidence is derived from monocentric case–control studies that, in most cases, lack external validation in large prospective cohorts, these differences can be attributed to potential differences in sample/patient numbers. In addition, co-morbidities should be considered. More importantly, there is a relevant lack of standardization in methods and analytical workflow, including sampling time, methods for miRNA quantification, as well as miRNA normalization parameters. Thus, the discovery of the ways in which one can avoid these limitations will be a major challenge in applying miRNAs for diagnostic and therapeutic purposes.


Two different mid-infrared (mid-IR) solid-state crystalline laser active media of Cr There is a continuous demand for affordable and reliable sources of middle infrared (mid-IR) laser radiation usable for all kinds of applications, including spectroscopy, medical treatment, biomedical non-invasive diagnostics, remote sensing of atmospheric constituents (trace gases), chemical and biological analysis, metrology, optical radars, optical communication in free space, optical switching, laser radar in eye-safe range, military applications (target designation or obstacle avoidance), and others ,2,3,4. Tμm . MoreoveA promising way, among others, is to utilize the Ae et al. ,6,7 intrinto ZnS ,9, ZnSe into ZnS ,11,12, Cinto ZnS , and rec1−xMnxTe ,15,16, Z1−xMnxSe ,18,19, Z1−xMnxTe ,21, Zn1−1−xMgxSe ,23, and 1−xMgxSe ,25, free1−xMgxSe ,27,28 or1−xMgxSe ,27,28), 1−xMgxSe ,30 regimr2+ ions  or over ith ZnSe .In several previous studies ,36,37,38ented in . This arIn this paper, the possibility of the energy transfer excitation mechanism for Cr,Fe:Znpared to , and theThe Cr,Fe:ZnThe transmission spectra of both crystals were measured by the FT-IR spectrometer  the electro-optically Q-switched flash-lamp pumped Er:YLF laser, (b) the electro-optically Q-switched flash-lamp pumped Er:YAG laser (both operated at room temperature—RT), and (c) the LNThe results of spectroscopic and laser output properties for 1.73, 2.94, and ∼4.05 The spectroscopic properties of both investigated materials in the temperature range of 78–300 K were measured. The results obtained for absorption and fluorescence measurements can be found in The absorption spectra of both crystal samples at 78 and 300 K, together with the used pumping laser’s oscillation spectra, are shown in The relative ratio between CrEach sample has been inserted at an angle of 45° to the incident laser beam of the Er:YLF laser at 1.73 It is good to note here that the CrThe temperature dependence of the CrThe FeIn comparison with , some reshown in ,39 for tt) is the intensity of Crt) is the intensity of CrUsing the data in ion from (1)ηT=1−d sample ,40. The As follows from Equation  using thThe investigated laser active crystalline media were placed gradually in a copper holder inside the evacuated cryostat, which was then cooled down by liquid nitrogen to a temperature of 78 K. The laser output properties, such as laser output energy, pulse duration, oscillation spectra, and beam profile, were measured. The temperature was controlled by a Cryogenic Temperature Controller . Three different excitation wavelengths of 1.73 , 2.94, aμm  were appr = −150 mm or r = −200 mm). To obtain the CrFor a better comparison, all investigated laser systems had similar cavity parameters—the laser cavity length of about ∼10 cm  OC with a reflectivity of RFor the direct excitation of Fesfer see b. Some dMoreover, the gain-switched Fe:ZnSe laser operated at 78 K, generating radiation at ∼4.05 In this paper, we present the comparison of two novel Cr,Fe:ZnThe maxima of absorption spectra, as well as whole absorption bands of both active ions, were shifted farther to the mid-infrared part of the optical spectrum in the case of the Mg sample. The difference between the FeThe temperature has a significant impact on all spectroscopic as well as laser properties of the studied materials (except for CrThe fluorescence lifetime of CrThe direct excitation of CrThe direct pumping of FeIn conclusion, both active laser crystalline media demonstrated an opportunity of mid-IR radiation generation under direct Creviously ,38, the Direct pumping of the Fe


Cancer is a horrific disease that, to date, has no cure. It is caused by various factors and takes many lives. Apoptosis is a programmed cell death mechanism and if it does not function correctly in cancer cells, it can lead to severe disease. There are various signaling pathways for regulating apoptosis in cancer cells. Flavonoids are non-artificial natural bioactive compounds that are gaining attention as being capable of for inducing apoptosis in cancer cells. Among these, in this study, we focus on flavones. Flavones are a subclass of the numerous available flavonoids and possess several bioactive functions. Some of the most reported and well-known critical flavones, namely apigenin, acacetin, baicalein, luteolin, tangeretin, and wogonin, are discussed in depth in this review. Our main aim is to investigate the effects of the selected flavones on apoptosis and cell signaling pathways that contribute to death due to various types of cancers. Cancer is one of the world’s most deadly diseases, is expensive and complicated to treat, and has many side effects due to the chemical drugs used in its treatments. The causes of cancer are diverse and can be divided into two groups: intrinsic and extrinsic risk factors. Intrinsic risks arise from accidental errors in DNA replication and cannot be modified. Extrinsic risks include both endogenous and exogenous factors. Endogenous risk factors that can be partially modified include aging, hormones, growth factors, inflammation, and DNA repair mechanisms. Exogenous risk factors are adjustable and include radiation, chemical carcinogens, tumor-causing viruses, and smoking .The hallmarks of cancer cells include the continuous maintenance of proliferation signals, avoidance of growth inhibitors, activation of invasion and metastasis, induction of angiogenesis, and the possibility of permanent proliferation. In addition, the abnormality of cellular energy metabolism, avoidance of immune destruction, tumor-promoting inflammation, genomic instability, and mutation have been suggested . The malApoptosis, along with necrosis and autophagy, is a form of programmed cell death in cancers . During In the course of cancer treatment, many studies have been conducted, and a sufficient understanding of apoptosis is considered to be important in dealing with cancer .To effectively prevent cancers through apoptosis and treat them without side effects, flavonoids as phenolic compounds play a significant role as natural biological response modifiers (BRM) . FlavonoAs an anticancer effect, the structure–activity relationship revealed in the comparison of flavonoids showed that the adjacent di-OH at the 3′ and 4′ of the B ring and the C2-C3 double bonds were important in the potent antiproliferative activity ,29. In p15H10O2 and the chemical structure is C6-C3-C6. The basic flavonoid structure contains a ketone group at C4 and has a double bond between C2 and C3 [Flavones are a subclass of flavonoids B and are2 and C3 ,38. Apig2 and C3 . Researc2 and C3 ,41, and 2 and C3 ,43,44, x2 and C3 , and lip2 and C3 . Antiath2 and C3 , nitric 2 and C3 , cyclo-o2 and C3 ,50, leuk2 and C3 , spasmol2 and C3 , NAD(P)H2 and C3 , GABA an2 and C3 ,55, pros2 and C3 ,57, anti2 and C3 , α-amyla2 and C3 ,60, glyc2 and C3 , aldose 2 and C3 , sirtuin2 and C3 , hepatop2 and C3 , antiarr2 and C3 ,66, and 2 and C3  are some2 and C3 , antibac2 and C3 , antifun2 and C3 , antipro2 and C3 , photo-p2 and C3 , cosmeti2 and C3 , and pho2 and C3   and is effective in treating breast, colorectal, lung, and ovarian cancers and glioma [Wogonin is called 5,7-dihydroxy-8-methoxyflavone F. The mo26 g/mol ,99. Wogod glioma ,100,101.In cancer cells, apoptosis is a very complex mechanism that is indispensable. If apoptosis does not function properly, cancer cells proliferate indefinitely . As showIn the intrinsic pathway, cytochrome c is released from the mitochondria by the Bcl-2 family, which combines with apoptotic protease activation factor-1 (APAF-1) and ATP to form an apoptosome complex by binding pro-caspase-9. It activates caspase-9 and caspase-3, -6, and -7 which are the executioners of apoptosis ,11,102. The extrinsic pathway is mediated by the death receptors including the Fas receptor, tumor necrosis factor receptor (TNF), and TNF-related apoptosis-inducing ligand receptor (TRAIL) . These dNumerous signaling pathways are involved in these two major (extrinsic and intrinsic) apoptosis pathways. The regulation of apoptosis is imperative in cancer cells as well as some of the major cell signaling pathways involved in the survival and death of cancer cells.2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) [3 is increased, AKT and phosphoinositide-dependent kinase 1 (PDK1), which are enzymes in the pleckstrin homology domain (PH), are gathered and AKT is activated by PDK1 and mTOR complex 2 (mTORC2) [The PI3K/AKT pathway, which can regulate apoptosis in cancer cells, plays a major role in mammalian cell proliferation, differentiation, autophagy, survival, and apoptosis . The lipe (PIP3) ,107. Whe(mTORC2) ,107. Thi(mTORC2) ,109,110.(mTORC2) ,112. In (mTORC2) .The Wnt pathway is a signaling pathway involved in cell proliferation, migration, stem cell differentiation, and various diseases including cancer. In this pathway, β-catenin belongs downstream of Wnt and plays a key role in apoptosis while triggering transcription by the Wnt ligand ,115,116.Abnormal activation of the janus kinase (JAK)/signal transducer and activators of the transcription (STAT) pathway promotes tumorigenesis . The cytThe mitogen-activated protein kinase (MAPK) pathway plays an important role in cell proliferation, differentiation, angiogenesis, tumor metastasis, and apoptosis . MAP kinAs a tumor suppressor, p53 is activated when DNA is damaged, causing cell cycle arrest and apoptosis. When this protein is mutated, cells that should die do not die and cancer occurs . In the Apigenin has exhibited anticancer effects on many cancer cells such as breast, prostate, liver, colorectal, skin, and lung cancers . ApigeniAcacetin induced apoptosis in cancer cells through various pathways such as mitochondria-mediated death signaling, caspase activation, the β-catenin pathway, and NF-кB/AKT signaling for breast, gastric, colorectal, and prostate cancers ,85,86,87Baicalein regulated apoptosis through various pathways for breast, liver, stomach, colorectal, lung, cervical, and ovarian cancers ,89. BaicLuteolin induced apoptosis in breast, lung, gastric, liver, and cervical cancers ,92,93. F2 [Tangeretin induced apoptosis in breast, gastric, prostate, and bladder cancers through various pathways ,96,97. T2 . In AGS 2 . In PC-3Wogonin induced apoptosis through various pathways in breast cancer, colorectal cancer, lung cancer, ovarian cancer, and glioma . WogoninComprehensively, Apigenin suppressed colorectal cancer and reduced tumor volume in prostate cancer without reducing appetite when ingested in Sprague–Dawley (SD) rats. Colorectal cancer was induced by azoxymethane (AOM), which is effective for the induction of colon carcinoma in SD rats. In these SD mice, apigenin treatment had the potential to suppress colorectal cancer by reducing aberrant crypt foci (ARF), which are clusters of abnormal tube-like glands, and increasing apoptosis . In addiAcacetin inhibited tumor growth through STAT3 regulation in DU145 prostate cancer cells in a nude mouse xenograft model .As a result of analyzing the gene expression of a lung cancer xenograft tumor mouse model, the genes when baicalein induce apoptosis, ITGB3 (+6.96) and TNFRSF25 (+3.4), were most significantly upregulated . In anotLuteolin reduced the weight and volume of gastric tumors in a rat model by the inhibition of Notch1 and β-catenin . When luTangeretin inhibited tumor growth in the MDA-MB-231 breast cancer cell nude mouse xenograft model . TangereAs a result of treatment with wogonin in BALB/C nude mice xenografted with A2780 ovarian cancer cells and HT-29 colorectal cancer cells, the tumor volume and weight were reduced .Cancer is still a disease whose treatments face many challenges. Anticancer research on polyphenols, such as flavonoids, has been actively conducted over the past few decades . Of courIn this review, the apoptosis of the external and internal pathways has been mainly investigated, but the endoplasmic reticulum (ER)-mediated apoptosis pathway is also emerging as an important apoptosis pathway. The endoplasmic reticulum is an important organelle of eukaryotic cells and is necessary for maintaining intracellular homeostasis; controlling the synthesis, secretion, and folding of membrane-bound proteins; and transporting calcium ions . When a Therefore, this endoplasmic reticulum-mediated apoptosis is very important and a pathway in which natural compounds, such as flavones, control ER stress  to act aThis review includes a basic introduction to the selected flavones and their anticancer effects. In addition, we investigated the various pathways involved in apoptosis and discussed the signaling pathways by which flavones induce apoptosis. Flavones could be promising compounds for anticancer action in the future after further research considering this review information. Lastly, we hope this review will serve as a valuable manual for anticancer-related research using flavones.


Poly(rC)-binding proteins (PCBPs), a defined subfamily of RNA binding proteins, are characterized by their high affinity and sequence-specific interaction with poly-cytosine (poly-C). The PCBP family comprises five members, including hnRNP K and PCBP1-4. These proteins share a relatively similar structure motif, with triple hnRNP K homology (KH) domains responsible for recognizing and combining C-rich regions of mRNA and single- and double-stranded DNA. Numerous studies have indicated that PCBPs play a prominent role in hematopoietic cell growth, differentiation, and tumorigenesis at multiple levels of regulation. Herein, we summarized the currently available literature regarding the structural and functional divergence of various PCBP family members. Furthermore, we focused on their roles in normal hematopoiesis, particularly in erythropoiesis. More importantly, we also discussed and highlighted their involvement in carcinogenesis, including leukemia and lymphoma, aiming to clarify the pleiotropic roles and molecular mechanisms in the hematopoietic compartment. They form complexes to participate in mRNA transport and stabilization, gene alternative splicing, translational enhancement, repression, and chromatin remodeling, thereby controlling the expressions of encoded proteins , from direct transcriptional to post-translational control. hnRNPs represent a superfamily of RNA binding proteins (RBPs) interacting with pre-mRNA proteins . Prior rproteins . Poly(rCproteins .in utero before E13.5 (Pcbp1 die early during the peri-implantation (E4.5–8.5) stage. Developing mutant Pcbp2 homozygotes proceed normally until E12.5–E13.5 owing to a significant loss in viability associated with combined cardiovascular and hematopoietic anomalies   and hnRNre E13.5 . Mutant nomalies , 12. PCBDuring the past few years, numerous studies have shown that PCBP family members, especially hnRNP K, PCBP1, and PCBP2, function as modulators in hematopoiesis through multiple-level molecular mechanisms. These include nucleic acid metabolisms such as pre-mRNA alternative splicing, specific mRNA stabilization, transcriptional and translational regulation, or critical iron chaperones delivering iron to target proteins. Considering their functional diversity and complexity, the primary role of PCBPs in regulating gene expression has gained interest in hematopoietic malignancies, such as leukemia and lymphoma. This review summarizes the existing knowledge regarding the structural and functional divergence of various PCBP family members. More importantly, we highlight their roles in normal hematopoiesis, especially erythropoiesis, as well as their involvement in carcinogenesis, including leukemia and lymphoma, over the past decade. Thus, we aim to elucidate the pleiotropic roles and molecular mechanisms of PCBPs in the hematopoietic compartment.22.1. PCBP family members are well-conserved, with the corresponding KH domains sharing a higher degree of homology than the KH domains within each protein (All PCBP family members primarily comprise triple hnRNP K homology (KH) domains  that recognize and combine C-rich regions of RNA, ssDNA, and dsDNA. The KH domain was initially found in hnRNP K as triple repeats  and was  protein . The KH  protein . The pri protein . Differe protein –19. In P protein , 19–21.  protein –24.. Two functionally independent NLS exist in both PCBP1 and PCBP2. A nine amino-acid segment of NLS I is mapped between KH2 and KH3 domains, and NLS II with 12 amino acids are localized in the KH3 domain  sequence that mediates transport from the cytoplasm to the nucleus, dictating differential subcellular localization of PCBPs 3 domain . PCBP1 a3 domain . Therefo3 domain . The locvia the KI domain that are involved in gene expression and signal transduction  domain located between KH2 and KH3 domains, which is absent in other PCBPs . The KI sduction . Immunopsduction . These ssduction .2.2Xu et al.  further increase the diversity of PCBPs’ structures and their potential functions, including protein phosphorylation, methylation, and SUMOylation. Generally, PTMs in certain regions can directly influence the subcellular localization of proteins or interact with nucleotides or proteins. hnRNP K possesses the most abundant PTM sites and has recently been comprehensively reviewed by u et al. . Herein 284/353 of hnRNP K, located in the KI or KNS region, promotes cytoplasmic accumulation of hnRNP K by affecting nucleocytoplasmic shuttling  leads to its nuclear localization and reduces its binding affinity to mRNA -binding activity . PCBP1 p to mRNA . TGF-β-aet mRNAs .33.1.Erythropoiesis, an important part of hematopoiesis, is a highly coordinated and precisely regulated developmental process. The committed erythroid progenitors, BFU-E and CFU-E from hematopoietic stem cells, undergo terminal differentiation into proerythroblasts and basophilic, polychromatophilic, and orthochromatic erythroblasts. They can enucleate to form reticulocytes, release them into the bloodstream, and mature into erythrocytes. With differentiation and maturation, erythroid precursors show a gradual cell and nuclear size reduction. Cell contents are rapidly and progressively replaced with an increased accumulation of hemoglobin . PCBPs h3.1.1Globin mRNAs accumulate 95% of total cellular mRNA during terminal erythroid differentiation ,. The ac3.1.2Alternative splicing programs produce multiple transcripts, resulting in diverse transcriptomic potential. During erythropoiesis, thousands of genes, including transcription factors and receptors, undergo alternative splicing to satisfy the physiological needs of differentiating erythroblasts .via the interaction with the U2 snRNP complex and canonical polypyrimidine tract binding protein, U2AF65, enhances splice acceptor activity and promotes target exon inclusion  splice acceptor sites located 5’ to the PCBP-enhanced exonic segment. PCBP1/2, nclusion .via the KH1 domain, along with TIA1, and promotes the recruitment and stabilization of U2 snRNP to a weak branch point by interaction with RBM39, U2AF65, and SF3b155. Thus, PCBP1 stimulates the spliceosome, complex formation, and subsequent inclusion of exon 16 , when they undergo prominent defects in the hematopoietic system . Further.5–E13.5,3.1.3in vitro and in vivo , is a key enzyme that initiates mitochondrial degradation during the final stage of reticulocyte maturation by catalyzing the deoxygenation of phospholipids in mitochondrial membranes, thus participating in their breakdown in mature reticulocytes , 66. ALO in vivo . PCBP2 cin vitro . The DICin vitro , 67–69.Previous studies have investigated how the mechanism of connection between the DICE-hnRNP K complex to the translation initiation machinery and interference with 60S subunit joining. hnRNP K interacts with ribosomal protein 19 (RPS19), which is localized at the head of the 40S subunit and extends into its functional center, thus inhibiting the 80S ribosome assembly . This prIn mature reticulocytes, the ALOX15 translational silenced state must be terminated by PTMs of hnRNP K. This modification includes the methylation, phosphorylation, and proteolytic cleavage of hnRNP K. During maturation, when the level of PRMT1 declines, methylated hnRNP K is replaced by a nonmethylated protein, abolishing the interaction between hnRNP K and RPS19, enabling 80S ribosome formation . The nonhnRNP K also regulates erythroid cell enucleation during terminal red blood cell maturation by inhibiting the expression of non-muscle myosin heavy chain (NMHC) IIA , an actin-binding protein involved in intracellular vesicle transport. Activated NMHC IIA functions as part of the contractile actinomyosin ring, which is a crucial structure in enucleation. NMHC IIA mRNA levels are constant during erythroid maturation, whereas protein levels increase, indicating translational regulation of NMHC IIA expression. UV-crosslinking and filter-binding assays demonstrate that KH3 of hnRNP K interacts with a specific U/CCCC-rich NMHC IIA mRNA 3′-UTR sequence element, mediating translational silencing in non-induced K562 cells. Simultaneously, translation inhibition was abolished in K562-induced maturation and hnRNP K-depleted K562 cells. K562 cell-induced model recapitulating enucleation and mitochondria degradation results in hnRNP K depletion , a transmembrane transporter of ferrous iron, which is also crucial for heme synthesis . The KH2 of FPN1 .via protein-protein interaction. Prolyl hydroxylases (PHDs) and asparaginyl hydroxylase (FIH1) can modify the hypoxia-inducible factor α (HIFα) protein for degradation via the VHL/proteasome pathway. Depleting PCBP1 or PCBP2 in cells leads to a loss of PHD2 activity and accumulation of active HIF1α for EPO transcription  is a proto-oncogene involved in multiple cancers that impacts many biological events, including cell proliferation, differentiation, and programmed cell death, when aberrantly regulated. MYC plays an important role in the maturation and expansion of myeloid and lymphoid cells. Uncontrolled MYC elevated expression is often observed in leukemia and lymphoma  mimicking the putative haploinsufficiency observed in AML patients were significantly smaller and had a decreased survival rate. The reduced hnRNP K expression led to the hematological malignancy development with a significant increase in neutrophils, basophilic granulocytes, platelets, and CD11b/LyG6 double-positive cells. The serum levels of interleukin (IL)-3 and IL-6, which promote the proliferation of myeloid cells, were also increased. Additionally, +/-Hnrnpk mice frequently harbor other hematologic malignancies, including T- and B-cell lymphomas  is one of the most common and aggressive malignancies of the hematopoietic system. hnRNP K haploinsufficiency has been discovered in AML patients, contributing to the disease process . Approxiymphomas . hnRNP Kymphomas . A reducymphomas . hnRNP Kymphomas , 110, isymphomas . A chromp21 gene , 112. hnp21 gene . C/EBPα p21 gene , was sigHowever, hnRNP K might be an oncogene due to its significant overexpression in the bone marrow of non-remission AML patients and drug-resistant cell lines , 90. hnRvia its KH1 domain to stabilize p27 mRNA and enhance its translation  is a myeloproliferative disorder caused by deregulated tyrosine kinase activity of p210-BCR/ABL oncoprotein, the product of t  translocation . CML provia distinct pathways through the activation of MYC mRNA translation and suppression of C/EBP α mRNA translation  (173/189/272 and Thr213) (via a pyrimidine motif (CUCCCCC) in its 5’-UTR that spaces an upstream open reading frame (uORF) (18-nt) from the main ORF  from CML-BC patients than in CML-CP cells . hnRNP Knslation . The con284/353)  and PCBP Thr213) , thereby Thr213) , account Thr213) , 120. PCmain ORF . Becausemain ORF , C/EBPα main ORF . miRNAs main ORF .4.3in vivo tumor burden. hnRNP K enhances autophagic activity in drug-resistant Ph+ ALL cells by iteration with the mRNA of Beclin1, an autophagy-related protein (hnRNP K overexpression was assessed in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) patients and imatinib-resistant ALL cell lines. hnRNP K knockdown increased the imatinib sensitivity of these tumor cells and decreased the  protein .4.4Malignant lymphomas constitute a heterogeneous group of neoplasms derived from cells of the immune system  . More spIn DLBCL patients without MYC genomic alterations, high hnRNP K expression is associated with poor overall survival, progression-free survival, and lack of response to chemotherapy. A transgenic mouse model with hnRNP K overexpression also exhibited lymphomagenesis and a reduced survival rate. Mechanistically, hnRNP K activates MYC expression through posttranscriptional and translational regulation, contributing to DLBCL pathogenesis , 93.Burkitt’s lymphoma is the most common B-cell lymphoma in children. Its genetic hallmark is the translocation t  involving the MYC gene and the immunoglobulin heavy chain (IGH) gene or its light chain variants, bringing the MYC oncogene under the influence of immunoglobulin gene enhancers. This translocation deregulates MYC expression, contributing to Burkitt’s lymphoma cell proliferation . hnRNP KA next-generation sequencing study identified recurrent somatic mutations in PCBP1 in Burkitt’s lymphomas . The mutations predominantly (83%) affect the PCBP1 KH3 domain, either by complete domain loss or amino acid changes. Missense mutations in two cases within NLSI or NLSII may solely lead to decreased PCBP1 expression within the nucleus. These mutations were predicted to alter various functions of PCBP1, including nuclear trafficking and pre-mRNA splicing . Notably5via modulation of mRNA stabilization, alternative splicing, translational repression, and activation, together with other additional factors, such as PABP-C, U2AF65, and RPS19. PCBPs are particularly relevant for erythroid differentiation and maturation. In addition to their involvement in nucleic acid metabolism, PCBPs act as iron chaperones, regulating the cytosolic iron levels for hemoglobin synthesis or activating crucial enzymes. Notably, the specific binding sites of PCBP1 and PCBP2 for ferrous iron are still unclear, and the exact mechanism by which PCBP1 delivers ferrous iron to its target proteins remains inconclusive.PCBPs, the triple KH domain proteins interaction with C-rich regions of RNA, ssDNA, and dsDNA and exert abundant biological functions of cellular events, including proliferation, differentiation, apoptosis, DNA damage, repair and stress, and immune responses . Herein,Post-transcriptional regulation is crucial in orchestrating gene regulatory networks in hematopoietic cell growth, differentiation, and tumorigenesis. The roles of other RNA-binding proteins in acute leukemia have recently been fully elucidated . Becausein vivo study demonstrated that hnRNP K is a crucial regulator of shaping leukemia cell resistance to imatinib (in vitro and in transplantation models. This suggests that BET-bromodomain inhibitors could be an effective therapeutic modality in the treatment of malignancies characterized by hnRNP K overexpression (Elucidating the roles of PCBPs in hematopoietic malignancies may provide a valuable therapeutic modality in the future. Certain studies have demonstrated that hnRNPK, PCBP1, and PCBP2 may serve as prognostic biomarkers and potential therapeutic targets. Although no established PCBPs-specific therapies currently exist, elucidating the role of PCBPs in hematopoietic malignancies may provide valuable insights. For instance, imatinib , partly pression .Besides, the roles of the other two family members, PCBP3 and PCBP4, in the hematopoietic compartment have not yet been revealed. Research on PCBP3 and PCBP4 in other contexts could offer valuable insights. For example, in neuronal cells, PCBP1, PCBP2, and hnRNP K work simultaneously to stimulate the µ-opioid receptor gene, while PCBP3 works as a repressor , 125. PCConclusively, the functional diversities of PCBP family members involved in the hematopoietic compartment and their pleiotropic molecule mechanisms will be revealed more clearly with further research.HZ, LS, RW, and XH contributed to the study design. HZ prepared and wrote the manuscript. ZW, GS and YC critically reviewed and revised the manuscript. All authors contributed to the article and approved the submitted version.


Primary soft-tissue lymphoma (PSTL) is a rare extranodal non-Hodgkin lymphoma, characterized by a mass growing within soft-tissue, which is connective tissue, adipose tissue, and skeletal muscle. Here, we describe a case of biphenotypic lymphoblastic lymphoma arising from soft tissue of the popliteal fossa in an 11-year-old boy. A pediatric review about PSTL revealed that anaplastic large cell lymphoma is the most common histological type and a biphenotypic lymphoblastic lymphoma has not yet been reported in childhood. Lymphoma should always be considered in patients presenting with a soft-tissue mass, and a comprehensive immunohistochemical evaluation, including B-cell, T-cell, and myeloid markers, is needed to make a correct diagnosis and establish the most suitable treatment. Primary extranodal non-Hodgkin lymphoma (PE-NHL) typically arises from tissue other than lymph nodes and even from sites that normally do not contain lymphoid tissue; the incidence rate ranges from 24% to 48% of all NHL ,2. A varWe describe a case of a pediatric PSTL localized in the popliteal fossa, with an outstanding histological characterization; a review about this topic was conducted thereafter.Detailed immunophenotyping was performed on paraffin sections from tissue sample; the staining panel included CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD11c, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD34, CD38, CD43, CD45, CD68, CD79a, CD99, CD117, CD163, myeloperoxidase (MPO), terminal deoxynucleotidyl transferase (TdT), cytoplasmic µ chains, vimentin, PAX5, FLI-1, lysozyme, Myf4, MND116, and EMA.As regards the molecular cytogenetic screening for chromosomal abnormalities associated with myeloid and lymphoid leukemias, total RNA was isolated using Trizol (Invitrogen); one microgram of RNA was reverse-transcribed into cDNA using the SuperScript II system (Invitrogen) according to the manufacturer’s instructions. Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed for the evaluation of all rearrangements.A literature review about pediatric PSTL was conducted using PubMed, Scopus, and Web of Science, combining the terms “soft-tissue” AND “non-Hodgkin lymphoma”, “connective tissue” AND “non-Hodgkin lymphoma”, and “skeletal muscle” AND “non-Hodgkin lymphoma”; 12 cases were retrieved.An 11-year-old boy suffered from a 2-months history of a growing mass in his right popliteal fossa; he did not complain of any other symptoms. Due to progressive increase in size of the popliteal mass, a magnetic resonance imaging (MRI) of the right knee was performed; it revealed a 5 × 4 × 10 cm lesion, localized in the distal region of the posterior thigh and in the popliteal fossa, isointense to muscle on T1-weighted images and with intermediate signal intensity on T2-weighted images, showing intense and not homogeneous contrast enhancement; concomitant edema in subcutaneous adipose tissue and in gastrocnemius muscle fascia was detected A,B. The Soft-tissues are uncommon primary sites for NHL, and most of the available information concerns the adult population. In adulthood, PSTL more often affects the extremities, especially the lower limbs, without any sex difference, diffuse large B-cell lymphoma (DLBCL) being the most frequent histological subtype .After literature search, no pediatric review about this topic was found; the discovered case reports are listed in In a patient presenting with a soft-tissue mass, the most common diagnostic hypothesis is soft-tissue sarcoma, especially desmoid tumors, rhabdomyosarcoma, synovial sarcoma, and Ewing’s sarcoma/pPNET; another differential diagnosis includes neuroblastoma. All these neoplasms belong to the group of small round blue cells tumors, characterized by some degree of cytomorphologic and immunophenotypical overlap , but witLBL is the second most common type of NHL in children and adolescents; in around 70–80% of cases, it is of T-lymphoblastic origin and in 20–30%, it arises from B lymphoblasts . For thePediatric PSTL is an extremely rare disease, characterized by a favorable prognosis in the majority of patients. In the presence of a soft-tissue mass, histological examination, including an extensive immunohistochemistry panel for lymphoma, and molecular genetic analysis are essential to make a correct diagnosis. In our case, unique because of both onset site  and histological characterization (biphenotypic LBL), the choice of a chemotherapy regimen active against both lymphoid and myeloid neoplastic cells showed to be highly effective.