Ovis aries) to provide insights into further research on the molecular mechanism and identify new biomarkers for genetic improvement.Understanding the molecular pattern of fertility is considered as an important step in breeding of different species, and despite the high importance of the fertility, little success has been achieved in dissecting the interactome basis of sheep fertility. However, the complex mechanisms associated with prolificacy in sheep have not been fully understood. Therefore, this study aimed to use competitive endogenous RNA (ceRNA) networks to evaluate this trait to better understand the molecular mechanisms responsible for fertility. A competitive endogenous RNA (ceRNA) network of the corpus luteum was constructed between Romanov and Baluchi sheep breeds with either good or poor genetic merit for prolificacy using whole-transcriptome analysis. First, the main list of lncRNAs, miRNAs, and mRNA related to the corpus luteum that alter with the breed were extracted, then miRNA–mRNA and lncRNA–mRNA interactions were predicted, and the ceRNA network was constructed by integrating these interactions with the other gene regulatory networks and the protein–protein interaction (PPI). A total of 264 mRNAs, 14 lncRNAs, and 34 miRNAs were identified by combining the GO and KEGG enrichment analyses. In total, 44, 7, 7, and 6 mRNAs, lncRNAs, miRNAs, and crucial modules, respectively, were disclosed through clustering for the corpus luteum ceRNA network. All these RNAs involved in biological processes, namely proteolysis, actin cytoskeleton organization, immune system process, cell adhesion, cell differentiation, and lipid metabolic process, have an overexpression pattern (Padj < 0.01). This study increases our understanding of the contribution of different breed transcriptomes to phenotypic fertility differences and constructed a ceRNA network in sheep ( B4GALNT2, BMPR1B, BMP15, and GDF9, control litter size and ovulation rates in the ewe [IL1B, IL1A, UCP2, STAR, and PTGS2) have been discovered to be associated with fecundity in the ewe [GDF9 has been recognized to be strongly associated with prolificacy in Finnsheep and other breeds [Enhancing fertility will improve the efficiency of animal production and reproduction. Expansion of reproductive performance will boost the lifetime of a sheep, reduce veterinary treatments and costs, lower insemination efforts, and shorten the lambing interval . Fertili the ewe ,8,9. In  the ewe . Earlier the ewe ,12,13, o the ewe . The Romr breeds . The sucr breeds . Therefor breeds . For exar breeds . HoweverOmics directs the usage and the high-throughput technologies’ output analysis to identify and interpret the genomics, transcriptomics, proteomics, metabolomics, and epigenetics levels . FurtherIn this study, we conducted multi-partite networks on reproductive traits, specifically litter size in the sheep breeds of high (Romanov) and low  fertility, with a litter size ranging from one to four, from different geographic regions of the farms, respectively. Romanov sheep show outstanding reproduction qualities: early sexual maturity, out-of-season breeding, and extraordinary fertility. However, the Baluchi sheep is a relatively low-prolificacy breed originally from Khorasan Razavi province, Iran, and excels in fleece weights and muscle growth . TherefoIn this way, a ceRNA network consisting of lncRNA–miRNA–mRNA was constructed based on lncRNA–mRNA and miRNA–mRNA interactions. In addition, the other networks including protein–protein interaction networks (PPI), metabolic pathways, and gene regulatory networks (GRNs) of the hub genes involved in the ceRNA network were conducted to explore the effects of functional modules and hub differentially expressed genes, miRNAs, and lncRNAs on prolificacy. Overall, our results suggest a group of genes or mRNAs, miRNAs, and lncRNAs using a computational approach that can play key and potential roles in transcriptional and post-transcriptional gene regulation under different conditions.The complete workflow for the sample collection, preparation, sequencing, and analysis of relevant species related to the corpus luteum in two sheep breeds is presented in n = 24) and one of low prolificacy  were collected from University of Tehran scientific park in Iran. The sheep included were as unrelated as possible based on records for the phenotype of litter size and the other reproductive traits. Data for the phenotype of litter size records are presented in A total of 48 ewes from two sheep breeds of high prolificacy  described by Herlihy et al.  and CL s2 . Some corpora lutea contained a central luteal cavity with an area of 335 mm2. A total of 59 biopsy attempts were made; 54 (92%) resulted in obtaining a luteal tissue specimen at the first attempt. The average size of the obtained biopsy core was 1 mm in diameter and 10.8 mm in length , and the average weight was 4.4 mg . CL samples were instantly frozen in liquid nitrogen and stored at −80 °C.On day 10 of the estrous cycle, CL biopsies were collected from each sheep as described previously ,44. BrieWhole RNA was extracted from CL (HF and LF) samples using a Trizol-based method . Whole RThe RNA samples were converted to cDNA libraries for sequencing based on the protocol of the Illumina TruSeq RNA Sample Preparation Kit . RNA-Seq libraries were amplified by 11 cycles of PCR. Library concentration and quality were determined by Qubit  and Bioanalyser 2100 . Each sample was sequenced on a single lane over a total of two flow cells on the Illumina HiSeq 2500 platform to generate 60 million 50-base paired-end reads, and FASTQ files were created using CASAVA (v1.9) (Illumina).A quality check of the raw sequence data was accomplished using FastQC software (v0.11.9) . For thihttp://ftp.ensembl.org/pub/release-103/fasta/ovis_aries/dna/ (accessed on 29 December 2020)) using HISAT2 software (v2.2.1) [Alignment sequences, mapping, and identification of known and novel RNAs of reads were conducted on the Ovis aries reference genome ((v2.2.1) . For transcript quantification, featureCounts software (v2.0.1) was utilized to calculate the total raw counts of mapped reads . Next, tBMP2, HNF4A, PLCB2, and RPS6KL1 were selected for analysis by qRT-PCR. The same RNAs extracted from the CL tissues at each breed were used for qRT-PCR validation. The primer pairs were designed using Geneious Prime software v2021.1 . The specificity of each PCR product was confirmed by melting curve analysis. All qRT-PCR assays were performed in triplicate reactions. The housekeeping genes RPL19 and GAPDH  were used as the internal control genes. The expression levels of target mRNAs were obtained based on RNAs extracted from the four ewes and were shown to be normalized to GAPDH.To validate the reproducibility of RNA-seq data, four DEGs including  v2021.1 . Quantithttps://biit.cs.ut.ee/gprofiler/gost (accessed on 12 March 2022)), GeneCards (www.genecards.org/ (accessed on 12 March 2022)), the STRING database [https://string-db.org (accessed on 12 March 2022)), and the DAVID [https://david.ncifcrf.gov/ (accessed on 12 March 2022)), which provide a set of functional annotation tools for the genes categorized using gene ontology (GO) terms.Gene set annotation enrichment analysis was also carried out using the online programs g: Profiler  (https:/database  (https:/he DAVID  (Databashttp://129.206.7.150/ (accessed on 23 December 2021)), a comprehensive atlas of microRNA–target interaction tools, which integrates 12 miRNA–target prediction tools. Further, lncRNA–mRNA interactions were predicted by the NONCODE database [http://www.noncode.org/ (accessed on 3 January 2021)) and the LNCipedia database [https://lncipedia.org (accessed on 3 January 2021)).The predicted and validated mRNA genes were identified using miRWalk 3.0 (database  (http://database  (https:/http://pathguide.org (accessed on 15 March 2022)) is a meta-database that provides more than 300 web-accessible network databases and biological pathways [https://genemania.org/ (accessed on 15 March 2022)) and the STRING database [https://string-db.org (accessed on 15 March 2022)). To describe the interaction between proteins using a probabilistic confidence score, the string uses eight major sources of interaction or association data, including co-occurrence, neighborhood, fusion, co-expression, experimental, text mining, and database [lncRNA–miRNA–mRNA ceRNA network was constructed based on miRNA–mRNA and lncRNA–mRNA interactions and online interaction databases. The Pathway Resource List  and the interactions between them are represented as nodes and edges, respectively. Gene Ontology terms with FDR < 0.05 were considered significantly enriched for the identified genes. Furthermore, constructed networks were compiled in simple interaction format (SIF) bowed to Cytoscape for topological analysis. Then, the statistical and topological significance of the network was assessed using the Network Analyzer plugin in Cytoscape software (v3.8.2.)   and, for clustering, ClusterONE  and MCODA summary of the RNA-seq data analysis pipeline and the steps of constructing of lncRNA–miRNA–mRNA ceRNA network is shown in p < 0.01)  . To assess the accuracy and reliability of differentially expressed genes identified by RNA-seq, four DEGs from two breeds were selected to perform qRT-PCR tests. The expression results for four genes assessed using RNA-seq and qRT-PCR are shown in p < 0.05)  A. Among To discover the mechanism of how lncRNAs regulate mRNA through sponging miRNA, a ceRNA network was constructed with a combination of predicted miRNA–mRNA and lncRNA–mRNA interactions. Based on the knowledge of interactions in databases, we could detect interactions for 312 nodes and 748 edges in the network. Moreover, the related files of networks are given in Network characteristics were calculated using the Network Analyzer plugin of Cytoscape for the ceRNA Network. Topological analysis recognizes the qualitative virtues of the complex biological systems. Network topology is used to examine the state of communication and information transfer of a node with other nodes of interactive networks. Topological parameters such as the average clustering coefficient of degrees, topological coefficient, average degree, betweenness centrality, and power–law distribution were evaluated. The distribution of the clustering coefficient is an important parcel of biological scale-free networks. Then, network density, characteristic path length, network centralization, and the clustering coefficient of the ceRNA network were compared with randomized model networks (Barabasi–Albert and Erdos–Renyi models), as presented in Finally, we utilized clustering on the lncRNA–miRNA–mRNA ceRNA network made by an integrated approach. Clustering algorithms are utilized to determine significant sub-networks or modules. The results were clustered by ClusterONE  and MCODThe whole-transcriptome profiling technique helps gain a more in-depth understanding of the functions of the corpus luteum, which may authorize the marker identification that is differentially expressed, for instance, between two breeds showing different litter size phenotypes. Although most of the investigations associated with pregnancy have been performed in sheep, only a few studies have used the whole-transcriptome approaches to the corpus luteum. Microarray-based transcriptomic research performed by Gray et al. (2006) identified several endometrial genes regulated by progesterone (from the corpus luteum). In a more comprehensive investigation performed by Brooks et al. , transcrIn 2011, Salmena et al. suggested the ceRNA (competitive endogenous RNA) hypothesis that lncRNAs and protein-coding RNAs can operate as competitive endogenous RNAs to communicate by binding to miRNA sites ,72. AccoHerein, we discovered several mRNAs/genes in the corpus luteum of high- and low-fertility sheep. Indeed, the role of these genes during fertility is not clearly understand yet, but some mRNAs/genes family members are believed to be involved in cell survival, proliferation, apoptosis, and other fundamental biological processes. The expression of these mRNAs/genes in both high-fertility and low-fertility in the corpus luteum could show their basal function cellular roles during the reproductive performance . Our resIn module 1, let-7b, let-7a, let-7c, and miR-199a suppressed NOXA1, SYNGAP1, ROCK2, and PLCB2 genes, and almost all miRNAs were up-regulated, except let-7c, which was down-regulated and genes were down-regulated. Moreover, in this module, ENST00000416770, ENST00000567819, ENST00000559520, and ENST00000618857 interacted with RPS6KA6, CAPN3, PLCB2, and BACE1, respectively. The hub-hub genes included CAPN3, PLS1, CNN1, ACTA1, and KLHL17, in which PLS1, CNN1, and ACTA1 genes were up-regulated and CAPN3 and KLHL17 genes were down-regulated. All of these hub-hub genes are involved in the actin cytoskeleton organization process. Calpain 3 (CAPN3), a muscle-specific member of the calpain large subunit family that specifically binds to titin, has been suggested to be related to muscle growth in cattle and broiler chickens. Gene Ontology (GO) annotations related to this gene include calcium ion binding, peptidase activity, meat traits, and disease resistance . PLCB2 gC3, SERPINE1, CFB, C5, and CFI genes were identified as the most important genes in that all of them were upregulated. The key biological process affected by these genes was the proteolysis process. The hydrolysis of proteins into amino acids or/and smaller polypeptides occurred by cleavage of their peptide bonds [In module 2, de bonds . Serpin de bonds . Complemde bonds . Moreovede bonds . Complemde bonds ,96. Compde bonds .C4BPB, SERPINE1, CFB, C4BPA, and MASP2. C3, C4BPB, SERPINE1, CFB, and C4BPA genes were upregulated and MASP2 gene was downregulated. C4BP gene binds strongly to apoptotic and necrotic cells and limits their complement activation. Complement component 4 binding protein β (C4BPB) and complement component 4 binding protein α (C4BPA) genes encode a member of a superfamily of proteins composed predominantly of tandemly arrayed short consensus repeats of approximately 60 amino acids. This gene has a major role in the innate immune system and immune response lectin induced complement pathways [MASP2) gene is associated with biological processes terms including acute inflammation and defense response. Activities related to this gene include calcium ion binding and peptidase activity [In module 3, let-7c suppressed the C3 gene and was down-regulated, and the mentioned gene was up-regulated. The hub-hub key genes involved in the immune system process were pathways . MBL assactivity . Therefoactivity .CDK3, KLHL17, MAPK15, and OLR1 genes and was downregulated. let-7b suppressed CDK3, GNB3, HOMER1, KLHL17, OLR1, and PRKACA genes and was upregulated. let-7a suppressed ERBB3 and NME2 genes and was up-regulated. miR-16b suppressed the DAPP1 gene and was up-regulated. let-7c suppressed the MYB gene and was down-regulated, and the MYB gene was up-regulated. let-7f suppressed CAPN3, CDH1, and SPEG genes and was down-regulated. miR-199a suppressed ALS2CL, MAPK15, and SERPINE1 genes and was up-regulated. Moreover, in this module, ENST00000559520, ENST00000586450, ENST00000501818, ENST00000455642, and ENST00000567819 interacted with PLCB2, MAP3K14, FABP7, HNF4A, and CAPN3, respectively. The hub-hub genes included ITGA2B, CDH1, SERPINE1, BMP2, TNC, NRCAM, CLDN3, CLDN4, MYB, and ERBB3. Among these, CDH1, SERPINE1, BMP2, TNC, NRCAM, CLDN3, CLDN4, MYB, and ERBB3 genes were upregulated and the ITGA2B gene was downregulated. Integrin subunit α 2b (ITGA2B) is a protein-coding gene that has a role in cytokine signaling in the immune system and RET signaling pathways and activities related to this gene including identical protein binding and fibrinogen binding. Moreover, for adhesion with this gene, integrins are known to participate in cell-surface mediated signaling [In module 4, miR-21, let-7b, let-7a, miR-16b, let-7c, let-7f, and miR-199a were identified. miR-21 suppressed ignaling . Estradiignaling ,103. Tenignaling ,105. Neuignaling . Claudinignaling . All of BMP2, MAP3K14, MAP3K7, and RPS6KA6 genes and was down-regulated. let-7b suppressed MAP3K14 and TRAF6 genes and was up-regulated. miR-16b suppressed the MAP3K7 gene and was up-regulated. let-7c suppressed the HSF4 gene and was down-regulated. let-7f suppressed the ATF3 gene and was down-regulated. miR-199a suppressed BMP2 and RPS6KA6 genes and was up-regulated. Moreover, in this module, ENST00000455642, ENST00000416770, and ENST00000586450 interacted with HNF4A, RPS6KA6, and MAP3K7, respectively. ATF3, HSF4, COL27A1, MYB, MAPT, SPIB, SFRP2, GREM1, and BMP2 genes were identified as hub-hub genes. Among these, ATF3, MYB, SPIB, SFRP2, and GREM1 genes were up-regulated and HSF4, COL27A1, and MAPT genes were down-regulated. The key biological process affected by these genes was cell differentiation. The process includes unspecialized cells, for example, functional features that characterize the cells, embryonic/regenerative cells, and tissues/organs of the mature organism. Differentiation includes the processes involved in the obligation of a cell to a specific fate and its further development to the mature state [MAP3K14 and RPS6KA6 interacted with ENST00000586450 and ENST00000416770, respectively. All of them were down-regulated and were suppressed by miR-16b and miR-199a, which were up-regulated. MAP3K14 encodes mitogen-activated protein kinase kinase kinase 14, which is a serine/threonine protein-kinase. This kinase binds to TRAF2 and stimulates NF-kappaB activity. It shares sequence similarity with several other MAPKK kinases. It participates in an NF-kappaB-inducing signalling cascade common to receptors of the tumour-necrosis/nerve-growth factor (TNF/NGF) family and to the interleukin-1 type-I receptor. Regardless, RPS6KA6 encodes protein that constitutively activate serine/threonine-protein kinase, which exhibits growth-factor-independent kinase activity and may participate in p53/TP53-dependent cell growth arrest signaling and play an inhibitory role during embryogenesis.In module 5, miR-21, let-7b, miR-16b, let-7c, let-7f, and miR-199a were identified. miR-21 suppressed re state . Activatre state . Collagere state . Microture state . Gremlinre state . In thisFABP7 and HNF4A genes and was downregulated. Moreover, in this module, ENST00000455642 and ENST00000501818 interacted with HNF4A and FABP7, respectively. Hub-hub genes were LPIN1, HSD17B1, PLA2G4B, CYP2S1, HNF4A, TTR, and CRABP2. Among these, HSD17B1, CYP2S1, HNF4A, and CRABP2 genes were upregulated and LPIN1 and PLA2G4B genes were downregulated. Lipin 1 (LPIN1) is a member of the lipin family of proteins that has an important role in animal and poultry lipid metabolism and its regulation. This gene may have value as a genetic marker for improving some traits such as meat production and carcass [HNF4A gene interacted with ENST00000455642 and was highly upregulated, and suppressed by let-7f. This gene is a transcriptional regulator that controls the expression of some genes during the transition of endodermal cells to progenitor cells, facilitating the recruitment of RNA pol II to the promoters of target genes and activating the transcription of CYP2C38. It represses the CLOCK-ARNTL/BMAL1 transcriptional activity and is essential for circadian rhythm maintenance and period regulation in the some tissues cells. Hydroxysteroid 17-β dehydrogenase 1 (HSD17B1) is a gene that has a key role in the super pathway of steroid hormone biosynthesis and metabolism pathway. Moreover, the HSD17B1 gene plays an essential role in the biological processes related to sheep ovulation [PLA2G4B) gene encodes a member of the cytosolic phospholipase A2 protein family and has a role in calcium ion binding and phospholipase activity. This gene has been identified as one of the important genes in interaction networks in sheep fertility [CYP2S1) encodes a member of the cytochrome P450 superfamily of enzymes. CYP2S1 gene is related to the reproductive processes as a regulatory factor in follicular development and ovarian development in the ovulation process in female mammals [In module 6, let-7f suppressed  carcass . The HNFvulation . Phosphoertility . Cytochr mammals . Therefo mammals . We offered a computational approach to an lncRNA–miRNA–mRNA ceRNA network using predicted and validated expression profiles of RNAs. Indeed, the use of the same breed might lead to more accurate results. However, these two sheep breeds are different, but in systemic studies, thresholds are usually considered that have the most genetic and phenotypic differences. The spatio-temporal differential expression in different tissues, especially CL, supports the potential role of RNAs in the transcriptional and post-transcriptional regulation of genes involved in prolificacy. The reports we presented here for the first time may be beneficial in discovering the basic molecular regulatory mechanisms of fertility in sheep. Although, further efforts are needed to discover the specific biological functional role of RNA modules during various stages of the reproductive traits. In addition, our findings showed that the integration of mRNAs, miRNAs, and lncRNAs based on the ceRNA networks, along with different data such as lncRNA interactions, miRNA target prediction, PPI, and other regulatory networks, can be considered a robust approach to provide greater insights into biological processes at the molecular level.This study applied a new approach to integrating different classes of RNA as an integrated network, and first reported transcriptome sequencing analysis of the corpus luteum in sheep. Integration of transcriptomic data for obtaining and identifying hub nodes with differences in expression levels led to the successful identification of 264 mRNAs/genes, 14 lncRNAs, and 34 miRNAs in the main process of prolificacy in the corpus luteum samples of Romanov compared with Baluchi breeds. Using lncRNA–miRNA–mRNA ceRNA network analysis and constructing the networks by merging the interactions and regulation networks, 6 key modules, 44 mRNAs/genes, 7 lncRNAs, and 7 miRNAs were identified as being involved in major biological processes including proteolysis, actin cytoskeleton organization, immune system process, biological adhesion, cell differentiation, and lipid metabolic process, and have an overexpression pattern. Overall, our results in this research demonstrate that integrated network analysis and the application of omics data of sheep corpus luteum generate novel insights into sheep fertility. Furthermore, a comparison of the transcriptomic level in these two breeds will be important for further genetic improvement of the trait and a better understanding of the molecular basis of reproduction in sheep as well as other mammals.


Mutations in the tumor suppressor p53 (p53) occur in ~50% of human cancers, the majority of which are missense mutations. Mutations in p53 not only impair the tumor suppressive function, but also confer missense mutant p53 (mutp53) with oncogenic activities independent of wild-type p53 (wtp53). Since p53 mutations are cancer-specific, several approaches targeting them have been taken to develop novel cancer therapies, including restoration or stabilization of wtp53 conformation from mutp53, rescue of p53 nonsense mutations, depletion of mutp53 proteins, and induction of p53 synthetic lethality or targeting of vulnerabilities imposed by p53 deficiencies (activated retrotransposons) or mutations (enhanced YAP/TAZ). Here, we summarize clinically available investigational and FDA-approved drugs that target p53 mutations for their mechanisms of action and activities to suppress cancer progression.Mutations in the tumor suppressor p53 (p53) promote cancer progression. This is mainly due to loss of function (LOS) as a tumor suppressor, dominant-negative (DN) activities of missense mutant p53 (mutp53) over wild-type p53 (wtp53), and wtp53-independent oncogenic activities of missense mutp53 by interacting with other tumor suppressors or oncogenes (gain of function: GOF). Since p53 mutations occur in ~50% of human cancers and rarely occur in normal tissues, p53 mutations are cancer-specific and ideal therapeutic targets. Approaches to target p53 mutations include (1) restoration or stabilization of wtp53 conformation from missense mutp53, (2) rescue of p53 nonsense mutations, (3) depletion or degradation of mutp53 proteins, and (4) induction of p53 synthetic lethality or targeting of vulnerabilities imposed by p53 mutations (enhanced YAP/TAZ activities) or deletions (hyperactivated retrotransposons). This review article focuses on clinically available FDA-approved drugs and drugs in clinical trials that target p53 mutations and summarizes their mechanisms of action and activities to suppress cancer progression. The tumor suppressor p53 was initially thought to be an oncogene since the originally cloned cDNA contained a missense mutation . Indeed,p53 is a transcription factor and regulates the transcription of numerous downstream target genes involved in apoptosis, cell cycle arrest, senescence, DNA repair, and cellular metabolism, thereby functioning as a tumor suppressor . The proAccumulating evidence indicates that p53 is the most frequently mutated gene in human cancers, with mutations in over 50% of human cancers . The majMET), which is in several phase 2 or 3 clinical trials. Although the tumor suppressive effects of APR-246 in mutp53-carrying tumors in mouse models have been shown to be successful, it is not yet approved by the Food and Drug Administration (FDA) [Several strategies to target p53 mutations have been taken . The firon (FDA) . The secon (FDA) ,14. The on (FDA) . This ston (FDA) ,17,18. Oon (FDA) ,20, inhion (FDA) , and inhon (FDA) ,22. HereMET), phenethyl isothiocyanate (PEITC), and arsenic trioxide (ATO/Trisenox).Most mutp53 lose the activity of wtp53 as a transcription factor and a tumor suppressor. However, accumulated evidence indicates that the tumor suppressive activity can be restored under specific conditions including temperature shift, exposure to synthetic peptides from 53BP2-derived “CDB3” and p53 C-terminal domain “Peptide 46”, and insertion of second-site mutations or an N-terminal deletion ,37,38. MMET  rescue the p53’s transcriptional activity from both DNA contact and structural p53 mutants [PRIMA-1 was identified as a small molecule compound that suppressed the growth of Saos-2 osteosarcoma cell line expressing p53R273H . PRIMA-1 mutants ,42. In m mutants ,42,43. A mutants  demonstr mutants . Specifi mutants .A phase 1b clinical trial (NCT04383938) was performed to examine the safety and efficacy of APR-246 combined with pembrolizumab, an immune checkpoint inhibitor, for patients with advanced/metastatic solid tumors . Due to Two clinical trials for p53-mutated myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML) with 20–30% marrow blasts (oligoblastic AML)   and for PEITC is extracted from cruciferous vegetables  and has been suggested to have anti-cancer effects . EpidemiA phase 2 clinical trial (NCT01790204) has been performed to examine whether PEITC-containing juice from watercress could reduce the number of oral cells having p53 mutations (not specified for p53R175H or R175L); however, the outcome of this study has not yet been reported.Arsenic trioxide  is an FDA-approved drug to treat acute promyelocytic leukemia (APL) that is characterized by the expression of PML-RARα fusion protein ,56. ATO Recently, Chen et al.  demonstrAnother interesting approach targeting p53 mutations is to rescue p53 nonsense mutations and restore wtp53 activity using aminoglycosides such as clinically available gentamicin. Approximately 10% of p53 mutations are nonsense mutations that lead to premature termination of protein translation ,59. The Stop codon readthrough in mammalian cells during protein translation by aminoglycoside antibiotics was first reported by Burke et al.  in 1985,Increasing evidence suggests that mutp53 is inherently unstable. However, genotoxic stress, including oncogenic stress and irradiation, stabilizes mutp53 to promote its GOF activities in tumors . CriticaHSP90 is an ATPase-dependent molecular chaperone that regulates protein folding and stabilization or degradation under physiological and stress conditions, thereby controlling proteostasis . HSP90 hSeveral HSP90 inhibitors have been developed  ,76,77,78Alexandrova et al.  reportedMany clinical trials of HSP90 inhibitors have been performed; however, most studies do not stratify the p53 status . Only onStatins are a group of drugs that lower the production of cholesterol by specifically inhibiting HMG-CoA reductase activity, a rate-limiting step of the mevalonate pathway . AlthougPreviously, our group found that several statins  induce CHIP-mediated degradation of mainly structural or conformational mutp53, based on a screen of ~9000 compounds using Saos2 (p53null) cells expressing a p53R175H-luciferase fusion protein . LovastaSimilarly, Ingallina et al.  showed tDespite the increasing evidence of mutp53 depletion by statins, only a few clinical reports have examined the statins’ effects on cancer progression or survival by stratifying the p53 mutation status, while the majority of clinical studies have been performed without stratifying the p53 status. For example, statin-user patients with lung adenocarcinoma had significant clinical benefits for improving overall survival compared with non-statin user patients only when tumors had p53 mutations, supporting the statins’ tumor inhibitory effects . On the Regarding clinical trials studying statin’s significance on cancer inhibition, some phase 3 clinical trials were performed in hepatocellular carcinoma , gastricAs described in Vorinostat/Zolinza/SAHA is an FDA-approved inhibitor of class I, II, and IV histone deacetylases and epigenetically regulates the malignant properties of multiple cancer types . The firA group from the MD Anderson Cancer Center conducted two phase 1 clinical trials investigating the combinatory effects of vorinostat and MLN9708  or pazopanib  in patients with advanced p53-mutated malignancies  ,29. PatiSynthetic lethality is a fatal phenomenon caused by the simultaneous occurrence of multiple genetic events, while a single event is tolerable . SynthetWee1 protein kinase inhibits Cdk1 and 2 that promote S and G2/M cell cycle progression, and hence Wee1 inhibitors prevent the activation of the G2/M cell cycle checkpoint. Indeed, a Wee1 inhibitor, PD0166285, which inhibits phosphorylation of Cdc2 and attenuates the G2/M checkpoint, sensitizes p53-deficient E6-overexpressing PA-1 cells to γ-irradiation, while it shows little effect on p53-proficient PA-1 cells . AnotherAdavosertib/AZD1775/MK-1775 has been tested in a total of 59 clinical trials. Among these, nine phase 2 trials have been performed with stratification of p53 status in cancers, including ovarian cancer , relapsed SCLC , untreated non-small cell lung cancer , NSCLC (NCT02087176), recurrent uterine serous carcinoma (NCT03668340), and colorectal carcinoma (FOCUS4-C). Specifically, in the NCT01164995 trial, adavosertib/AZD1775 enhanced carboplatin efficacy in p53-mutated ovarian cancer with an overall response rate of 43% , a modesAdditionally, in the FOCUS4-C trial, Seligmann et al.  reportedOther drugs known to cause p53 synthetic lethality include UCN-01 (PKC inhibitor) and BI-2536 (PLK1 inhibitor). These drugs have been tested in phase 2 clinical trials, including UCN-01 for leukemia, lymphoma, SCLC, metastatic melanoma, metastatic kidney cancer, metastatic pancreatic cancer, fallopian tube cancer, ovarian cancer, and primary peritoneal cavity cancer, as well as BI-2536 for small cell lung cancer, advanced or metastatic NSCLC, refractory or relapsed acute myeloid leukemia, advanced unresectable pancreatic cancer, prostate cancer, and recurrent or metastatic solid tumors. However, none of these trials stratified the p53 status in tumors.LINE-1 (Long Interspersed Element 1) is a family of retrotransposon elements present in the human genome, consisting of ~15% of the genome . Active Lamivudine (3TC/Epivir/Zeffix/DELSTRIGO) is an FDA-approved synthetic nucleoside RT inhibitor used for the treatment of HIV and HBV, and it specifically reduces human LINE-1 retrotransposition . RecentlBased on these findings, Rajurkar et al.  have iniThe Hippo signaling pathway regulates various cellular and tissue homeostatic processes, such as cell survival, proliferation, differentiation, cell regeneration, and organ size, while its dysregulation significantly contributes to malignant progression and drug resistance . YAP , one of the most frequently used bisphosphonates that inhibit osteoclast activity and so are used to treat hypercalcemia, osteoporosis, Paget’s disease, multiple myeloma, and cancers with bone metastasis ,142. IndA phase 2 clinical trial (NCT03358017) is underway to examine the effects of the combination of atorvastatin and zoledronic acid on the standard anthracyclines/taxanes-based neoadjuvant chemotherapy in triple-negative breast cancer. This trial not only investigates changes in the YAP/TAZ activity by the treatments, but also assesses the anti-tumor effects of the mevalonate pathway blockade associated with neoadjuvant standard chemotherapy by stratifying p53 protein levels (low ≈ wtp53 vs. high ≈ mutp53). Together, the use of FDA-approved drugs that block the mevalonate pathway may inhibit cancer progression by targeting the activities of two oncogenic factors, mutp53 and YAP/TAZ.The majority of p53 mutations are missense mutations; however, it remains unclear whether some mutants behave similarly, or each mutant has a different biological character. Missense mutp53 is roughly classified into two categories based on recognition by p53 conformation-specific antibodies under non-denaturing conditions: one is the DNA contact type that retains a relatively intact p53 structure, and the other is the conformational, structural, or misfolded type with robust p53 structural changes. Both mutant types generally lose the p53’s DNA-binding activity. However, these mutant types have different thermodynamic stability of the core DNA-binding domain ,147. HenIncreasing evidence indicates that even wtp53 sometimes acts like mutp53 under specific conditions, referred to as “pseudo-mutp53”. This finding is detected in cancer-associated fibroblasts (CAFs), in the absence of specific molecular chaperones, and in a subpopulation of pre-leukemic hematopoietic stem or progenitor cells with mutations in DNMT3A  ,153,154.Two p53 reactivators, APR-246 and ATO, are in clinical trials. Although both stabilize wtp53 conformation and reactive p53 in tissue culture and mouse models, it is yet unclear if p53 reactivation by these drugs is sufficient to inhibit cancer progression as a single agent in human clinical trials. Indeed, the experimental and clinical outcomes of APR-246 are varied ,24,25,26Depleters of mutp53 include the HSP90 inhibitors and statins mentioned above. HSP90 inhibitors cause MDM2- and CHIP-mediated degradation of both DNA contact and conformational mutp53, whereas statins induce CHIP-mediated degradation of mainly conformational mutp53 ,149. BotDrugs that induce p53 synthetic lethality often inhibit proteins or pathways involved in G2 or M (mitotic) cell cycle checkpoints ,112,155.Mutations in p53 are cancer-specific and, hence, are ideal molecular targets for targeted cancer therapy that is expected to cause minimum side effects. Several drugs have been developed to target p53 mutations. Although biological effects using cell culture and mouse models have been demonstrated, the clinical safety and efficacy of the majority of drugs targeting p53 mutations need to be determined. To improve the efficacy of mutp53-targeted therapy and reduce side effects, the exact mechanisms of action and efficient strategies to induce cell death specifically in p53-deleted or -mutated cells by these drugs need to be elucidated.


Homer1a expression, regulated by both cAMP-response element binding protein (CREB) and circadian clock genes, may be the final common pathogenetic mechanism in depression.Several studies have reported separate roles of adenosine receptors and circadian clockwork in major depressive disorder. While less evidence exists for regulation of the circadian clock by adenosine signaling, a small number of studies have linked the adenosinergic system, the molecular circadian clock, and mood regulation. In this article, we review relevant advances and propose that adenosine receptor signaling, including canonical and other alternative downstream cellular pathways, regulates circadian gene expression, which in turn may underlie the pathogenesis of mood disorders. Moreover, we summarize the convergent point of these signaling pathways and put forward a pattern by which  Major depressive disorder (MDD) is one of the most prevalent forms of mental illness. It is a complex and heterogeneous disorder associated with high individual suffering, increased risk of suicide, and a severe economic burden for society . SeveralPer1 and Per2 were involved in the antidepressant action of an adenosine A1 receptor (A1R) agonist [Numerous studies have demonstrated a role of adenosine receptors in the development of depression and antidepressant therapies –5. Moreo agonist . In addi agonist –12, is d agonist .In the present article, we review relevant recent advances linking adenosine receptors, circadian clock, and mood and propose that adenosine signaling regulates circadian clockwork and Homer1a, which may be a potential final common mechanism involved in the neurobiology and treatment of depression.1R, A2AR, A2BR, and A3R. In general, it was proposed that A1Rs promote antidepressant-like effects, while A2ARs’ activation enhances depression-like behaviors in rodents [2B and A3 receptors, at present, we could not find any reports on their role in mood disorders [There are numerous studies on adenosine signaling and depression, which have been recently reviewed extensively by others –5. The cisorders , 15.1R signaling [1Rs are necessary for the antidepressant action of SD and that their activation leads to rapid antidepressant-like effects [1R in calcium/calmodulin-dependent protein kinase type II (CaMKII) forebrain neurons [1R led to pronounced acute and chronic resilience toward depressive-like behavior in various tests, while A1R knockout mice displayed an increased depressive-like behavior and were resistant to the antidepressant effects of SD [1R activation are mediated by the synaptic plasticity protein Homer1a, which is upregulated by various antidepressant treatments such as SD, imipramine, ketamine, and A1R activation [1R in the cortex and hippocampus, we found that depending on the brain region of A1R upregulation, the mice show different resistance to depression-like behavior, and that enhanced Homer1a expression in the hippocampus increases stress vulnerability [Several non-pharmacological antidepressant treatments including sleep deprivation (SD), electroconvulsive therapy (ECT), and deep brain stimulation (DBS) enhance Aignaling . Hines e effects . Our gro neurons , 11, 17.ts of SD . Furthertivation , 12. Usi1R may elicit also manic or hypomanic episodes in patients with bipolar disorder [However, activation of Adisorder . It has disorder . Therefo2AR overexpression in hippocampus, cortex, and striatum show increased depression-like behavior [2AR KO mice exhibit reduced depression-like behaviors, such as decreases in the immobility time in forced swimming test and tail suspension test [2AR antagonist istradefylline (KW6002) showed an antidepressant-like action on learned helplessness model rats [2AR and mood have also been released. Tsai et al. reported that they did not find any association of A2AR (1976C > T) genetic polymorphism with mood disorders [2AR in the pathogenesis of mood disorders; rather, other A2AR variants must also be extensively studied. Moreover, A2ARs have also been linked with depression, suicidal behavior, and impulsivity based on indirect evidence at a statistical association level [In contrast, it has been reported that rats with Abehavior . Vice veion test , 22. Thedel rats . Howeverisorders . Howeveron level . For inson level . The act of A2AR 976C > T Period (Per1 & Per2) and Cryptochrome (Cry1 & Cry2) genes. The PER and CRY proteins interact and translocate to the nucleus, where they act as negative regulators inhibiting CLOCK/BMAL1 transcription [Circadian clocks govern a wide range of biochemical, physiological, and behavioral processes. In mammals, the circadian master pacemaker is located in the suprachiasmatic nucleus (SCN) . The circription . An addicription , 30.CLOCK, BMAL1, NPAS2, Per3, and NR1D1, play a role in mood disorders, mainly based on statistical analyses [A large number of studies have demonstrated the relationship between the circadian clock and depression –39, withanalyses –53. In aanalyses .Per2 and Bmal1 expressions in SCN were either phase-delayed or phase-advanced in the chronic stress group. Taken together, these reports suggest that stress and/or MDD might differently affect the circadian clockwork in particular brain areas and that further investigation on region-specific circadian mechanisms are needed.Several brain regions relevant to psychopathology of depression, including the prefrontal cortex, hippocampus, amygdala, lateral habenula (LHb), and nucleus accumbens (NAc), possess an oscillating molecular clock –57. IncrPer1, Per2 and Cry2, by rapid antidepressant therapies SD and low-dose ketamine, using comparative transcriptomics analyses [A potential role has been recently proposed for the circadian clock in the mechanism of rapid antidepressant treatments, like SD and ketamine . Duncan analyses . Furtheranalyses , a phenoanalyses . Preclinanalyses , 62–65, analyses , 66.CLOCKΔ19 and Per2Brdm1 mice exhibit hyperdopaminergic state and mania-like phenotypes [Per1 knockout mice show depression-like behavior in forced swim test [Per1 in the LHb [1R agonism shows antidepressant-like effects, it may potentially induce manic or hypomanic episodes and vice versa [The role of circadian rhythm in mood regulation is bidirectional, affecting both depression and mania –75. For enotypes –82, whilwim test , directl the LHb . Therefoce versa , 84. As ce versa . HoweverTaken together, these reports support a causal relationship between the circadian system and mood. However, alternative hypotheses have been proposed, and whether the disruption of circadian clocks are causes or consequences of mood disorders remains undetermined. Accordingly, Lazzerini Ospri et al. provide a model suggesting that mood may be an output of circadian rhythm by probability . However1R antagonism can reverse this effect [hPer2 and mBmal1 [Light is the most potent resetting stimuli of the circadian clock. In addition to glutamate, adenosine appears to be a strong candidate for modulating SCN activity . Indeed,s effect , 89. Ades effect  and accos effect , 92. Liks effect , 94. Cons effect , 96. Mors effect , 97, 98.d mBmal1 . In humad mBmal1 .1R and A2AR downstream signaling, including classical pathways and some alternative cascades, implicated in the regulation of the cellular circadian system and mood. Moreover, transcriptional factor CREB phosphorylation and induction of the synaptic protein Homer1a appear to be a convergent point of various pathways [In the following chapter, we will review and discuss Apathways , and plapathways , 103 andpathways .1R activates the phospholipase C (PLC)β—inositol triphosphate (IP3) pathway in order to induce the release of calcium from endoplasmic reticulum and subsequently activates extracellular regulated protein kinase (ERK) [Homer1a, Per1, and Per2 genes to regulate their transcription  , 104. Af-Tai-Wan . Additio-Tai-Wan –113. Mor-Tai-Wan . Thus, t1R and A2AR and plays a key role in the mammalian circadian clock [1R can suppress the cAMP pathway through inhibiting adenylate cyclase (AC) via its Gi; contrarily, the adenosine A2A receptor can stimulate cAMP pathway through activating AC via its Gs -CRE signaling pathways to modulate the expression of Per1 and Per2 genes in mice [Recently, Jagannath et al. revealed that adenosine could regulate the circadian clock through activating the adenosine A in mice . They fo in mice . Thus, a in mice .2AR is colocalized postsynaptically in dopamine areas, including the striatum and NAc [Adenosine receptors are also involved in the modulation of other neurotransmitter systems. For example, the A and NAc . Indeed, and NAc . Likewis and NAc . The dop and NAc .Per1/2 have been elucidated .Apart from circadian genes 1R signaling, may be a convergent point of several non-pharmaceutical treatments of MDD [Homer1 gene is bimodally regulated by CREB via the CRE site and by the CLOCK/BMAL1 complex via E-box, demonstrating an important crosstalk between CREB and the circadian clock, and thus showing a pivotal role of Homer1a in integrating signals from both adenosine signaling and circadian rhythms [Homer1 and Per genes, receiving signals from both CREB and the CLOCK/BMAL1 complex, which is inhibited by PERs, may be a potential common mechanism of various antidepressant therapies . At the same time, caffeine, an antagonist of adenosine receptors, may have a stronger antagonistic effect on A2AR than A1R, resulting in an antidepressant effect [Homer1 and Per genes, and the final Homer1a protein expression level may depend on the probability of circadian output, which may match the alternative hypothesis model provided by Lazzerini Ospri et al. [We deduce that, on one hand, this conflicting effect of adenosine might be due to the preferential activation of its receptors, since At on A1R , whereast effect , 22, 139i et al. .As discussed above, circadian gene expression is differentially affected by chronic stress, depression, or antidepressant treatments in different brain regions. Thus, the different effects of adenosine signaling on the circadian clock, Homer1a expression, and mood might also be brain region-specific , 133.1R/A2AR signaling converges on the transcriptional factor CREB. After CREB is activated, it can bind to both CRE/AP-1 sites on Per1/2 gene promoters, or the CRE site on the Homer1 promoter, thus modulating their expression. In turn, Per/Cry complexes translocate to the nucleus and inhibit BMAL1 activity. Since Homer1 expression is bimodally regulated by BMAL1 and CREB, we deduce that Homer1a expression might be inhibited by Per indirectly , and CREB . This paAbove all, identification of the common pathogenesis of MDD will help us to better understand the underlying pathogenesis of mood disorders and to explore novel antidepressants or mood stabilizers with fewer side effects.


SDC1). Microarray data analysis showed that of 34 placentally expressed proteoglycans, SDC1 production is markedly the highest in the placenta and that SDC1 is the most upregulated gene during trophoblast differentiation into the syncytiotrophoblast. Furthermore, placental transcriptomic data identified dysregulated proteoglycan genes in pre-eclampsia and in fetal growth restriction, including SDC1, which is supported by the lower concentration of syndecan-1 in maternal blood in these syndromes. Overall, our clinical and in vitro studies, data analyses, and literature search pointed out that proteoglycans, as important components of the placenta, may regulate various stages of placental development and participate in the maintenance of a healthy pregnancy. Moreover, syndecan-1 may serve as a useful marker of syncytialization and a prognostic marker of adverse pregnancy outcomes. Further studies are warranted to explore the role of proteoglycans in healthy and complicated pregnancies, which may help in diagnostic or therapeutic developments.Proteoglycan macromolecules play key roles in several physiological processes , all of which are important for placentation and healthy pregnancy. However, their precise roles in human reproduction have not been clarified. To fill this gap, herein, we provide an overview of the proteoglycans’ expression and role in the placenta, in trophoblast development, and in pregnancy complications , highlighting one of the most important members of this family, syndecan-1 ( Proteoglycans are macromolecules consisting of a protein core to which glycosaminoglycan (GAG) side chains are covalently attached . Most prEmerging data at the RNA and protein levels have shown that more than a dozen of the nearly 50 proteoglycan core proteins ,12 are eOf note, the extremely rapid development of the placenta highly resembles the formation of malignant tumors. Indeed, among the properties shared by placental trophoblast cells and cancer cells are the capabilities to invade healthy tissues, form new vessels, and promote an environment that is protected from the immune system . Since pHowever, only a few studies attempted to determine the proteoglycans’ diverse roles in the placenta, and a systematic effort to provide a global view on placental proteoglycans has not been carried out. Therefore, herein we performed a systems-level analysis on proteoglycan expression in the placenta in normal and complicated pregnancies, focusing on syndecan-1 as one of the most important members of the proteoglycan family in the placenta.SDC1), followed by glypican-3 (GPC3)—both found on the surface of the parenchymal cells—decorin , and collagen, type XV, alpha 1 .To screen for proteoglycan genes expressed by the term placenta, BioGPS microarray data ,50 were GPC3, SDC1, and COL15A1—had relative expression orders of magnitude: 441-fold, 390-fold, and 198-fold higher expression in the placenta than the median of other tissues, respectively. Among these genes, GPC3 and SDC1 also met an additional criterion, being predominantly expressed in the placenta, previously defined in papers by Than et al. and Szilagyi et al. [As a second approach, the placental expression for each gene was transformed as a ratio relative to the median expression in other tissues b. Nine pi et al. ,52. Of ni et al. .SDC1 mRNA expression patterns from the qRT-PCR data were consistent with the microarray data. The highest expression was observed in the placenta, representing an approximately three-fold increase over the second-highest expression in the liver, while it has low expression in other tissues. These data suggest that proteoglycans, including SDC1, are important structural and molecular components of the placenta.To validate BioGPS microarray data, syndecan-1 expression results were quantified by qRT-PCR, using a TaqMan Assay and a human 48-tissue cDNA panel c. SDC1 mSDC1 showed the highest difference in the course of syncytial trophoblast differentiation with a 10.9-fold increase in expression on day 2 (CD44 and transforming growth factor-beta receptor III (TGFBR3) were also upregulated with a 2.1-fold and 2.6-fold increase, respectively, while leprecan-1—also known as prolyl 3-hydroxylase 1 (P3H1)—(3.4-fold) and serglycin (SRGN) (−3.5-fold)—was downregulated. To validate the microarray results, the expression of SDC1 mRNA was measured by qRT-PCR in cell lysates, and the protein level was monitored in cell culture supernatants by ELISA. The maximum mRNA expression of syndecan-1 was detected in the cells on day 2 after seeding with an approximately 120-fold increase in expression , and P3H1 were downregulated in the syncytiotrophoblast, while edgeR analysis of the same dataset revealed the downregulation of collagen type XII alpha 1 chain (COL12A1), P3H1, syndecan-4 (SDC4), and structural maintenance of chromosomes 3 (SMC3) as well as the upregulation of GPC3 [SDC1 expression compared to other trophoblast types in the placenta , biglycan (BGN), GPC3, and heparan sulfate proteoglycan 2 (HSPG2), showed higher expression in extravillous trophoblasts in both datasets  (SDC1 (Upregulated expression of clusters a–c retris (GPC4) d and SDC4) , SDC1, brevican (BCAN), transforming growth factor-beta receptor III (TGFBR3), versican (VCAN), and agrin (AGRN) with 2.4-, −2.2-, 1.7-, −1.6-, 1.5-, and 1.5-fold changes, respectively , and chondroitin sulfate proteoglycan 4 (CSPG4) were significantly upregulated [DCN and SRGN were upregulated in villous cytotrophoblasts; GPC3 and SRGN were downregulated, while DCN, LUM, SDC4, and SMC3 were upregulated in the syncytiotrophoblast; HSPG2 and syndecan-2 (SDC2) were downregulated, and SRGN was upregulated in extravillous trophoblasts  and sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) [BGN and GPC3 to be upregulated in fetal growth restriction.In the case of fetal growth restriction, four microarray studies ,73,74,75(SPOCK1)  and founAt the protein level, the expression of five proteoglycans  of the nine measured was significantly decreased in the placentas of women with pre-eclampsia compared to gestational-age-matched controls . RegardiSDC1 expression was not significant in microarray and single-cell RNA-seq analyses, syndecan-1 expression levels in the placenta, using immunohistochemistry and/or qRT-PCR, were shown to be reduced during pre-eclampsia, as reported by a few publications [Although reduced ications ,60,79. Iications ,61,80,81ications , and oneications . Althougications . This isications ,84,85,86ications ,88,89. Iications ,91. Inteications . This alIn the case of fetal growth restriction, the mRNA and protein expression levels of both syndecan-1 and syndecan-2 were significantly decreased in diseased placentas compared to the controls . HoweverSDC1 production is markedly the highest. Moreover, during villous trophoblast differentiation into the syncytiotrophoblast, SDC1 has the highest upregulation among all proteoglycans. Furthermore, SDC1 gene expression (microarray and RNA-seq data), although not always significantly, is found to be downregulated in pre-eclampsia and fetal growth restriction, which corroborates with a significantly lower concentration of syndecan-1 protein in the maternal blood in these obstetrical syndromes. These findings, along with data on the role of syndecan-1 in regulating cytoskeleton remodeling, cell adhesion, growth factor signaling, anchorage-dependent growth, and invasiveness in different cell systems [Microarray data showed that of the proteoglycans expressed in the placenta,  systems ,93,94, sOn the fetal side of the placenta, the critical involvement of syndecan-1 in trophoblast syncytialization  has beenInterestingly, the role of syndecan-1 also emerges in trophoblast invasion in association with ADAM12. ADAM12, upregulated during extravillous trophoblast differentiation, was suggested to exert its promigratory function in extravillous trophoblasts by inducing integrin beta 1-mediated cellular spreading . As syndSDC1 knockdown in endometrial epithelial cells resulted in the decreased expression of several anti-apoptotic proteins, a cellular inhibitor of apoptosis (cIAP)-1 and -2, X-linked inhibitor of apoptosis (XIAP), survivin, clusterin, heme oxygenase (HO-2), and heat shock proteins HSP27 and -70. Correspondingly, active caspase-3 was increased more severely in knockdown cells after treatment with implantation-related stimuli [SDC1 knockdown further increased Bad expression. On the contrary, anti-apoptotic Livin decreased due to decidualization and knockdown of SDC1. Implantation stimuli provoked an increase of abundant proteins in all different cell types  in varying degrees. Bad was upregulated in all four cell types, whereas anti-apoptotic cIAP-1 and XIAP were downregulated only in knockdown cells [On the maternal side of the placenta, it was demonstrated by the workgroup of Hess that syndecan-1 is involved in the control of apoptosis both in human endometrial epithelial  and in s stimuli . In endo stimuli . Pro-apown cells . Altogetwn cells ,101.SDC1 knockdown led to significant changes in cytokine, chemokine, growth factors, and angiogenic factor , CXCL8, C-C motif chemokine ligand 2 (CCL2), hepatocyte growth factor (HGF)) expression profiles of human endometrial stromal cells [Of note, the same workgroup found that al cells . Incubatal cells .Of importance, the syndecan-1 ectodomain can be shed by various matrix metalloproteinases in a highly regulated fashion, and soluble syndecan-1 can compete with the intact one for extracellular ligands. The multiple roles of syndecan shedding, including inflammation, wound healing, and tumor progression, were reviewed by Manon-Jensen et al. . It woulSDC1 to be primarily expressed by the placenta among all human tissues and primarily in the syncytiotrophoblast within the placenta, where it is localized to the cytoplasm and the apical cell surface, the largest interface between the maternal and fetal compartments. Microarray data showed that of 34 proteoglycans expressed in the placenta, SDC1 production is markedly the highest. Moreover, during villous trophoblast differentiation into the syncytiotrophoblast, SDC1 has the highest upregulation among all proteoglycans. We also analyzed transcriptomic data of the placenta from various obstetrical syndromes and identified SDC1, among other dysregulated proteoglycan genes, in pre-eclampsia and fetal growth restriction. In line with these findings, syndecan-1 concentrations are decreased in the maternal blood in these syndromes, where placental development and malfunction are at the center of the disease. Since syndecan-1 supports proper implantation and angiogenesis by the regulation of chemokine and angiogenic factor secretion in favor of the implanting embryo, it may have a key role in the development of placental dysfunction in obstetrical syndromes, where these functions are severely affected.In this review, we aimed to characterize the expression and potential functions of all proteoglycans at the maternal-fetal interface in healthy and diseased pregnancies. Importantly, we detected In conclusion, proteoglycans are important components of the placenta and regulate various steps of placental development as well as participate in the maintenance of healthy pregnancy. Syndecan-1, with the highest expression among proteoglycans, may serve as a useful marker of syncytialization and may also be a diagnostic/prognostic marker of adverse pregnancy outcomes, such as pre-eclampsia and fetal growth restriction. Further studies are warranted to explore the role of proteoglycans, including syndecan-1, in healthy pregnancies and in pregnancies with adverse outcomes to better characterize molecular signaling pathways and to reveal potential therapeutic targets.We downloaded the human U133A/GNF1H microarray data on 79 human tissues from the BioGPS database . In the Microarray data of trophoblast differentiation into the syncytiotrophoblast were downloaded from GEO (Acc. No.: GSE130339) . MicroarSingle-cell RNA-seq data (pTPM) were downloaded from the Human Protein Atlas . The 19 4Cl solution . The cells were then washed and resuspended, layered over 20–50% Percoll gradients, and centrifuged at 1200× g for 20 min. Negative selection was used to collect trophoblast-containing bands and to exclude non-trophoblastic cells, using anti-CD14 (20 μg/mL) and anti-CD9 (20 μg/mL) mouse monoclonal antibodies , MACS anti-mouse IgG microbeads, and MS columns . Next, the primary trophoblast cells were plated on a collagen-coated 12-well plate (BD Biosciences) in Iscove’s modified Dulbecco’s medium  and completed with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). Primary trophoblasts were kept in IMDM containing 1% penicillin/streptomycin and 5% non-pregnant human serum  to test the effect of trophoblast differentiation on selected genes’ expression. Every 24 h, the medium was replenished, and all 24 h cells were harvested for total RNA between days 1 and 7.Placentas (n = 6) were collected prospectively from normal pregnant women at term, delivered by cesarean section, at Hutzel Women’s Hospital of the Detroit Medical Center , and were then processed at the Perinatology Research Branch, NICHD/NIH/DHHS, for in vitro trophoblast experiments. For cytotrophoblast isolation, we used the modified method of Kliman et al. . One hunSDC1 (Hs00896423_m1) and RPLP0  were used for gene expression profiling on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems-Thermo Fisher Scientific). All experiments were run in triplicate.Total RNA was isolated from cells in 12-well plates each day with RNeasy Mini Kit and RNase-Free DNase Set . RNA concentrations were measured with a NanoDrop1000 Spectrophotometer (Thermo Fisher Scientific). Total RNA (500 ng) was reverse-transcribed with SuperScript III First-Strand Synthesis Kit (Invitrogen-Thermo Fisher Scientific). TaqMan Assays (Applied Biosystems-Thermo Fisher Scientific) for SDC1 and RPLP0 were also used for gene expression profiling on Human Major Tissue qPCR Arrays containing first-strand cDNAs from 48 different pooled tissues (n = 3), OriGene Technologies, Inc., Rockville, MD, USA) on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems-Thermo Fisher Scientific). All experiments were run in triplicate.The same TaqMan Assays for Syndecan-1 concentrations in supernatants of primary trophoblast cell cultures (n = 5) were measured with a human syndecan-1 sandwich ELISA Kit , according to the manufacturer’s instructions. The sensitivity of the assay was <2.56 ng/mL, and the coefficients of intra-assay variation and inter-assay variation were 7.6% and 6.8%, respectively. All experiments were run in triplicate.t-test was used for the analysis of SDC1 expression profiling with the Human Major Tissue qPCR Array. One-Way ANOVA test with Dunnett’s post-hoc test was used for the analysis of qRT-PCR and ELISA results. Results were considered statistically significant at * p < 0.05, ** p < 0.01, and *** p < 0.001.Statistical analysis was performed using GraphPad Prism 5.0 . Unpaired


Cucumis sativus L.) is one of the most popular cultivated vegetable crops but it is intrinsically sensitive to cold stress due to its thermophilic nature. To explore the molecular mechanism of plant response to low temperature (LT) and the mitigation effect of exogenous nitric oxide (NO) on LT stress in cucumber, transcriptome changes in cucumber leaves were compared. The results showed that LT stress regulated the transcript level of genes related to the cell cycle, photosynthesis, flavonoid accumulation, lignin synthesis, active gibberellin (GA), phenylalanine metabolism, phytohormone ethylene and salicylic acid (SA) signaling in cucumber seedlings. Exogenous NO improved the LT tolerance of cucumber as reflected by increased maximum photochemical efficiency (Fv/Fm) and decreased chilling damage index (CI), electrolyte leakage and malondialdehyde (MDA) content, and altered transcript levels of genes related to phenylalanine metabolism, lignin synthesis, plant hormone (SA and ethylene) signal transduction, and cell cycle. In addition, we found four differentially expressed transcription factors  and their target genes such as the light-harvesting complex I chlorophyll a/b binding protein 1 gene (LHCA1), light-harvesting complex II chlorophyll a/b binding protein 1, 3, and 5 genes , chalcone synthase gene (CSH), ethylene-insensitive protein 3 gene (EIN3), peroxidase, phenylalanine ammonia-lyase gene (PAL), DNA replication licensing factor gene (MCM5 and MCM6), gibberellin 3 beta-dioxygenase gene (GA3ox), and regulatory protein gene (NPRI), which are potentially associated with plant responses to NO and LT stress. Notably, HD-ZIP and b-ZIP specifically responded to exogenous NO under LT stress. Taken together, these results demonstrate that cucumber seedlings respond to LT stress and exogenous NO by modulating the transcription of some key transcription factors and their downstream genes, thereby regulating photosynthesis, lignin synthesis, plant hormone signal transduction, phenylalanine metabolism, cell cycle, and GA synthesis. Our study unveiled potential molecular mechanisms of plant response to LT stress and indicated the possibility of NO application in cucumber production under LT stress, particularly in winter and early spring.Cucumber ( Cucumis sativus L.) is a typical profitable and nutritious vegetable crop across the world. Despite being a thermophilic species, it is one of the most popular greenhouse-cultivated crops in winter and early spring. However, cucumber plants routinely encounter chilling (0–12.5 °C) during the winter or early spring production in regions with cold climates such as in northern China which appears to be the main limiting factor for the growth, productivity, and quality of greenhouse-grown cucumber [−2, more than five times that of cultivated in the open field, and the productivity of cucumber cultivated in greenhouse decreased by 3–4 kg·m−2 due to low temperature (LT) stress [S-transferases (GSTs) and cytochromes P450 [Cucumber (illing 0–2.5 °C du) stress . Moreove) stress ,8,9,10. mes P450 ,12,13.2S)-induced tolerance to chilling stress in cucumber [ADC1-associated putrescine pathway, probably by enhancing the expression of the CBF gene [With the development and application of “omics” technologies, transcriptomics analysis has provided a new experimental tool to investigate genome function and its physiological regulation mechanism of stress tolerance in crops. In addition, as a comprehensive and accurate tool, high-throughput RNA sequencing (RNA-seq) provides an opportunity to quantify the transcriptome. Transcriptome profiling based on the RNA-seq is used to analyze gene expression patterns and the molecular mechanism in plant development, response to abiotic stresses and to map the response pathways ,15,16. Acucumber . In a trCBF gene . More reCBF gene . Those rCBF1, CBF2, and CBF3 expression, followed by the expression of COR genes under LT stress [peroxidases and GST, was up-regulated after cold treatment in VaWRKY12-overexpressing grapevine calli compared to the control calli [MYB was extremely up-regulated in the purple kale and induced by LT stress [Transcriptional regulation plays a bridging role in plant responses to abiotic stress, and transcription factors (TFs) cover a significant part of the genome and can interact with promoters of different abiotic stress-related genes and initiate the expression of genes . Among tT stress . FurtherT stress , NAC [25T stress , HD-ZIP T stress , bHLH [2T stress , CBF [29T stress  familiesol calli . In addiT stress . FurtherT stress . HoweverOur previous studies at the physiological level have revealed that NO improves the LT tolerance of cucumber by inducing the photosynthetic and antioxidant systems ,10. HoweThe severity of cucumber leaf damage was apparently higher in LT treatment than in NO + LT treatment A,B. WhilNine cDNA libraries were established from Jinyan No. 4 cucumber seedlings under normal conditions (Control), low-temperature stress (LT), and a combination of exogenous NO and low-temperature stress (NO + LT) for RNA-seq analysis. The clean reads of each sample ranged from 42.6 M to 55.9 M, with an average amount of 50.5 M. The quality of Q30 bases ranged from 92% to 93%, with an average of 92.9% . In summp-value less than 0.01 (padj < 0.01) were chosen for GO enrichment analysis. The results showed that there were 4609, 4915, and 159 DEGs that obtained GO annotations in the three comparisons of LT vs. Control, NO + LT vs. Control, and NO + LT vs. LT, respectively. In addition, 2439, 2632, and 81 DEGs were enriched in the biological process (BP), 835, 863, and 44 DEGs were enriched in cellular component (CC), and 3320, 3428, and 116 DEGs were enriched in molecular function (MF) in the three comparisons, respectively. Furthermore, the MF contains the most DEGs, followed by BP, and the CC with the least DEGs. Taking p < 0.05 as the criterion of significant GO enrichment, the significantly enriched GO terms of each comparison were selected and plotted as a bubble diagram  was performed to further elucidate the metabolic pathways and key regulatory genes involved in response to LT stress and exogenous NO. The significantly enriched pathways with Arabidopsis protein sequence for homology comparison after converting the cucumber gene ID to the Arabidopsis gene ID. Then the corresponding Arabidopsis gene IDs were input into the Plant TFDB  software to predict the TFs. Moreover, The TFs enrichment function was used to perform upstream transcription factors enrichment, and p < 0.01 was assigned as a significantly different enrichment. As shown in In order to investigate the effect of LT stress and NO + LT treatments on transcription factors (TFs) in cucumber, the TFs encoded by the DEGs and the upstream TFs that regulate the DEGs were mined in this study , Table 1http://chromatindynamics.snu.ac.kr:8080/EatupTF, accessed on 22 April 2021) software to predict the regulatory network. A total of 14 upstream TFs were identified, of which only four TFs including MYB63, WRKY21, b-ZIP, and HD-ZIP were differentially expressed, while b-ZIP and HD-ZIP TFs were specific activation TFs that respond to NO under LT stress. The four TFs potentially regulated 18, 12, 26, and 7 DEGs, respectively  and light-harvesting complex II chlorophyll a/b binding protein (LHCB), located at the photosynthetic system I (PSI) and the photosynthetic system II (PSII), respectively, are the most important part of the light-harvesting complex (LHC) in photosynthesis to absorb light energy. As shown in The biosynthesis of flavonoids starts from the phenylalanine metabolic pathway. The cinnamic acid is catalyzed by chalcone synthase (CHS) to form chalcone, and chalcone is catalyzed by chalcone isomerase (CHI) and forms naringenin. Naringenin is catalyzed by flavanone 3-hydroxylase (F3H) to produce flavanone alcohols. Flavonols, the main product of metabolism, enter other different flavonoid synthesis pathways to form different flavonoid substances. In this study, the CHS and CHI were down-regulated in both LT vs. Control and NO + LT vs. Control, while had no significant difference in NO + LT vs. LT B. These Phenylalanine ammonia-lyase (PAL) can catalyze the formation of trans-cinnamic acid (t-CA), the precursor substance of salicylic acid (SA), from phenylalanine. Exogenous NO could up-regulate the expression of PAL B, which Ethylene can bind with the ethylene receptor (ETR) located on the endoplasmic reticulum membrane, resulting in the inactivation of the negative regulatory component receptor constitutive triple response 1 (CTR1). The inactivated CTR1 complex can no longer phosphorylate the downstream signal components ethylene-insensitive protein 2 (EIN2), thus causing activation of EIN2. Then the carboxyl end of EIN2 protein (EIN2 CEND) is cleaved followed by entry to the nucleus. EIN2 can promote the accumulation of EIN3/EIL1 in the nucleus by inhibiting the ubiquitination and degradation of transcription factors EIN3-binding F-Box 1_2 (EBF1_2), and then EIN3/EIL1 transcription activates the expression of ethylene-responsive factor1 (ERF1) and other downstream target genes ,33. In o9 is the initial product of gibberellin, which can be catalyzed to synthesize bioactive gibberellins GA1 and GA4 by GA3ox. In this study, the GA3ox was down-regulated by exogenous NO under LT stress. In addition, the GA3ox was regulated by HD-ZIP  and qPCR methods are shown in To further validate our transcriptome results, transcripts of 12 key genes involved in plant hormone signal transduction, phenylalanine metabolism, and cell cycle were analyzed by qRT-PCR assays. The expression levels of these 12 genes under each treatment were calculated based on the 22+), hydrogen sulfide (H2S), protein kinases, phosphatases, and transcription factors had been identified in cold-stress signaling pathways [Low temperature (LT), a major environmental constraint, severely affects plant growth and development. It also can lead to a substantial reduction in production, and even plant death . To copepathways ,34,35,36The RNA-seq technology has the advantage of global representation and precise measurement of the expression level of each gene in a sample by mapping short DNA sequences on a reference . PreviouThe GO and KEGG enrichments are used to analyze the function of genes and the metabolic pathways of plants in response to abiotic stress ,37,38. ICoffea canephora, Solanum lycopersicum, Camellia sinensis, and tall fescue [BoPAP1, an orthologous gene of BoMYB1, was induced by LT and subsequently activated a subset of anthocyanin structural genes [Dof is up-regulated in response to cold stress in grapevine [As a core of transcriptional regulatory networks, TFs are responsible for the control of gene transcription, technically, TFs also act as the on/off switch of gene expression and are responsible for inducing and repressing genes by binding directly to the promoters region of the target genes in a sequence-specific mode, as well as responding to signal transduction, thereby regulating their function under abiotic stress ,42. Thusl fescue ,45,46,47al genes , NAC, MYal genes , and Dofrapevine . In thisArabidopsis have demonstrated a close relationship between these enzymes and lignin accumulation in secondary cell walls [PAL and peroxidases were induced by exogenous NO under LT stress, while CHS and CHI were down-regulated by both LT stress and NO + LT treatment in cucumber seedlings. Furthermore, PAL was regulated by HD-ZIP, and peroxidase was regulated by both WRKY21 and MYB63 TFs. This result was consistent with the previous study that suggested that some TFs including MYB, WRKY, and HD-ZIP could control the lignin biosynthesis [WRKY genes were identified according to the cucumber  genome (v3.0) [WRKY46 conferred cold tolerance to transgenic plants and positively regulated the cold signaling pathways in an ABA-dependent manner in cucumber seedlings [Mutations that inhibit phenylalanine ammonia-lyase (PAL) synthetase are usually associated with significant changes in the levels of many phenylpropanoids . As a prll walls . In thisynthesis . In addie (v3.0) , and WRKeedlings . We founGA3ox1 and GA3ox2, two key biosynthetic enzymes for active GA [GA3ox under LT stress in cucumber seedlings. SA and ethylene have also been indicated to interact with NO to regulate plant growth and improve the abiotic stress tolerance in plants. For example, SA can raise the content of NO, and NO interacts with SA to play a crucial role in regulating stomatal closure in Arabidopsis leaves and alleviate cadmium toxicity in rice [ERF1, MKK4_5, ETR, EIN3 and NPR1, TGA, PR1 genes. In addition, exogenous NO can also regulate the content of GA by up-regulating the expression level of GA3oX, and similar results were reported previously [EIN3, NPR1, and GA3oX were regulated by WRKY21, b-ZIP, and HD-ZIP TFs, respectively. According to these results, we supposed that the WRKY21, b-ZIP, and HD-ZIP were activated by LT stress, and these TFs initiated the mechanisms of the hormone response to stress by inducing the expression of genes related to ethylene, GA, and SA. In addition, exogenous NO could delay the growth of cucumber plants by participating in HD-ZIP-regulated GA metabolism under LT stress, thereby improving the LT tolerance of cucumber. A similar study reported that LT stress led to a decrement in bioactive GAs, thus inhibiting rice seed germination [NO interacted with phytohormones involved in the regulation of plant growth and development, pathogen defense, and abiotic stress responses in plants . A studyctive GA . We foun in rice ,58; The  in rice . In thiseviously . Furthermination .MCM5 and MCM6 were up-regulated by exogenous NO under LT stress in this study. In addition, the MCM5 and MCM6 were regulated by HD-ZIP. A previous study reported that MCM6 shuttled between cytoplasm and nucleolus in a cell cycle-dependent manner, and the subunits of the MCM2-7 were coordinately expressed during Arabidopsis development and were abundant in proliferating and young tissues [MCM5 and MCM6, and finally promoted cell cycle transition, cell mitosis, and accelerated the production of the new cell to resist the damage of LT to cucumbers in this study.The ability of plant cells to undergo transformation and regeneration is associated with cell cycle activity , while t tissues . CombineLHCB1, LHCB3, LHCB5, and LHCA1, and these genes were also negatively regulated by MYB63 under LT stress. In addition, exogenous NO down-regulated the expression levels of LHCB1, LHCB3, LHCB5, and LHCA1 by negative regulating the b-ZIP. Similar results were also reported by a previous study [LHCA1-4 subunits are reduced under Fe deficiency, and it impairs the plant light-harvesting capacity, resulting in decreased photosynthetic efficiency in rice seedlings. Consistent with these results, we propose that LT reduced the photosynthesis by down-regulating the expression levels of LHCB1, LHCB3, LHCB5, and LHCA1 gene, and NO could not improve the photosynthesis of cucumber seedlings at the transcriptional level.The light-harvesting chlorophyll a/b binding (LHC) proteins, namely antenna proteins, play a significant role in capturing solar energy, as well as in photoprotection under stress conditions . When plus study , which f−2·s−1 average photosynthetic photon flux density (PPFD) across replications for daytime hours. When the second true leaf is fully expanded, that is, after fifteen days of pre-culture, different treatments were carried out. The cucumber seedlings were divided into three groups, two of which were pre-treated with distilled water and the other group was pre-treated with 200 μmol·L−1 SNP . After the cucumber seedlings were pre-treated with distilled water and SNP for 2 days. SNP-treated group and one of the distilled water-treated group were placed in an incubator  with the temperature of (10 ± 1) °C/(6 ± 1) °C (day/night), approximately 75% relative humidity, 14/10 h (light/dark) photoperiod, and 100 μmol·m−2·s−1 average PPFD at 10 o’clock in the morning on the third day, and another group of cucumber seedlings treated with distilled water grew in another incubator with normal conditions. The experiment for transcriptome was defined as: (1) Control, cucumber seedlings treated with distilled water and grew in normal conditions for 24 h; (2) LT, cucumber seedlings treated with distilled water and then grew under LT stress for 24 h; (3) NO + LT, cucumber seedlings pre-treated with SNP and then grew under LT stress for 24 h. The experiment was arranged in a completely randomized design with three replicates.Cucumber seeds of Jinyan No. 4  were germinated at 28 °C for 24 h in dark and then sown in vermiculite at (25 ± 1) °C/(20 ± 1) °C (day/night). After the two cotyledons were fully expanded, healthy seedlings were selected and transplanted into plastic containers  filled with a mixture of peat and vermiculite (2:1 by volume). Since 7 days after transplantation, 50 mL of Hoagland’s nutrient solution was irrigated every four days. The conditions were maintained as follows: (25 ± 1) °C/(18 ± 1) °C (day/night), approximately 75% relative humidity, 14/10 h (light/dark) photoperiod achieved with supplemental lights, and 300 μmol·mThe chilling damage index (CI) was measured after low-temperature stress of 24 h according to the method described by . The degThe malondialdehyde (MDA) content was determined in terms of thiobarbituric acid reactive substances (TBARS) according to the method described by Heath and Packer . The relTotal RNA of samples were extracted from frozen leaf tissues of cucumber seedlings and then its purity and integrity were detected using Nanodrop 2000 and 2% agarose gel electrophoresis, respectively. The quality-controlled mRNA samples were used for strand-specific RNA library construction. mRNA was purified from total RNA and enriched by A-T complementary pairing with poly-T oligo-attached magnetic beads. Then the fragmentation buffer was added to break the mRNA into short fragments. mRNAs were used as a template, six-base random hexamers were used to synthesize one-strand cDNA, and then the buffer, dNTPs, and DNA polymerase I were added to synthesize two-strand cDNA. AMPure XP beads were used to purify the double-strand cDNA. The purified double-stranded cDNA is then repaired, A-tailed, and ligated with a sequencing adapter, then AMPure XP beads were used for fragment size selection, and finally, PCR enrichment was performed to obtain the final cDNA library. The quality-controlled libraries were used for transcriptome sequencing. Paired ends of 250–300 bp sequencings were carried out on the HiSeq platform.https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 15 April 2021), removing poly-N, low-quality, and adaptor sequences [http://cucurbitgenomics.org/, accessed on 15 April 2021), and Salmon was used to map the high-quality sequences with cucumber genome and transcripts at the same time to obtain gene expression (TPM) [2 (Fold Change)| > 1 and the Padj < 0.01 as the standard to identify the differential expression genes in each comparison. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was carried out using the clusterProfiler package in R. KEGG annotation information was obtained from the KAAS comparison, and the cluster Profiler package was used for GO enrichment and KEGG pathway analysis [http://chromatindynamics.snu.ac.kr:8080/EatupTF, accessed on 15 April 2021).The quality of clean reads (clean data) was evaluated using Fastqc (equences . Cucumbeon (TPM) . DESeq2 on (TPM) . The |loanalysis . The traanalysis . Furtherhttp://bioinfo.ut.ee/primer3-0.4.0/, accessed on 4 September 2021) and the pair of primers were presented in Actin was used as the reference gene. qRT-PCR was performed using SYBR® Premix Ex Taq™ . The PCR reactions were carried out on the iCycler iQ Multicolor Real-time PCR Detection System . The relative expression level of each gene was calculated by 2−ΔΔCt with three technical replicates [The total RNA was isolated using Trizol reagent according to the manufacturer’s instructions, and DNase I was used to remove any gDNA contamination. The reverse transcriptase  was used to reverse-transcribe pure RNA to cDNA according to the manufacturer’s protocol. The genes involved in the phytohormone signal transduction, cell cycle, and phenylalanine metabolism pathways were selected for qRT-PCR analysis. The primers were generated in the online software Primer 3.0  was used for statistical analyses. Data was performed by two-tailed analysis of variance (ANOVA), and values represent the means ± SE of at least three replicates. LHCA1, LHCB1, LHCB3, LHCB5, CSH, EIN3, peroxidase, PAL, MCM5, MCM6, GA3ox, and NPRI, to modulate plant responses to exogenous NO under LT stress. It is worth mentioning that HD-ZIP and b-ZIP specifically responded to exogenous NO under LT stress. These results demonstrate that exogenous NO improved the LT tolerance of cucumber plants by modulating the transcription of some key TFs and their downstream genes, thereby regulating photosynthesis, lignin synthesis, plant hormone signal transduction, phenylalanine metabolism, cell cycle, and GA synthesis. Our study unveiled potential molecular mechanisms of plant response to LT stress and indicated the possibility of NO application in cucumber production under LT stress, particularly in winter and early spring.In the present study, the transcriptome analysis of cucumber seedlings revealed the molecular mechanism of plant response to LT and the mitigation effect of exogenous NO on LT stress in cucumbers. The transcript level of genes related to the cell cycle, photosynthesis, flavonoid accumulation, lignin synthesis, active GA, phenylalanine metabolism, phytohormone ethylene, and SA signaling were significantly regulated by LT stress. Exogenous NO can improve the LT tolerance of cucumber seedlings by affecting Fv/Fm, chilling damage index, electrolyte leakage, lipid peroxidation, and changing the phenylalanine metabolism, lignin synthesis, plant hormone (SA and ethylene) signal transduction, and cell cycle pathway. In addition, four differentially expressed transcription factors MYB63, WRKY21, HD-ZIP, and b-ZIP were identified which can regulate their target genes such as