Unc-5 netrin receptor A (UNC5A), a netrin family receptor, plays a key role in neuronal development and subsequent differentiation. Recently, studies have found that UNC5A plays an important role in multiple cancers, such as bladder cancer, non-small cell lung carcinoma, and colon cancer but its pan-cancer function is largely unknown. Herein, the R software and multiple databases or online websites (The Cancer Genome Atlas (TCGA), The Genotype-Tissue Expression (GTEx), The Tumor Immune Estimation Resource (TIMER), The Gene Set Cancer Analysis (GSCA), Gene Expression Profiling Interactive Analysis (GEPIA), and cBioPortal etc.) were utilized to examine the role of UNC5A in pan-cancer. UNC5A was found to be highly expressed across multiple human cancer tissues and cells, was linked to clinical outcomes of patients, and was a potential pan-cancer biomarker. The mutational landscape of UNC5A exhibited that patients with UNC5A mutations had poorer progress free survival (PFS) in head and neck squamous cell carcinoma (HNSC) and prostate adenocarcinoma (PRAD). Furthermore, UNC5A expression was associated with tumor mutation burden (TMB), neoantigen, tumor microenvironment (TME), tumor microsatellite instability (MSI), immunomodulators, immune infiltration, DNA methylation, immune checkpoint (ICP) genes, and drug responses. Our results suggest the potential of UNC5A as a pan-cancer biomarker and an efficient immunotherapy target, which may also guide drug selection for some specific cancer types in clinical practice. Cancer poses a huge economic and disease burden worldwide due to its high morbidity and mortality rates ,2. SurgeUnc-5 netrin receptor A (UNC5A), encoding an axon-guiding molecule, is located on the 5q35 chromosomal region and functions as a receptor of netrin family  with key roles in neuronal development and differentiation ,15. PrevHerein, multiple online databases and R packages were used to evaluate the role of UNC5A in human cancers. We systematically illustrated the UNC5A mRNA expression profile, prognostic value, mutation patterns, gene enrichment of UNC5A and co-expressed genes, immune infiltration, relationships with various immunomodulators, tumor mutation burden (TMB), neoantigen, immune checkpoint (ICP) genes, tumor microenvironment (TME), and tumor microsatellite instability (MSI). Finally, we assessed the methylation status of UNC5A in pan-cancer and its correlations with drug sensitivity. Our findings indicated the potential of UNC5A as a prognostic biomarker and immunotherapy target, which may also influence drug sensitivity, thus, guiding the clinical use of drugs against tumors.http://biogps.org/#goto=welcome (accessed on 30 August 2022)). The results were visualized using Graphpad Prism ). A p-value < 0.05 was considered statistically significant.The Tumor Immune Estimation Resource (TIMER) platform, a comprehensive tool for the systematic analysis of immune cell infiltrates in multiple cancer types, comprises different gene expression profiles between normal and tumor tissues from The Cancer Genome Atlas (TCGA) database ,23. Therp-value < 0.05 was considered statistically significant.The pan-cancer clinical information was collected from The Genotype-Tissue Expression (GTEx) and TCGA databases, and the relationships between UNC5A expression and progression-free interval (PFI), overall survival (OS), disease-free interval (DFI), and disease-specific survival (DSS), were assessed by Logrank test in R environment. Moreover, the prognosis predictive utility of UNC5A in pan-cancer was also analyzed in R environment through “timeROC” package (version 0.4). A https://www.cbioportal.org/ (accessed on 23 September 2022 )) via Logrank test [p-value < 0.05 indicated a statistically significant result.The mutation status of UNC5A, as well as the progress free survival (PFS) across cancer types with or without UNC5A gene mutations, were determined on the cBioPortal database ) was used to construct the protein-protein interaction (PPI) network for UNC5A and its related genes. Subsequently, the Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment and Gene Ontology (GO) enrichment analyses of UNC5A and related genes were conducted. First, we used the KEGG rest API (https://www.kegg.jp/kegg/rest/keggapi.html (accessed on 2 October 2022)) to obtain the latest gene annotations for the KEGG pathway. After that, the KEGG pathway was analyzed through R package “clusterProfiler”  [The STRING online platform  ). As forhttp://sangerbox.com/home.html (accessed on 2 October 2022)) through Spearman correlation analysis. Differences with a p-value < 0.05 were considered statistically significant. In addition, in this study, correlations between variables were defined as follows: r < 0.3, weak correlation; r ≥ 0.3 and < 0.6, moderate correlation; and r ≥0.6, strong correlation.The correlations between UNC5A expression and different immunomodulators , immunoinhibitor, and immunostimulators), ICP genes, TMB, MSI, neoantigen, and TME were evaluated online on SangerBox ) online website. First, the association of four methyltransferases, including DNMT3b, DNMT3a, DNMT2, and DNMT1, with UNC5A expression were analyzed through the Gene Expression Profiling Interactive Analysis online website ) via Pearson correlation analysis; after that the results were visualized in R software [Over 750 small molecule drugs from Cancer Therapeutics Response Portal (CTRP) and Genomics of Drug Sensitivity in Cancer (GDSC), along with integrated 10,000 multi-dimensional genomic data across 33 cancer types from TCGA can be found on The Gene Set Cancer Analysis (GSCA) (software . SubsequUNC5A mRNA was highly expressed in colon adenocarcinoma (COAD), head and neck squamous cell carcinoma (HNSC), liver hepatocellular carcinoma (LIHC), kidney renal papillary cell carcinoma (KIRP), cholangiocarcinoma (CHOL), stomach adenocarcinoma (STAD), kidney renal clear cell carcinoma (KIRC), breast invasive carcinoma (BRCA), kidney chromophobe (KICH), and prostate adenocarcinoma (PRAD), whereas its expression was low in uterine corpus endometrial carcinoma (UCEC), thyroid carcinoma (THCA), and lung squamous cell carcinoma (LUSC) A, accordp = 7.2 × 10−4), LUSC , KIRC , and LUAD , while it served as a protective factor in lower grade brain glioma    A. Kaplan× 10−12) B–F. On t× 10−12) G.p = 0.01) and testicular germ cell tumors  but served as a protective factor for pheochromocytoma and paraganglioma , and LGG , as shown in For DFI, Cox regression showed that a high UNC5A level was a risk factor for LUSC , KIRC , and uveal melanoma  but a protective factor for LGG   A. Moreov× 10−13) B,C,E,F, × 10−13) D.p = 5.1 × 10−4), ACC , and UVM  but a protective factor for LGG , and PCPG   A. Kaplan × 10−6) B–E, wher × 10−6) F,G.Subsequently, time-dependent ROC curves were plotted to examine the prognostic value of UNC5A expression in the prediction of 1-, 3-, and 5-year survival in terms of DSS, OS, and PFI in pan-cancer. UNC5A is a candidate prognostic biomarker for multiple cancer types. For example, the AUC values for predicting the corresponding 1-, 3-, and 5-year OS, DSS, and PFI of patients with KICH were , , and , respectively. The two cancer types (KICH and UVM) with the highest AUC values are shown in p = 0.0127) and PRAD (p = 9.596 × 10−3)  D,E.There was a strong interaction between UNC5A and the co-expressed genes  A. FunctiNeoantigen, MSI, and TMB are strongly linked to tumorigenesis and progression and can predict tumor immunotherapeutic efficacy ; therefoImmune and stromal cells are crucial in TME. SangerBox was utilized to assess the association of UNC5A levels with StromalScore and ImmuneScore in pan-cancer. UNC5A expression correlated positively to both ImmuneScore and StromalScore in HNSC, rectum adenocarcinoma (READ), KIRC, THCA, UVM, esophageal carcinoma (ESCA), LAML, BLCA, COAD, LUAD, OV, STAD, prostate adenocarcinoma (PAAD), LUSC, and skin cutaneous melanoma (SKCM), while negatively in GBM, LGG, and PCPG D,E. MoreThe immune infiltration level of UNC5A in pan-cancer was examined utilizing the TIMER database. UNC5A level was significantly related to the infiltration of B cells in 10 diverse cancer types, CD4+ T cells in 21 various cancer types, CD8+ T cells in 9 cancers, neutrophils in 18 cancers, macrophages in 16 cancers, and dendritic cells in 18 cancers. Notably, UNC5A was related to all the above six immune cell types in HNSC, LIHC, LUSC, and THCA . DetaileNext, we evaluated the relationships between UNC5A expression and immunomodulators  and various ICP genes across cancers. The results exhibited that UNC5A expression was substantially associated with the majority of immunomodulators in READ, COAD, BLCA, HNSC, and LUSC etc., while it was negatively related to immunomodulators in TGCT A. FurtheDNA methylation is crucial in tumor onset progression, as confirmed in numerous studies ,31. ThusThe relationship between UNC5A level and drug sensitivity was investigated based on CTRP and GDSC databases, suggesting a significant association between UNC5A level and sensitivity to multiple drugs. A high level of UNC5A expression exhibited low drug sensitivity for various targeted or chemotherapeutic agents such as Bosutinib, Cytarabine, Dabrafenib, Pluripotin, and Trametinib. Conversely, a high level of UNC5A expression exhibited high drug sensitivity for Austocystin D, Belinostat, and Pifithrin-mu A,B. ThesOwing to the rapid development in bioinformatics, researchers can now use various analytical software and online databases (such as the R software and TCGA database) to explore potential biomarkers, signaling pathways, and therapeutic targets, and assess the relationship between specific genes with drug responses in cancer. Although several studies have reported the role of UNC5A in breast cancer, colorectal cancer, non-small cell lung cancer, bladder cancer, and gliomas, its significance in other human cancers remains unclear. Herein, the R software and multiple databases  were used to examine UNC5A’s role in pan-cancer. Herein, UNC5A was highly expressed across various human cancers tissues and cells and was linked to the clinical outcomes of patients and may serve as a potential biomarker in pan-cancer. The mutational landscape of UNC5A exhibited that patients with UNC5A mutations had poor PFS in HNSC and PRAD. UNC5A expression was associated with TMB, MSI, neoantigen, TME, immune infiltration level, immunomodulator, ICP genes, DNA methylation, and drug responses.High UNC5A expression was found in 14 human cancers. Notably, previous studies have shown that UNC5A is significantly highly expressed in adjacent non-cancerous bladder tissues ; howeverTME is a complex system, where the TIME is an essential constituent intimately correlated with tumor immunity and is implicated in tumor progression ,42,43. TDNA methylation is a crucial epigenetic change implicated in tumorigenesis and development ,47. GeneAlthough we comprehensively analyzed UNC5A expression in pan-cancer, some limitations warrant further consideration. First, the data utilized in this study were derived from open sources, and in vitro/in vivo experiments, and other public databases are needed to validate our conclusion. Second, UNC5A is highly expressed and related to clinical outcomes in pan-cancer; however, its potential mechanism of action has not yet been elucidated. Thus, further investigations are needed to evaluate the potential action mechanism of UNC5A in the diagnosis and patient prognosis of cancers. Finally, in this study we found that there is a tight relationship between UNC5A and tumor immunity. However, how UNC5A’s relation to tumor immunity has not been elucidated, which should be further explored in future studies.In summary, we present the first comprehensive role of UNC5A in pan-cancer. As shown in


OSCA genes are identified in the Triticum aestivum genome. A phylogenetic analysis reveals the close clustering of the OSCA proteins of Arabidopsis thaliana, Oryza sativa, and T. aestivum in all the clades, suggesting their origin before the divergence of dicots and monocots. Furthermore, evolutionary analyses suggest the role of segmental and tandem duplication events (Des) and purifying selection pressure in the expansion of the OSCA gene family in T. aestivum. Expression profiling in various tissue developmental stages and under abiotic and biotic stress treatments reveals the probable functioning of OSCA genes in plant development and the stress response in T. aestivum. In addition, protein–protein and protein–chemical interactions reveal that OSCA proteins might play a putative role in Ca2+-mediated developmental processes and adaptive responses. The miRNA interaction analysis strengthens the evidence for their functioning in various biological processes and stress-induced signaling cascades. The current study could provide a foundation for the functional characterization of TaOSCA genes in future studies.The hyperosmolality-gated calcium-permeable channels (OSCA) are pore-forming transmembrane proteins that function as osmosensors during various plant developmental processes and stress responses. In our analysis, through in silico approaches, a total of 42  Calcinditions . Howeverpathways . The cytnd pumps ,6.2+-permeable channels involved in the influx of Ca2+ from the apoplast to the cytoplasm. They are usually activated in response to hyper-osmotic conditions, dehydration, and mechanical stress in plants [2+-permeable channels work as osmosensors in animals and bacteria [The hyperosmolality-gated calcium-permeable channels (OSCAs) are the membrane-localized Can plants ,8,9. Simbacteria ,11. The bacteria ,13.OSCA genes have been identified in various plant species, including Arabidopsis thaliana (15 genes); Oryza sativa L. Japonica, Oryza sativa L. ssp. Indica, and Oryza brachyantha (11 genes); Oryza glaberrima (12 genes); Pyrus bretschneideri (16 genes); Vigna radiata (13 genes); Zea mays (12 genes); Gossypium hirsutum (35 genes); Gossypium arboretum (21 genes); Gossypium raimondii (22 genes); and Glycine max (21 genes) [OSCA genes in various plant developmental processes and stress responses. For instance, Yaun et al. characterized OSCA1 in A. thaliana and revealed that it mediates an osmotic-stress-evoked Ca2+ concentration increase and plays a crucial role in osmosensing [Z. mays provided drought stress tolerance in A. thaliana [OsOSCA genes of O. sativa performed redundant functioning in Ca2+ signaling induced in response to hyperosmolality and salt stresses [G. hirsutum enhances salt and drought resistance by modulating the activity of antioxidant enzymes [thaliana  genes; Ohneideri  genes; V1 genes) ,18,19,20osensing . ZmOSCA2rboretum  genes; Gaimondii  genes; astresses . The GhO enzymes ,25. ThesOSCA genes in plants, there was a grave need for an inclusive characterization of the genes in an important staple food crop, T. aestivum L. Herein, a total of 42 TaOSCA genes were identified following various in silico approaches in the T. aestivum genome, which were used for further analyses, including duplication event, Ka/Ks, phylogenetic, gene, and protein structure analysis, using various bioinformatic approaches. To gain insight into the roles of TaOSCA genes, an expression analysis was carried out in various tissue developmental stages and biotic and abiotic stress conditions using high-throughput RNA seq data and qRT-PCR. Furthermore, protein–protein, protein–chemical interaction, and miRNA interaction analyses were performed to explore their diverse roles. This work could provide new directions for the functional characterization of TaOSCA proteins in future studies.Due to the immense importance of TaOSCA genes were identified in the T. aestivum genome by performing a BLAST search using the already reported 15 and 11 OSCA sequences of A. thaliana and O. sativa, respectively. The identified TaOSCA genes could assemble into 14 homoeologous groups based on sequence similarity (≥90%) among them and their subgenomic  locations. Then, their nomenclature was conducted following the standard wheat symbolization protocol (TaOSCA genes were derived from subgenome D (15), followed by subgenome B (14) and subgenome A (13). A chromosomal distribution analysis showed that all the identified TaOSCA genes were scattered on 15/21 chromosomes, including 1A, 1B, 1D, 2A, 2B, 2D, 3A, 3B, 3D, 4A, 4B, 4D, 5A, 5B, and 5D. A maximum number of four genes were found on chromosomes 1A, 1D, 4B, and 5A each, while a minimum of one gene was found on chromosome 3A  residues . The lonTaOSCA gene family, the duplication events were analyzed. A total of three duplication events were predicted, which were segregated into two segmental duplication events (SDEs) (TaOSCA4-A-TaOSCA6-B and TaOSCA1-B-TaOSCA2-D) and one tandem duplication event (TDE) (TaOSCA12-B-TaOSCA13-B) in T. aestivum , while a value of more than one shows the presence of positive selection pressure. In our analysis, the Ka/Ks ratio of all the identified duplicated gene pairs was less than one, which suggested that they had endured negative or purifying selection pressure. In addition to this, the time of divergence was also estimated, which was in a range from 13.0 to 32.9 MYA .p > 0.05 , drought (DS), combined heat–drought (HD), and salt stress using available high-throughput RNA sequence data [TaOSCA genes were selected for analysis which showed more than two-fold down- or upregulation in at least one of the abiotic stress conditions. This criterion led to the identification of a total of 31 TaOSCA genes, which displayed differential expression in response to the DS, HS, and HD stress conditions  of HS and HD. However, only a few TaOSCA genes, such as TaOSCA6-D and TaOSCA12-B, etc., exhibited upregulation at an early hour (1 h) of the HS and HD conditions. TaOSCA12-B displayed significant upregulation after every hour of the DS, HS, and HD treatments (TaOSCA8-B was highly upregulated (69-fold) at DS 6 h as compared to the rest of the genes. The genes TaOSCA3-A, TaOSCA3-B, TaOSCA7-B, TaOSCA10-B2, TaOSCA10-D, and TaOSCA13-A were downregulated after each hour of the DS, HS, and HD treatments , and at 12 h of salt stress, TaOSCA14-B would be highly upregulated (10-fold) as compared to other genes  and Puccinia striiformis (Pst), using high-throughput RNA seq data [OSCA genes were differentially expressed after Bgt and Pst infestation  was performed after CaCl2 treatment using qRT-PCR , ARFC1 (ADP-ribosylation factor C1), LEA4-1 (late embryogenesis abundant 4-1), C/VIF2 , CYP71B5 (cytochrome p450 71b5), FLA6 , GLR2.3 (glutamate receptor 2.3), GRV2 (gravitropism defective 2), NiaP (organic cation/carnitine transporter 7), PLP4 (patatin-like protein 4), UPS4 (ureide permease 4), VSR1 (vacuolar-sorting receptor 1), and YLMG1-2 (YGGT family protein) A. MoreovA protein–chemical interaction analysis suggested the interaction of 37 TaOSCA proteins with 15 different chemicals: 5-hydroxytryptophan, beta-D-glucopyranoside, AR-11, AR-21, cytidine triphosphate, emodin anthrone, glucose, guanosine triphosphate, hyperforin, isobutyrophenone, maltose solution, MgATP, pyrophosphate, Tyr-Met-Lys, and uridine 5′-triphosphate B. FurtheOSCA transcripts of T. aestivum  [OSCA genes could be attributed to their complex polyploid genome. Further, a positive correlation between the number of genes and the ploidy level of the genome was observed. The comparable number of the homeologous grouping (14) of the TaOSCA genes of T. aestivum to the total number of OSCAs in the diploid plant species [T. aestivum have also been reported as compared to those of other crops [The extensive BLAST search identified a total of 42 tum (35) ,18,19,20grouping  of the T species ,17,18 fuer crops ,34. The grouping  of the TT. aestivum, both SDEs and TDEs were predicted as key factors behind the expansion of the TaOSCA gene family. Similar duplication events in the OSCA gene family have also been reported in Pyrus bretschneideri (five SDEs), V. radiata (three SDEs), and G. hirsutum (16 SDEs) [TaOSCA paralogous genes, which was in agreement with the paralogous OSCA genes in other plants, such as Pyrus bretschneideri, V. radiata, and G. hirsutum, [TaOSCA genes probably earlier than the hybridization events of the A, B, and D subgenomes [Duplication events play vital functions in the expansion of gene families, introducing genetic variability, and gaining neo-functionality in paralogues genes . In T. ahirsutum  SDEs [1616 SDEs) ,19. Duri16 SDEs) . The Ka/irsutum, ,19. Furtbgenomes .OSCA genes of A. thaliana, O. sativa, and T. aestivum in all the clades indicated their evolvement before the division of dicotyledons and monocotyledons. The tight clustering of the homeologous genes of T. aestivum could be due to their high sequence homology. Moreover, the close clustering of paralogous TaOSCA genes pointed toward their conserved nature, which was in accordance with the purifying selection.The phylogenetic analysis suggested the clustering of OSCA proteins into four clades as reported in earlier studies ,19,32. TTaOSCA genes were performed to reveal their roles in the growth and development of vegetative and reproductive tissues, as well as in the stress response. The specifically higher expression levels of certain genes in particular tissue developmental stages suggested their precise roles in said tissue development. For instance, the higher expression of the majority of TaOSCA genes in the early developmental stages of root tissue indicated their vital function in Ca2+ absorption, which is highly required during the early stage for the proper growth and development of plants because Ca2+ is an integral part of the cell wall and membrane and also plays a significant role in various organelles and in signaling [TaOSCA genes in leaf and stem tissues further indicated their roles in vegetative tissue development. Moreover, the specifically greater expression levels of certain TaOSCA genes in particular developmental stage spike and grain tissue indicated their involvement in reproductive tissue development and the reproductive health of plants. Similarly, the differential expression of OSCA genes has also been reported in the cereal crop rice, where certain genes, such as OsOSCA1.1, OsOSCA1.2, OsOSCA2.4, OsOSCA3.1, and OsOSCA4.1, exhibited higher expression levels in the roots, shoots, mature stems, mature flag leaves, stamens, pistils, and mature seeds [TaOSCA genes in various developmental stages of grain indicated their roles during grain filling. This could be due to either the positive involvement of Ca2+ during grain development or the fact that increased osmotic potential is responsible for the enrichment of TaOSCA transcripts. Earlier research has also suggested the roles of several OSCA genes in O. sativa in caryopsis development and seed imbibition [OSCA genes in vegetative and reproductive tissues has also been reported in P. bretschneideri [OSCA need to be established individually in future studies.Expression studies on the ignaling . Similarre seeds . The spebibition . Furtherhneideri . MoreoveTaOSCA genes under abiotic stresses, such as heat, drought, salt, and biotic stresses including fungal (Bgt and Pst) infestation, suggested the putative roles of TaOSCA genes in defense responses. The majority of TaOSCA genes exhibited differential responses during drought and salinity stresses, which is in agreement with previous reports [OSCA genes in O. sativa, OsOSCA1.2, OsOSCA2.1, OsOSCA2.4, OsOSCA2.5, and OsOSCA3.1, showed upregulation after ABA, drought, PEG, and salinity treatments. Further, OsOSCA4.1 is upregulated after PEG, salinity, and ABA treatments, while OsOSCA1.1 is solely upregulated after PEG and salinity stress. The OSCA1 of A. thaliana has also been reported to play a precise role in osmotic stress-induced Ca2+ signaling [OsOSCA genes  mediated Ca2+ signalling in osca1 mutant of A. thaliana during hyperosmolality and salt stress [ZmOSCA genes of Z. mays are upregulated in response to ABA, PEG, and NaCl treatments, and ZmOSCA2.3 and ZmOSCA2.4 exhibited significantly higher upregulation under these stresses [ZmOSCA2.4 conferred drought resistance in the mutant of A. thaliana [TaOSCA genes in T. aestivum in response to biotic stresses has suggested their involvement in biotic stress-induced Ca2+ signaling. Thor et al. [2+ influx in the cytoplasm of guard cells in Arabidopsis during PAMPs-triggered stomatal immunity, which established their role in Ca2+ signaling under biotic stress. The induced expression of TaOSCA genes at different hours of CaCl2 treatment in the present study further suggests their crucial participation in Ca2+ homeostasis and signaling. Several previous studies already established the roles of OSCAs in Ca2+ homeostasis in response to different stress conditions [The modulated expression of various  reports ,19,20,32 reports ,13. For ignaling . It has t stress ,23. Simistresses . Furtherthaliana . The eler et al.  recentlynditions ,23,24,25TaOSCAs were analysed by co-expression, protein–protein interaction, and protein–chemical interaction to reveal their roles in downstream signaling during developmental stages, metabolism, and stress conditions. The co-expression analysis and GO mapping indicated the involvement of TaOSCA genes in a wide range of developmental processes as well as in stress responses. For instance, the co-expression of TaOSCA genes with UDP-glucose 6-dehydrogenase 4-like, cellulose synthase, embryogenesis-associated proteins, and Rubisco revealed their role in cell wall synthesis, reproductive development, and photosynthesis [TaOSCA genes with Ca2+-binding protein, Ca2+ uptake proteins, Ca2+-dependent protein kinase, Ca2+ transporter ATPase, and WRKY transcription factor showed their role in Ca2+-related signaling cascades and Ca2+ homeostasis during these stresses [cis-regulatory element DRE1 (drought-responsive element 1) in the promoter region of TaOSCA genes also indicated their functioning in drought and osmotic stress responses [TaOSCA genes with transcripts encoding various kinases, such as serine-threonine kinase, well-associated receptor kinases, receptor-like kinases, and phosphatidylinositol 4-phosphate 5-kinase, further suggested their role in the defense-related signaling cascades [TaOSCA genes with chitinase CLP-like and pathogenesis-related thaumatin-like protein 3.5 indicated their involvement in Ca2+-mediated downstream signaling for the activation of defense-responsive genes during biotic stresses [TaOSCA genes after Bgt and Pst infestation.The interaction analyses of ynthesis ,39. Duristresses . The occesponses . The co-cascades . During stresses , which w2+ influx into the cytosol [2+-mediated stomatal closure. The interaction of TaOSCA proteins with LEA and C/VIF2 proteins indicated their role in adaptive responses during drought stress conditions [TaOCSA genes in root developmental stages also endorse the above statement. The interaction of TaOSCA with MgATP further suggested their probable functioning in Ca2+ signaling and homeostasis [2+ concentration by modulating endo/exocytosis or by signaling cascades during pollen tube growth indicated their function in reproductive tissue development [TaOSCA genes in spike developmental stages. Guanosine triphosphate was also reported to be involved in signal transduction, vesicular transportation, and cell proliferation, etc. [Similar to the co-expression analysis, the protein–protein interaction analysis of TaOSCAs also suggested their linkage with various biological as well as stress-related signaling pathways. For instance, the interaction of TaOSCA proteins with GLR showed their functioning in stomata closure via Ca cytosol ,43, becanditions ,45,46. Anditions ,48. The eostasis . MgATP ieostasis ,51. Inteelopment , which ion, etc. .TaOSCA genes, their interaction with the known miRNAs [T. aestivum was investigated in the present study. The interaction of TaOSCAs with miR156 and miR172, associated with grain development [TaOSCA genes in plant reproductive development as indicated by expression and co-expression studies. Additionally, their interaction with some drought stress-related miRNAs, such as miR528, miRNA172, miRNA395, and miRNA530 [miRNAs control gene expression at the post-transcriptional and translational level through the regulatory mechanism known as RNA interference ,54. Manyn miRNAs  of T. aeelopment  and florelopment , respectmiRNA530 ,63,64, aTaOSCA gene family in T. aestivum suggested their diverse roles from development to the stress response, which could be attributed to their involvement in Ca2+ homeostasis. The duplication event analysis suggested that segmental duplications are the major factors responsible for the OSCA gene family expansion. The phylogenetic analysis of OSCA proteins among different plant species indicated the close evolutionary relatedness between dicotyledons and monocotyledons. The modulated expression patterns of TaOSCA genes suggested that they play essential roles in plant development as well as in adaptive responses. The co-expression and interaction analysis suggested the involvement of TaOSCA genes in Ca2+-mediated developmental processes and stress responses. TaOSCA interaction with numerous known miRNAs associated with different biological functions suggested the miRNA-mediated regulation of TaOSCA genes during different biological processes. The current study revealed numerous functions of TaOSCAs, but the specific roles of each of the genes need to be further established in future research.In conclusion, comprehensive analyses of the −10 to identify the TaOSCA genes in the T. aestivum genome. The OSCA protein sequences of A. thaliana and O. sativa were used as queries against the protein model sequences of T. aestivum, retrieved from the Ensembl Plant  and IWGSC (IWGSC RefSeq assembly v2.0)  databases. Furthermore, the SMART server [T. aestivum.The bidirectional BLAST hit approach was adopted with e-value 10T server  and ConsT server  approachTaOSCA gene were downloaded from the Ensembl Plant server. Further, TaOSCA genes were mapped on their respective chromosomes using the MapInspect tool (http://mapinspect.software.in-former.com/accessed on 1 October 2021). The proposed guidelines for wheat gene symbolization  were followed for the nomenclature of the TaOSCA proteins. The homeologous grouping of TaOSCA genes was performed on the basis of ≥90% sequence similarity among them, as performed in the previous analysis [The data regarding the chromosomal and subgenomic location of each analysis ,34.https://services.healthtech.dtu.dk/service.php?SignalP-5.0, accessed on 30 August 2022) and TMHMM-2.0  server, respectively.The Expasy tool was used to compute the various properties, for instance, the protein length, MW, and pI of each TaOSCA protein . Subcellhttps://www.ebi.ac.uk/Tools/msa/mafft/, accessed on 3 June 2022), and TaOSCA genes with 80–90% sequence similarity were considered duplicated genes. Further, the predicted duplicated genes were categorized into tandem and segmentally duplicated genes based on the distance among them as described in earlier reports [To analyze the duplication events, a sequence similarity matrix was conducted using the MAFFT software , synonymous substitution per synonymous site (Ks), and Ka/Ks ratios of software . Further5 × 10−9 . Moreove5 × 10−9 .A. thaliana, O. sativa, and T. aestivum were aligned via the ClustalW algorithm . Further, the neighbor-joining method was applied to construct the phylogenetic tree OSCA protein using the MEGA X software with 1000 bootstrap replicates [For the construction of a phylogenetic tree, firstly full-length OSCA protein sequences of plicates .TaOSCA genes was performed to gain insight into the functional involvement of these genes in plant development and defense. The high-throughput RNA-seq data were downloaded from the publicly available URGI at https://urgi.versailles.inra.fr/files/RNASeqWheat, accessed on 24 February 2019. In the case of tissue developmental expression profiling, the RNA seq data were produced from three development stages of five tissues, including root, leaf, stem, spike, and grain tissues, and were present in biological replicates [An expression analysis of the plicates ,28. The plicates  were purplicates .TaOSCA genes, expression profiling was carried out using high-throughput RNA-seq data generated after 1 and 6 h of HS, DS, and HD in duplicates from leaf tissue [To study the effects of abiotic stresses on f tissue , and 6, f tissue .TaOSCA genes, expression profiling was carried out using high-throughput RNA seq data generated in triplicates after 24, 48, and 72 h of their inoculation [http://www.cs.umd.edu/hcil/hce/, accessed on 4 January 2022) and the Euclidean distance method was used for hierarchical clustering [Moreover, to study the effects of the two fungal pathogens Bgt and Pst on culation . Using tustering .T. aestivum (cv. Chinese spring) were surface sterilized with sodium hypochlorite (1.2%) in 10% ethanol and washed with double autoclaved water. The washed seeds were placed overnight at 4 °C for stratification. Further, these seeds were kept at room temperature for germination. Afterwards, these germinated seedlings were grown in a plant growth chamber at 22 °C, 60% relative humidity, and in 16 and 8 hours of light and dark periods, respectively. Further, the seven-day-old seedlings were treated with CaCl2 (20 mM) Murashige and Skoog (MS) growth media. The samples were collected after 6, 12, 24, and 48 h in liquid nitrogen. RNA was taken out of root tissues using the Spectrum TM Plant Total RNA kit . The TURBO DNA-free™ Kit  was used to remove the traces of DNA contamination from the RNA samples. Moreover, both the quality and quantity of RNA samples were examined using agarose gel electrophoresis and a Nanodrop spectrophotometer, respectively. The cDNAs were produced from 1 microgram of RNA using Superscript III First-Strand Synthesis SuperMix . qRT-PCR was performed with CFX96 Real-Time PCR  using SYBR Green and gene-specific primers of selected OSCA genes by following a previously established procedure [n = 3) which were further illustrated in the terms of mean § standard deviation (SD).Firstly, the seeds of rocedure . An ADP-rocedure . All theTaOSCA genes were predicted using CoExpress v.1.5. server. The Pearson correlation coefficient with a correlation power of 1 [The co-expressing partners of wer of 1  and threwer of 1  was usedwer of 1 .http://stringdb.org) [http://stitch.embl.de/, accessed on 16 June 2022) [https://cytoscape.org/download.html) was used for the construction of interaction networks. The interacting miRNAs of TaOSCA genes were predicted with the psRNATarget  database [T. aestivum miRNA sequences [The protein–protein and protein–chemical interaction were performed using the STRING (gdb.org)  and STITne 2022)  servers,database  using thequences .


To understand the molecular mechanisms involved, we performed a transcriptome-wide analysis. Here we show that 1715 up- and 1624 down-regulated genes were involved in this biological process. Through the protein–protein interaction (PPI) network analyses performed in the STRING database (v11.5), we found 299 genes tightly associated in 14 clusters. Two major clusters of up-regulated proteins fundamental for life growth and development were found: 29 ribosomal proteins (RPs) interacting with 6 PEBP family members and 117 cell cycle (CC) proteins. The PPI network of up-regulated transcription factors (TFs) revealed that at least six TFs–MYB43, TSF, bZIP27, bZIP43, HAT4 and WOX9–may be involved during MTs development. The PPI network of down-regulated genes revealed a cluster of 83 proteins involved in light and photosynthesis, 110 in response to hormone, 74 in hormone mediate signaling pathway and 22 related to aging.Potato microtuber (MT) development through in vitro techniques are ideal propagules for producing high quality potato plants. MT formation is influenced by several factors, i.e., photoperiod, sucrose, hormones, and osmotic stress. We have previously developed a protocol of MT induction in medium with sucrose (8%  Solanum tuberosum L.) is the most important staple food worldwide just after maize, rice, and wheat with an approximate production of around 360 million tons in 2020 on a surface area of 16.5 million hectares [Potato  mediated by radial divisions, expanding to further differentiate into starch accumulating parenchyma. Tubers function in vegetative propagation  and as storage enabling survival during winter months and/or prolonged periods of abiotic stress  and are a means to bypass sexual reproduction.The molecular mechanisms of the potato tuberization process have been well documented and depend on light , temperature, and nutrient supply ,4,5.Plant biotechnology techniques have been applied to produce potato tubers in vitro called microtubers (MTs). The MTs process occurs when axillary buds are cultured in medium containing high content of sucrose, hormones, and different light quality/darkness. MTs have advantages in terms of storage, transport, and cultivation, due to their reduced size and weight, requiring less space compared to seedlings, with a higher multiplication rate producing seed potato faster and cheaper than other methods ,7,8,9.S. tuberosum var Alpha culturing stolon explants in MS medium supplemented with 8% SUC, high content of gelrite 6.0 g/L, and cytokinin (CK) 2iP 10 mg/L under darkness. The rationale of this protocol was that two-component and cytokinin (CK) signaling interacts with homeobox TFs; ribosomal proteins (RPs), cell cycle (CC), carbon metabolism, auxin responsive factors, and stem cell maintenance genes were involved in this process [We developed a protocol for MTs induction in potato Several transcriptomic analyses during potato tuberization have revealed conserved molecular machinery during this process: PEBP members (florigen and tuberigen genes), photoperiod, transcriptional regulation, protein synthesis, cell cycle regulation, metabolic pathways, secondary metabolism, starch and sucrose metabolism, hormone signaling, among others ,14,15,16Moreover, a few papers have published reliable gene ID in order to compare results. Sharma et al.  publisheLight modulates the molecular mechanisms during potato tuberization (photomorphogenesis); little attention has been given to the molecular events in the dark. To elucidate the molecular mechanisms of MTs development under these conditions, we performed a transcriptomic-wide analysis.https://string-db.org/cgi/input?sessionId=bbmDlM5pze5B&input_page_active_form=multiple_identifiers) with the highest confidence (0.860). Differential expressed genes (DEG) obtained from the analysis were screened in a bioinformatic tool STRING database v11.5  versus control line (non-MTs); 2896 were up-regulated and 1843 down-regulated.Gene Ontology (GO) enrichment analysis of up-regulated genes identified 2713 genes with annotated GO terms. GO biological process includes 440 significantly enriched GO terms. Both up- and down-regulated biological process were related to protein phosphorylation, regulation of transcription, transmembrane transport, carbohydrate metabolic process, and proteolysis. Specific up-regulated biological processes include: microtubule-based movement, translation, fatty acid biosynthetic process, lipid transport, DNA replication, gycolitic process, positive regulation of transcription, cell wall modification, DNA repair, defense response, carboxylic acid metabolic process, protein dephosphorylation, gluthatione metabolic process, and the amino acid metabolic process. Specific down-regulated biological processes include: photosynthesis, light harvesting, cell wall biogenesis, cellular glucan, and the xyloglucan metabolic process .GO cellular components include 82 significantly enriched GO-terms; 64 were up-regulated and those specific of MTs were: ribosome, nucleosome, microtubule, extracellular region, and cytoplasm. Specific down-regulated cellular components include: cell wall, extrinsic component of membrane, photosystem I and II .GO molecular function includes 132 significantly enriched GO-terms; the seven main functional subgroups are: GO:0003824 catalytic activity (963), GO:0005488 binding (998), GO:00116491 oxidoreductase activity (243), GO:0043167 ion binding (604), GO:0016787 hydrolase activity, GO:0098772 molecular function regulator, and GO:0008092 cytoskeletal protein binding (67).Overall, 112 up-regulated and 145 down-regulated TFs were found in the transcriptomic-wide analysis of MTs induced under dark conditions. Up-regulated TFs are related to C2H2-type zinc finger (20), F-box (15), bHLH (13), AP2 ERF (13), MYB (10), GATA (6), ZF-HD protein (3), Basic leucine zipper (5), bZiP (5), B3 DNA binding d2ggomain (5), NF-Y (4), WRKY (3), TCP (2). The PPI network of up-regulated TFs made on the STRING data base v11.5 with medium confidence revealed that 23 out of 112 TFs interact , while 1Overall, 145 TFs were found to be down-regulated in MTs development. These TFs are related to bHLH (38), Homeobox (30), AP2/ERF (21), SANT/Myb domain (19), Myb domain (19), Zinc finger C2H2 superfamily (18), F-box (14), B-box-type zinc finger (11), WRKY (9), CCT domain (8), B3 DNA binding domain (7), Transcription factor TCP (6), BTB/POZ domain (6), and SKP1/BTB/POZ (6).The PPI network of down-regulated TFs revealed that 58 out of 112 TFs interact , while 9The list of down-regulated TFs interacting in the network and levels of gene regulation are shown in the heat-map .The PPI network of up- and down-regulated TFs revealed both sets of TFs found interact in separated clusters–one forming MTs and the another corresponding to down-regulation TFs involved in shoot-stolon differentiation, gametophytes, plant organ development, and hormone signaling .From 1699 up-regulated genes, 299 were tightly associated in two main clusters . Two funOverall, 271 out of 1616 down-regulated genes were present in the PPI network with high confidence (0.700). Since MTs were induced in darkness, the network consists in one cluster related to photosynthesis (69) tightly interacting with several functions . These fOverall, six PEBP family members were present in MTs development, three FT orthologs of PEBP family are present in potato, StSP6A, StSP5G (PGSC0003DMT400041726), and StSP3D (PGSC0003DMT400041725), two CEN1-BFT (PGSC0003DMT400030575), CEN1-BFT (PGSC0003DMT400037143), and a self-pruning ATC (PGSC0003DMT400018307). The TF FD (PGSC0003DMT400061403) a bZIP transcription factor-27 self-pruning was found interacting with ATC . Gene ontology (GO) from functional annotation revealed a relationship between florigen genes present in the PPI network and RPs. bZIP27 FD (PGSC0003DMT400061403) was associated in 35 out of 59 GO-terms in which RPs were present . In the case of StSP6A, it was associated in 12 out of 51 GO-terms in which RPs were present . StSP3D (PGSC0003DMT400041725 and PGSC0003DMT40004172a) were associated in 12 out of 51 GO-terms in which RPs were present . CEN1/BFT/TFL1C genes (PGSC0003DMT400030575 and PGSC0003DMT400037143) were associated in 3 out of 17 GO-terms in which RPs were present . The list of PEBP family members and levels of gene regulation are shown in the heat map  and CDC20-CDKA (PGSC0003DMT400071215) were found among the up-regulated transcripts in this work. The plant CDC2 is homologous to CDKA and in mammals to CDK1 and CDK2. CDKA function has been characterized as a regulatory protein at the G1/S and G2/M checkpoints. In addition, among the up-regulated transcripts related to Cyclins, we found 10 cyclins, which correspond to cyclin-A (four transcripts), cyclin-B (three transcripts), and cyclin-D (three transcripts). Furthermore, in the PPI network analysis the CDKA (PGSC0003DMT400071215) protein interacts with all the cyclins A and B in the network . The lisTHY-1 (PGSC0003DMT400001937). The THY-1 interacts with LOG10, PGSC0003DMT400057413) which their transcriptional activation is CK-dependent. Further, PCNA2 interacts with SWI/SNF2 (PGSC0003DMT400034983), RNR1 (PGSC0003DMT400060658), and TSO2 (PGSC0003DMT400052217). The E2F3 transcription factor also interacts with BRCA1 (PGSC0003DMT400023253)  interacts with E2F3 (PGSC0003DMT400042347), which in turn interacts with 0023253) .Among the proteins present in the CC cluster, we also found three histone proteins: H2A (PGS003DMT40009751), H2A.1 (PGS0003DMT40003677), and H2AXB (PGSC0003DMT00034958). Those three proteins interact with the plant protein BRCA1 that in turn interacts with ORTHRUS 2 (PGSC000DMT400084297) and PCNA2 (PGSC0003DMT400078207). Moreover, ASF1 (PGSC0003DMT400078369) interacts with PCNA2 and with THY-1.The PPI network analyses shed light on to protein interactions and allow a glimpse as to their biological and functional relationship. In this regard, we found that PEBP family members including the tuberigen StSP6A interact with RPs via RPL11 (PGSC0003DMT400031869), then with RPL29e (GSC0003DMT400069847) and UBQ1 (PGSC0003DMT400000364) within the cluster of RPs. UBQ1 interacts with the first protein of CC, PCNA2 (PGSC0003DMT400078207) . PCNA2 iThe list of the transcripts genes found in the DEG analysis under MTs developmental darkness condition were directly related to cytokinin response, osmotic stress, one-carbon, carbon metabolism, glycolysis/gluconeogenesis, citrate cycle, fatty acid metabolism, thylakoids, acyl-carrier proteins, redoxins, response to oxidative stress, immunophilins, ribosomal proteins, sulfur metabolism, mitotic cell cycle, E2F activation, cytoskeleton, motor proteins, methylation, histone binding, sterol biosynthetic process and terpenoid backbone biosynthesis, amino sugar and nucleotide sugar metabolism, disulfide isomerase activity, LRR; leucine rich repeats, and sucrose metabolism, which are available in Validation of the transcriptomic-wide analysis was made by selecting 19 DEG and analyzing their regulation by quantitative reverse transcription PCR (qRT-PCR), using the primers described in The genes selected to validate de transcriptomic-wide analysis of MTs development were: StSP6, ATC, UBQ1, FKBP12, RPL29, RPL11, PRXQ, MDH1, SHM1, METK2, TPI, TPP, SPP, BRCA1, RNR1, TSO2, THY-1, LOG10, SWI/SNF, PCNA2, and E2F3. EFa1, SEC3 were used as endogens.MTs development were induced from a stolon in medium containing sucrose 8%, gelrite 6 g/L, a CK 2iP 10 mg/L under darkness. In this environment, light modulating/repressing signals from leaves to underground were absent.The molecular mechanisms of tuber development depend on genes related to light perception, temperature, and nutrient supply. These mechanisms have been associated mainly with phytochromes (PHYB and PHYF), homeobox TFs (BEL5 and POTH1), florigens belonging to the PEBP gene family , St14-3-3, StFDL1, TAC complex, StABI5 (StABL1), and several molecular regulators controlled under length day conditions  a tuber-specific and sucrose-responsive element binding factor. MYB44 confers resistance to abiotic stresses dependent on ABA. Enrichment analysis with 50 interactors revealed that MYB44 is involved in auxin signaling pathways and tissue vascularization. MYB44 interacts with an AFR protein (PGSC0003DMT400004486), F-box/kelch-repeat skip25-like. AFR protein interacts with another tuber-specific and sucrose-responsive element binding factor TSF (PGSC0003DMT000794169). Enrichment analysis with 50 interactors revealed that TSF is involved in cell fate specification and commitment, multicellular organism development, cell cycle regulation, spliceosomal and ribonucleoprotein complex. TSF interacts with bZIP43 (PGSC0003DMT400003516); it is expressed in inflorescence meristem during petal differentiation and expansion stage. An enrichment analysis with 50 interactors revealed that bZIP43 is involved in histone modification, h3-k9 methylation, ribonucleoprotein complex, 14-3-3 interaction, and amino acid biosynthesis. bZIP43 interacts with a bZIP27-like, self-pruning TF (PGSC0003DMT400061403), required for positive regulation of flowering. Enrichment analysis with 50 interactors revealed that bZIP27 is involved in structural component of ribosomes, cell cycle regulation, cyrcadian rhythm, protein transport, methylation. bZIP43 interacts with a wuschel-related homeobox 9-like (WOX9), COMPOUND INFLORESCENCE (PGSC0003DMT400027850). WOX9 is required for meristem growth and development, and acts through a positive regulation of wuschel. Mutant phenotypes include embryo lethality, reduced root development, and smaller meristems. Phenotypes can be rescued by the addition of sucrose in the growth media. Overexpression can partially rescue the triple mutants of CK receptor phenotype, as a downstream effector of CK signaling. Enrichment analysis with 50 interactors revealed that WOX9 is involved in ribosome, cell cycle regulation, cell fate commitment, meristem growth and maintenance, translation initiation factor 4f, and flower development.bZIP43 also interacts with a homeobox-leucine zipper protein HAT4 (PGSC0003DMT400067540). HAT4 acts as mediator of the red/far-red light effects on leaf cell expansion in the shading response. It is also involved in the negative regulation of cell elongation and proliferation processes, such as root formation and secondary growth of the vascular system. HAT4 TF interacts with 6 bhlh TFs. Enrichment analysis with 50 interactors revealed that HAT4 is involved in flower development, meristem maintenance, photoreception, sugar signaling, and abiotic stimulus.The above information about PPI network of up-regulated TFs during MTs development may explain partially some molecular events required to develop MTs: vascularization (MYB44), proliferation process (HAT4), cell cycle, ribosomes (bZIP27), cell fate commitment and meristems (WOX9), light regulation (HAT4), and epigenetic regulation (bZIP43) present during MTs differentiation. The rest of TFs in the PPI network may participate during MTs development . FurtherMTs development revealed that 57 down-regulated TFs interact in a PPI network . The morThe PPI network revealed a cluster of 18 homedomain TF superfamily/16 MYB interacting with two BTI TF (PGSC0003DMT400039384) and (PGSC0003DMT400069392) involved in gametophyte development. The MYB TF are involved in multicellular organism development, stamen maturation and dehiscence, seed coat, lateral organ boundaries, and root and xylem differentiation . The gamAnother cluster interacting with the gametophyte is composed by TCP TFs. These TFs are involved in leaf development, branching, circadian rhythm, embryonic growth, floral organ morphogenesis and hormone signaling. Recently, Nicolas et al. , demonstTang et al. , reporteHomo sapiens, PEBP1 (RKIP) has been implicated in several cancers, probably acting as a metastasis suppressor [FLOWERING LOCUS (FT)-like, the TERMINAL FLOWER1 (TFL1)-like, and CENTRORADIALS (CEN)-like. The MOTHER OF FT AND TFL1-like (MFT), and the BROTHER OF FT AND TFL1 (BFT)-like are present as a separate subclade within the CEN group [FT paralogues are related to both sexual and vegetative reproduction. SP3D, an FT orthologue, controls flowering, while SP5G and SP6A, two other PEBP genes, act as a repressor and inducer of tuber formation, respectively [FT/TFL1 defines the plant growth habit as indeterminate or determinate by modulating the pattern of formation of vegetative and reproductive structures in the apical and axillary meristems [SP6A, StSP5G (PGSC0003DMT400041726), SP3D (PGSC0003DMT400041725), and two orthologs of CEN1-BFT (PGSC0003DMT400030575). In the PPI network analysis, we found that all PEBP protein family members interact with the universal plastid RPL11 (PGSC0003DMT400031869). PEBPs lack a DNA-binding domain but can exert the function as transcriptional co-regulators through complexing with TFs, regulating the expression of downstream target genes [The PEBP gene family is well-conserved across species from animals to plants, which play central roles in several biological processes. In ppressor . In addippressor , and a cppressor . In flowEN group . In potaectively . The baleristems . In our S. tuberosum have revealed the presence of RPs during the tuberization process [Purwestri et al.  and Wang process ,16,39,40 process , we founThe ABI5-like 1 (StABL1) bind to SP6A in a 14-3-3 manner forming an alternative tuberigen activation complex (aTAC), thereby resulting in early tuberization . In our CENTRORADIALS (CEN) represses the initiation of floral meristems in several species. The DHvCEN5 barley plants developed spikelet initiation in both photoperiods; GO enrichment analysis demonstrate that HvCEN had a negative impact on to chromatin modification, cell cycle regulation, CK signaling, and cell growth through RPs [TERMINAL FLOWER-1/CENTRORADIALS gene family (CEN) has shown to function as a negative regulator of tuberization. At the protein level, CEN competes with SP6A for the interaction with other members of the TAC complex [CEN overexpression induced a lower rate of sprout growth, lower content of CKs, and higher content of abscisic acid (ABA) [ough RPs . A membe complex . Moreoveid (ABA) .FT was found in this work–the SP5G (PGSC0003DMT400041726) which was up-regulated. The function of this FT is to blockade the tuberization process. In the absence of light/darkness, SP5G might not repress SP6A. In addition, it has been shown that PHYB and PHYF co-stabilized CONSTANS-like 1 protein (COL1), binds SP5G, and represses SP6A and tuberization [SP3D was up-regulated and that this protein is involved in flowering, and it has been shown that florigens can promote tuberization; therefore, the SP3D might be able to promote this process [Another transcript for a rization . We also process ,48.S. tuberosum have revealed the transcriptional activation of the RPs coding genes in the tuberization process [Several transcriptomic analyses in  process ,20,39,40 process  found a  process ,54,55,56The up-regulation of RPs in Arabidopsis is activated by different biotic and abiotic factors ,61,62,63In our study, a high content of sucrose (8%), a CK 2iP 10 mg/L, and osmotic stress conditions were applied under darkness conditions. Hummel et al.  found thArabidopsis, 87 transcripts for RP of the 474 identified responded specifically to CKs; CK-responsive RPs related genes encode proteins for both plastidic and cytoplasmatic localization [In our study, 12 transcripts for RPs were directly related to CKs activation , 12 tranlization .Arabidopsis, Karunadasa et al. [Arabidopsis, 87 out of 474 respond to CKs [CKs binds directly to ribosomes in higher plants with high affinity ,69. CKs a et al.  demonstra et al. , and 12 d to CKs . The CK-Camellia sinensis L.), RPL32 is up-regulated in drought stress [The expression levels of RPs are altered under stress conditions ,62,63,75t stress .Saccharomyces cerevisiae, the repression of nine RPs 60S genes  resulted in G2/M phase arrest. The repression of nine other 60S RPs  and 22 40S genes  caused arrest in the G1 phase [In G1 phase .Overall, 14 of 38 proteins analyzed in yeast were present in the potato RPs network: RPL1, RPL18, RPL25, RPL35, RPL40, RPS2, RPS3, RPS6, RPS9, RPS13, RPS15, RPS17, RPS29, and RPS30. Accordingly, they could contribute to the activation of CC during potato MTs development.Maitra et al.  demonstrWe proposed that RPs present in our analysis, in addition to being essential in the progression of CC in MTs development, could also participate in the one-carbon pathways that ultimately comprise a series of interrelated metabolic pathways that include the methionine and folate cycles–critical for cell function–providing 1C units (methyl groups) for the synthesis of DNA, polyamines, amino acids, creatine, and phospholipids .Tuber development relies on the temporal and spatial control of cell proliferation and cell growth to correctly develop an organ that will help the potato plant to survive under adverse conditions. Potato tubers develop from stolons, from which swelling growth occurs in the subapical region of the meristem. Initial expansion and radial cell division of cells in the pith and cortex, in combination with restricted longitudinal growth is followed by cell division and enlargement in random orientations in the perimedullary region once the swelling has reached a diameter of 2 to 4 mm, continuing until the tuber reaches its full size. In this study, genes involved in CC regulation were present, cyclins/CDKs, E2F factors and histone binding proteins.Overall, 2 cyclin-dependent kinase (CDKs); CDC2 (PGSC0003DMT400075349) and CDKB1;2 (PGSC0003DMT400071215), 10 cyclins: 4 cyclin-A, 3 cyclins-B and 3 cyclin-D were found. CDKB1;2 (PGSC0003DMT400071215) interacts with cyclins A and B present in our network. CKs in the presence of sucrose activate the expression of cyclins-D that have been shown to be essential in CC regulation. Yang et al.  demonstrStCYCD3;1 and StCYCD3;3 were more expressed in above-ground organs whereas StCYCD3;2 was expressed more abundant in undergrounds organs. The above expression levels were when cytokinins like zeatin and BAP combined with sucrose induced higher expression compared to 2,4-D. In our work, StCYCD3;2 was found interacting within the PPI network with a cyclin-dependent kinase B2 (CDKB) (PGSC0003DMT400025910). The levels of expression found were similar; StCYCD3;2 was 3.5, while StCYCD3;1 was 3.03, and StCYCD3;3 was a 2.85 fold change.Rhee et al.  analyzedH. sapiens, PEBP1 (RKIP) is a metastasis suppressor in aggressive cancers [RKIP accelerate DNA synthesis and G1/S transition entry by inducing the expression of CDC6, MCM2,4,6,7, cyclinD2, D1, and E2. It also enhances G2/M transition and down-regulates G2/M checkpoints like AURORA-B, cyclin G1, and slow G2/M transition time. This led to a higher proliferation rate compared to control cells [In  cancers  and cell cancers . Silenceol cells .H. sapiens.In our work, the components of G2/M checkpoints were present, showing similarities with the function of PEBP1(RKIP) in The CC regulation of mitosis is summarized as follows: The CYCB-CDKA complex (PGSC0003DMT400071215) interacts with MAD2 (PGSC0003DMT400045025) (mitotic arrest deficient), MAD2 interacts with CDC20 protein (PGSC0003DMT400047585) (APC metaphase/anaphase complex). MAD2 and CDC20 are checkpoint proteins locally catalyzed by kinetochores, the central player in the distribution of genetic material to the daughter cells during mitosis .TUB1 expression is linked to light induction [MAD2 interacts with EB1c (PGSC0003DMT400064236) (microtubule stability). MAD2 interacts with TUB1 (PGSC0003DMT400037093) (tubulin formation). In Arabidopsis, nduction . TUB1 highest expression levels are found in dark conditions. The TUB1 transcriptional behavior in previous reports is in accordance with our data, since our work was carried out in dark conditions [In hypocotyl explants, the nditions . MAD2 interacts with TUB8 (PGSC0003DMT400028800). During the initiation of tuber formation, there are changes in the expression of tubulins at the tip of the stolons .MAD2 interacts with AURORA2 (PGSC0003DMT400065470), a serine/threonine protein kinase. It is abundant in tissues with high division, such as roots and flowers, but low in leaves and stems. In mitosis it is intimately linked with the cytoskeleton  required for cytokinesis .MAD2 interacts with KRP-130 (PGSC0003DMT400078142). KRP-130 are involved in microtubule dynamics, morphogenesis, chromosome segregation, organelle transport, and vesicles .KRP-130 interacts with BUB1 (PGSC0003DMT400082809); BUB1 binds to the kinetochore and has an important role in establishing the mitotic spindle checkpoint and chromosome aggregation . BUB1 interacts with the CDKA-CYCA complex (PGSC0003DMT400071215). BUB1 interacts in the same way with the complex of CDKA-CYCA. CDKA (PGSC0003DMT400071215) interacts with four B cyclins. CDKA has been identified at the G1/S and G2/M checkpoints  and is m1 to the S phase in the cell cycle. We found 31 E2F factors in this analysis. The activation of genes such as PCNA2, RNR1, TSO2, THY-1, SWI/SNF2, ORC6, ORC4, ORC5, SPT16, MCM6, SMC3, MCM2, CHR11, POLA2, and PSF2 demonstrates that DNA replication was carried out correctly, validating that the conditions evaluated are optimal for the development of MTs. Abnormal induction of the S phase re-entry in mature leaf cells is observed in overexpression of E2F in Arabidopsis [The E2F proteins form a family of TFs that regulate the transition from the Gbidopsis . Unregulbidopsis .PCNA2. CKs are perceived through the activation of LOG10 to the synthesis of dihydrofolate reductase. Key proteins were found during mitosis, such as those of the cytoskeleton, microtubules, tubulins, motor proteins, and chromosome stability, as well as the proteins of the synthesis phase. Like PCNA2, RNR1, TSO2, THY-1, SWI/SNF2.Abiotic stress is perceived through SWI/SNF2 and its interaction with Three histone proteins are present in the CC cluster; histone H2A (PGS003DMT40009751), histone H2A.1 (PGS0003DMT40003677), and histone H2AXB (PGSC0003DMT00034958). Chromatin structure is essential for normal cell cycle progression, chromosome segregation, and centromere function .H2A cause an increase in ploidy phenotype, and an increase rate of chromosome loss and defects in completing the G2/M phase of the cell cycle. Altered centromeric chromatin structure and mutations in genes encoding kinetochore components revealed the role for H2A in the proper centromere-kinetochore function [Loss of function mutations in function .H2AX Ser139Ala, that partially disrupts the phosphorylation site, suppressed the normal G2/M cell cycle arrest following DNA damage by ionizing radiation. The G2/M cell cycle arrest by DNA damage is promoted by checkpoints such as BRCA1 [H2AXB (PGSC0003DMT00034958) regulates the DNA damage response via BRCA1 (PGSC0003DMT400023253) to arrest mitotic cell cycle and ensure an appropriate cell division during MTs development.Histones also regulate a DNA damage response by epigenetic modifications. The expression of a loss of function mutant of as BRCA1 . Taken tThese three proteins interact with BRCA1, a heterodimeric ubiquitin E3 ligase, required for the accumulation of ubiquitin conjugates at sites of DNA damage and for silencing at DNA satellite repeat regions. BRCA1 interact with E3 ubiquitin-protein ligase ORTHRUS 2 (PGSC000DMT400084297) and PCNA2.Histone chaperone ASF1 (PGSC0003DMT400078369) interacts with PCNA2 with a combined score of 0.970 and with THY-1 with a score (0.860). ASF1 play an important role in chromatin replication, maintenance of genome integrity, and cell proliferation during plant development. Histones prevent DNA from becoming tanged, protect it from DNA damage, and play important roles in gene regulation and DNA replication.MTs development under high content of sucrose/gelrite, 2iP and darkness is activated by PEBP members  and the TF FD. PEBP members interact with RPs to modulate CC regulation (DNA synthesis and mitosis) .To validate the results of the transcriptomic-wide analysis and the conclusions reached, we performed an analysis by RT-qPCR of 21 genes interacting within the PPI network.2/s of irradiance. Stolon explants derived from propagated shoots were used for MTs induction. Potato cv. Alpha plantlets were propagated by shoot proliferation under in vitro conditions in MS medium , supplemPotato MTs induction protocol was made according to Herrera-Isidron et al. . Stolon To isolate RNA from MTs, tissue samples were collected after 15 days of incubation; at this time, MTs initiate to develop .w/v) gels. Total RNA was isolated by using Trizol , RNA concentration was measured by its absorbance at 260 nm, ratio 260 nm/280 nm was assessed, and its integrity confirmed by electrophoresis in agarose 1%  in Real-Time PCR Systems . http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and preprocessed to remove sequence adapters and low-quality bases using the software Trimmomatics [Final product cDNA samples for sequencing were sent to GENEWIZ, Plainfield, NJ, USA. Illumina HiSeq 2500  was used for sequencing. Sequenced reads were tested for quality using the FastQC software package  with the STAR aligner v.2.5.2b. [Adapter removal was performed using the Trimmomatic v0.3.6 program . RNA-seq.2.5.2b. . In this.2.5.2b. . A quantS. tuberosum, homologous genes present in the S. tuberosum genome in Sol genomics network [S. tuberosum greater than 60% in protein sequence with A. thaliana were considered. Oligonucleotides were designed to qPCR  [A gene network with high confidence (0.860) was performed with the STRING database v11.5 , based o network . Gene id network , NCBI [1 network  databasenalysis)  gene expA PPI network of up-regulated TFs revealed that at least six TFs–MYB43, TSF, bZIP27, bZIP43, HAT4, WOX9–may be involved during MTs development.Two fundamental biological process essential for life and highly conserved through organisms were found interacting tightly: RPs comprising 29 and CC 117 proteins.PEBP members interact with RPs and CC process to activate MTs development under high content of sucrose and gelrite, 2iP under darkness.Further experiments by yeast two-hybrid screening approach of genome edited up- and down-regulated PEBP members with RPL11 are required to demonstrate our model of MTs development under darkness.


Tumor cells obtain various unique characteristics, which known as hallmarks of cancers, including sustained proliferative signaling, apoptosis resistance, and metastasis. These characteristics are crucial for tumor cells survival and for supporting their rapid growth. Studies have revealed that tumorigenesis is also accompanied by alteration in mechanical properties. Tumor cells could sense various mechanical forces, such as compressive force, shear stress, and portal vein pressure, which in turn could affect tumor progression. Piezo1 is a mechanically sensitive ion channel protein that can be activated mechanically, and is closely related to various diseases. Recent studies showed that Piezo1 is overexpressed in numerous tumors and is associated with poor prognosis. Furthermore, previous studies revealed that Piezo1 mediates these cancer hallmarks, and thus links up mechanical forces with tumor progression. Therefore, the discovery of Piezo1 provides a new insight for elucidating the mechanism of tumor progression under a mechanical microenvironment.Tumor cells alter their characteristics and behaviors during tumorigenesis. These characteristics, known as hallmarks of cancer, are crucial for supporting their rapid growth, need for energy, and adaptation to tumor microenvironment. Tumorigenesis is also accompanied by alteration in mechanical properties. Cells in tumor tissue sense mechanical signals from the tumor microenvironment, which consequently drive the acquisition of hallmarks of cancer, including sustained proliferative signaling, evading growth suppressors, apoptosis resistance, sustained angiogenesis, metastasis, and immune evasion. Piezo-type mechanosensitive ion channel component 1 (Piezo1) is a mechanically sensitive ion channel protein that can be activated mechanically and is closely related to various diseases. Recent studies showed that Piezo1 mediates tumor development through multiple mechanisms, and its overexpression is associated with poor prognosis. Therefore, the discovery of Piezo1, which links-up physical factors with biological properties, provides a new insight for elucidating the mechanism of tumor progression under a mechanical microenvironment, and suggests its potential application as a tumor marker and therapeutic target. In this review, we summarize current knowledge regarding the role of Piezo1 in regulating cancer hallmarks and the underlying molecular mechanisms. Furthermore, we discuss the potential of Piezo1 as an antitumor therapeutic target and the limitations that need to be overcome. Mechanical forces are pervasive in living organisms . For dec2+) ions to pass through the cell membrane, with a slight preference for Ca2+ [2+, which in turn acts as the second messenger of signal transduction and controls various physiological processes [Piezo-type mechanosensitive ion channel component 1 (Piezo1), originally called Fam38A, is a core component of the mechanically activated ion channel ,10,11. Ifor Ca2+ ,16,17. Pfor Ca2+ , shear ffor Ca2+ , substrafor Ca2+ , and tisfor Ca2+ ; therebyrocesses . Hence, rocesses .2+ to pass through the cell membrane and in turn affects various downstream signaling pathways, is closely linked with multiple hallmarks of malignancies.Recently, Piezo1 has been shown to participate in mediating multiple cancers  24,25,2,43,44,45Mammalian Piezo1 is an evolutionarily conserved ion channel protein containing more than 2500 amino acid residues with a molecular weight of about 300 kDa. Piezo1 forms an about 900 kDa homotrimer with a trimeric propeller-like structure containing a central cap and three distal blade-like structures as its extracellular domains, together with 42 transmembrane segments and three long beam-like structures that are located at the intracellular region . TopologThe transmembrane region comprises transmembrane segments forming three peripheral wings and a central pore module . The corTumor progression is accompanied by a significant increase of solid tumor structural components, such as cancer cells, stromal cells, and extracellular matrix constituents, which lead to the increase of tumor stiffness, and thereby generate solid compression forces . Solid cLimitless cell proliferation is one of the most obvious and important cancer hallmarks . Normal 2+ influx. The yes-associated protein (YAP) signaling cascade, which is regulated by Piezo1 during the differentiation of human neural stem cells and the development of zebrafish hearts, is also upregulated by Piezo1/MAPK signaling pathway, thereby promoting tumor cell proliferation [Abundant evidence shows that Piezo1 regulates the expression of crucial genes in the growth signaling pathway to control proliferation. MAPK is a major component of numerous signal transduction cascades that transmit upstream signals to downstream response molecules by sequential phosphorylation . MAPK caferation . Intriguferation .Piezo1 suppresses Ca2+ influx, leading to the decrease of PI3K expression as well as AKT and mTOR phosphorylation [Using bioinformatics analysis based on the Chinese Glioma Genome Atlas (CGGA) dataset and the Cancer Genome Atlas (TCGA) network, Zhou et al. showed that Piezo1 is positively correlated with phosphatidylinositol-3-OH kinase (PI3K) and AKT2, which play important roles in regulating various cellular functions, such as growth, proliferation, and protein synthesis . Piezo1 rylation . This, irylation . Howeverrylation . TogetheCyclin D, Cyclin E, and CDK2 [Cyclin A, CDK1, and CDK2, which are necessary for the S phase. Consequently, this negative regulation accelerates cell cycle progression [Retinoblastoma protein (Rb) is a tumor suppressor that represses the transcription of genes required for the transition from the G1 to S phase, such as and CDK2 . Rb bindand CDK2 . Rb phosgression .p21 as well as B-cell lymphoma-2 (Bcl-2)-associated X (Bax) [p53 mutation could be found in more than 50% of tumor patients, and in patients with wild-type p53, its aberrant regulation is frequently found. Hence, increasing p53 protein accumulation has attracted attention as a potential antitumor therapeutic strategy. Given that Piezo1 suppression could increase p53 transcription level and reduce tumor growth [Increased Rb phosphorylation has been found in various tumors, including cervical cancer, gastric cancer, and colorectal carcinoma, leading to a decrease in its inhibitory effect and subsequently promoting tumorigenesis ,76,77,78 X (Bax) ,79,80. Tr growth , targetiApoptosis is an autonomous programed cell death controlled by a series of genes, which involves a cascade of the activation of related factors, such as p53, Bcl-2 family, and caspase family . ApoptosBax and/or suppresses that of antiapoptotic factors, such as Bcl-2, thus blocking apoptosis [Evading apoptosis, even in the presence of DNA damage and genomic instability, is an important hallmark of cancer ,87. Impapoptosis . Piezo1 poptosis . Gao et poptosis . Howeverpoptosis . Similarpoptosis ,42. TherSolid tumor cells grow in a severe microenvironment with hypoxia, high matrix stiffness, and a lack of nutrients . To over2+ influx, as HIF-1α is a Ca2+-sensitive factor [VEGF, and ultimately accelerates tumor angiogenesis and progression [2+ influx, promoting the expression levels of MMP2 and MMP1, which in turn promote sprouting angiogenesis [Piezo1 can induce tumor angiogenesis by sensing mechanical forces and transmitting downstream signals. Recent studies have shown that Piezo1 can induce tumor angiogenesis in an HIF-1α-dependent manner. Piezo1, HIF-1α, and VEGF are highly expressed in tumor tissues . Piezo1 e factor . Furthergression . Additioogenesis . Taken tLocal invasion and distant metastasis are characteristics of malignant transformation. The complex process, in which tumor cells are subjected to various mechanical courses, such as compressive force, shear stress, and portal vein pressure, requires tumor cells to acquire a migration–invasion phenotype. This phenotype enables tumor cells to invade the basement membrane and propagate through the blood or lymph vessels . Indeed,Epithelial-mesenchymal transition (EMT) is a critical step in initiating the metastasis of transformed cells, in which epithelial cells lose their epithelial phenotypes, such as cell polarity and basement membrane detachment, while acquiring the characteristics of mesenchymal cells, including high migration and invasion, anti-apoptosis, and ECM degradation potentials. Recent studies showed that Piezo1 suppresses E-cadherin expression while promoting those of snail, E-cadherin, N-cadherin, and vimentin, which are typical markers of EMT, suggesting the role of Piezo1 in regulating EMT ,37. Hipp2+ influx mediated by Piezo1 and activating downstream Src signaling transduction. This leads to increased MMPs expression and, subsequently, ECM degradation [Furthermore, tumor cells secrete ECM remodeling enzymes, such as membrane type 1 MMP (MT1-MMP), MMP-1, and MMP-2. These enzymes promote the degradation of various proteins that compose ECM; thereby disrupting histological barrier for tumor cell invasion . Using tradation . Piezo1 radation ,111. Furradation . CollectTumor cells migrate forward in the ECM by changing their morphology . Piezo1 Piezo1 deletion variants in non-small cell lung cancer (NSCLC) lead to down-regulation of its expression, and, furthermore, the low level of Piezo1 is associated with poor prognosis in NSCLC patients [However, the implications of Piezo1 in lung cancer contradict the other reported tumors, such as colon cancer, gastric cancer, and hepatocellular carcinoma . Huang epatients . Moreovepatients ,39. McHupatients . HoweverIn recent years, the regulatory functions of mechanical forces on immunity and inflammation have received increasing attention . Despite+ memory T cells and CD8+ T cells accumulation, thereby promoting tumor progression [4+ memory T cells and CD8+ T cells, which was associated with poor prognosis in breast cancer patients [Tumors usually present in a state of immunosuppression and chronic inflammation, which are also essential hallmarks of cancer. Solid tumors contain many immune cells that recognize and destroy new tumor cells during cancer immunosurveillance ,121. Howgression . Piezo1 gression . Clinicapatients . These sAs a critical component of mechanical conduction, Piezo1 has been reported to control physiological and pathological processes, such as innate immunity, bone formation, and various cancers . Piezo1 2+ signals in tumor cells extensively affect its downstream pathways, developing new drugs targeting its major downstream pathways is also a potential alternative approach. Moreover, Yoda1 and Jedi2, which are highly specific activators of Piezo1, can induce Piezo1 overexpression that causes the downstream factors alteration, thereby contributing to the study of its downstream signaling pathways [However, the lack of specific inhibitors renders the clinical application of targeting Piezo1 . While rpathways ,127,128.Together, Piezo1 not only links up the physical changes surrounding the biological behaviors of tumor cells, but also has potential as a biomarker and antitumor therapeutic target.


Primary biliary cholangitis (PBC) is a chronic, progressive cholestatic liver disease in which the small intrahepatic bile ducts are destroyed by autoimmune reactions. Among autoimmune diseases, which are polygenic complex traits caused by the combined contribution of genetic and environmental factors, PBC exhibits the strongest involvement of genetic heritability in disease development. As at December 2022, genome-wide association studies (GWASs) and associated meta-analyses identified approximately 70 PBC susceptibility gene loci in various populations, including those of European and East Asian descent. However, the molecular mechanisms through which these susceptibility loci affect the pathogenesis of PBC are not fully understood. This study provides an overview of current data regarding the genetic factors of PBC as well as post-GWAS approaches to identifying primary functional variants and effector genes in disease-susceptibility loci. Possible mechanisms of these genetic factors in the development of PBC are also discussed, focusing on four major disease pathways identified by in silico gene set analyses, namely, (1) antigen presentation by human leukocyte antigens, (2) interleukin-12-related pathways, (3) cellular responses to tumor necrosis factor, and (4) B cell activation, maturation, and differentiation pathways. Primary biliary cholangitis (PBC) is a chronic cholestatic liver disease characterized by the destruction of the intrahepatic bile ducts, portal hypertension, the development of biliary cirrhosis, and ultimately, hepatic failure. Histologically, PBC is characterized by chronic, non-suppurative destructive cholangitis, ductopenia, interface hepatitis, and fibrosis ,2,3,4. A+ T cells, CD8+ T cells, and B cells) and innate immune responses , against biliary epithelial cells (BECs) .,45.MMEL1IL28B and ITPA genes are known to be strongly associated with response to treatment and side effects of pegylated interferon-α and ribavirin therapy for chronic hepatitis C [L2TFL1, FOXP4, TMEM65, OAS1, KANSL1, DPP9, RAVER1, and IFNAR2 were reported as susceptibility loci for severity to coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [CTSZ/NELFCD, which has not been reported as a PBC-susceptibility locus, was associated with the progression of PBC by comparing an advanced jaundice/liver failure group with a non-advanced, early-stage group of Japanese PBC patients [Generally, a GWAS aims to comprehensively search for genetic factors that contribute to the onset of a disease. However, by using the same methodology, it is also possible to comprehensively explore genetic factors associated with disease severity, response to treatment, and side effects. The atitis C ,47,48,49S-CoV-2) . As for patients .STAT4, ULK4, and KCNH5 were identified as novel Japanese PBC susceptibility loci in a study utilizing regional gene mapping, a method used to detect polygenic effects [GWAS data can also be used to examine the effects of multiple variants that are located within a chromosomal unit region but contribute only marginally to pathogenesis. For example,  effects   or expression regulatory regions, such as gene expression promoters and enhancers located in the intronic or intergenic region, and they do not alter the amino acid sequence of the relevant gene product. Primary functional variants usually control the expression of the mapped gene in which they are located. However, as DNA adopts a higher-dimension structure during transcription that associates with physically distant gene regions, primary functional variants often control the expression of other genes that can be more than several hundred kilobases apart . In thisTo date, we have identified 11 primary functional variants in 60 PBC susceptibility gene loci . In ordeTNFSF15, NFKB1, MANBA, FGFR1OP, and CCR6, respectively, were identified as primary functional variants that regulate the expression of each mapped gene [IKZF3, TIMMDC1, and POU2AF1, respectively, regulate the expression of ORMDL3, POGLUT1, and COLCA1/COLCA2, respectively [CD28, which was reported as a susceptibility locus for lymphocyte and eosinophil counts, multiple sclerosis, ulcerative colitis, celiac disease, rheumatoid arthritis, asthma, and PBC, has a shared disease-related primary functional variant  that regulates the CD28 alternative splicing that generates loss-of-function isoforms (CD28i and CD28∆ex2) [Using the allele knock-in version of a cell line constructed using genome-editing techniques such as CRISPR/Cas9, the endogenous effect of a primary functional variant can be evaluated without interference from other variants that show high LD. These analyses can also be used to elucidate the mechanism of pathogenesis. Among primary functional variants in each PBC susceptibility locus, approximately 40% were found to be associated with the expression of each mapped gene  .HLA class II locus showed a relatively high OR, similarly to other autoimmune diseases, all other PBC susceptibility gene loci outside HLA showed an OR of less than 2.0. However, by analyzing disease-susceptibility gene loci in detail by gene set analysis, it is possible to obtain a more comprehensive understanding of disease pathways. In such cases, gene set analysis using only mapped genes may be insufficient, because primary functional variants can sometimes be associated with the expression of genes other than the mapped genes. Gerussi et al. summarized the PBC susceptibility genes as of 2021 and classified the contribution of candidate causal genes, as well as mapped genes, into interleukin (IL)-12-mediated signaling, the cellular response to tumor necrosis factor (TNF), and the activation, maturation, and differentiation of B cells [Compared with monogenic diseases that are caused by mutations in a single gene within a patient’s DNA sequence, each GWAS lead variant in the disease-susceptibility gene locus will show a weak association with the risk of a polygenic complex disease. Although the  B cells . In addition, functional enrichment analysis based on protein–protein interactions using STRING  was founIL12A, IL12B, IL12RB2, STAT4, and TYK2 (shown as red nodes in p = 0.0034) [IL12B, NFKB1, IRF5, and IRF8 (shown as green nodes in p = 0.0472) [IRF5 was not a PBC susceptibility mapped gene, but an e-QTL gene. Therefore, “Regulation of interleukin-12 production (GO: 0032655)” in the Gene Ontology database did not reach a level of statistical significance when using PBC susceptibility genes alone.Interleukin-12 induces interferon (IFN)-γ production. By the enrichment analysis using STRING, the “Interleukin-12-mediated signaling pathway (GO: 0035722)” in the Gene Ontology database includes mapped PBC susceptibility genes and e-QTL genes such as  0.0034) . In addi 0.0472) . HoweverTNFSF15), promotes IFN-γ production through activated CD4+ T cells. This suggests that TL1A is positioned in the pathway, leading to IL-12 receptor (IL12R) stimulation and IFN-γ production [Death domain receptor 3 , the receptor for TNF-like cytokine 1A  [TNFSF8, LTBR, PSMA6, and SMPD3 were not PBC susceptibility mapped genes, but e-QTL genes. Therefore, “Cellular response to tumor necrosis factor (GO: 0071356)” in the Gene Ontology database did not reach a level of statistical significance when using PBC susceptibility genes alone.TNF-α itself plays a role in disrupting the barrier function of tight junctions in cholangiocytes without inducing major structural changes . Additio 0.0041) . However+ IgM+ B cells by engaging with the ligand TNFR superfamily member 8  [TNFR superfamily member 1A  is a receptor for TNF-α. Activated TNFRSF1A initiates caspase-8-mediated apoptosis via receptor-interacting serine/threonine–protein kinase 3 (RIPK3) . TNF supas CD30) . Althougas CD30) . This paas CD30) . The lymas CD30) . TNF-α aCD28, FCRL3, IKZF3, ID2, PPP2R3C, and NDFIP1 (shown as blue nodes in p = 0.0156). The “B cell activation (GO: 0042113)” in the Gene Ontology database includes genes such as IL7R, CXCR5, CCR6, IKZF3, FCRL1, and PLCL2 (shown as green nodes in p = 0.0383). Finally, the “B-lymphocyte cell line (BTO: 0001522)” in the Gene Ontology database includes genes such as HLA-DRB1, HLA-DQB1, WDFY4, SPIB, IL7R, and IL21R (shown as red nodes in p = 0.0139) [ID2, PPP2R3C, FCRL1, and PLCL2 were not PBC susceptibility mapped genes, but e-QTL genes. Therefore, “Regulation of B cell activation (GO: 0050864)” and “B cell activation (GO: 0042113)” in the Gene Ontology database did not reach a level of statistical significance when using PBC susceptibility genes alone.By the enrichment analysis using STRING, the “Regulation of B cell activation (GO: 0050864)” in the Gene Ontology database includes genes such as  0.0139) . However+ CXCR5+ cells, express more IL-7 receptor (IL7R)-α and secrete a higher proportion of IL-21, thereby inducing greater IgG production than CCR9− CXCR5− helper T cells [POU class 2 homeobox associating factor 1 (POU2AF1) and its cofactor, Spi-B transcription factor (SPIB), are essential molecules in the differentiation and maturation of B cells into plasma cells . IKAROS  T cells . Fc rece T cells ,78. ThesIFNG and CD40L [Based on gene ontology analyses, PBC susceptibility mapped genes and e-QTL genes contain a signal transduction system leading to the production of interferon-γ (IFN-γ), which is involved in immune and inflammatory reactions, and genes involved in B cell differentiation. These genes are assumed to play an important role in the development of PBC. In support of these data, an integrated pathway analysis of Japanese PBC susceptibility genes and analyses of PBC patient liver biopsy transcriptome data indicated that genes with characteristic expression patterns in PBC patients are primarily regulated by upstream factors such as nd CD40L .An in silico drug efficacy screening study using disease-susceptibility genes identified by GWAS has recently been reported for several diseases ,80,81. TIn this study, we summarized the PBC susceptibility genes reported as at December 2022 and discussed the latest post-GWAS approaches to identifying primary functional variants  and effector genes. The understanding of the mechanisms by which these genetic factors mediate the pathogenesis of PBC has been expanded through the use of in silico gene set analyses, e-QTL and STRING database searching, and studies examining knock-in versions of cell lines constructed using genome-editing techniques such as CRISPR/Cas9.p-values less than those of the GWAS lead variants located in the same LD blocks. Protein kinase C β (PRKCB) and chromosome 3q13.33  were identified as the susceptibility gene loci for PBC in the Japanese population using high-density association mapping [Recent advances in informatics and genetic statistics methodologies have led to new analytical methods that utilize GWAS data. For example, SNP imputation analysis can be used to infer the genotypes of variants not loaded on a GWAS array by combining with thousands of individual whole-genome sequencing data. High-density association mapping, which involves case-control association studies for all of the variants in the disease-susceptible gene regions, can detect variants with  mapping ,42. SNP imputation analysis can also be used for meta-analysis combining GWAS data that were analyzed by different GWAS arrays. In a recent genome-wide meta-analysis (meta-GWAS) of five European and two East Asian cohorts, SNP imputation analysis was performed ,45. By pWhole-genome sequencing analysis of individual PBC patients can aid in the identification of disease-causing rare variants that have not been identified by GWASs and subsequent SNP imputation analyses. At present, this analysis has succeeded in detecting rare pathological variants in many Mendelian diseases and some immune-related diseases, such as cold medicine-related Stevens–Johnson syndrome . Whole-gAdditionally, for more accurate post-GWAS analysis, protein–QTL analysis, an approach derived from a novel proteomics assay capable of measuring thousands of human protein analytes, enables accurate determination of correlations between variants and expression levels, without the uncertainty of e-QTL data that results from occasional non-correlations between mRNA and protein levels .In the near future, re-analysis of existing GWAS data may yield important findings that have thus far been overlooked in the pathogenesis of multifactorial diseases such as PBC.