Molecular Mechanism Of ESRP2 Regulating CAMK1 Invasion And Metastasis In Renal Cancer Cells Through Alternative Splicing Of IRF3

Background:With the continuous development of transcriptome sequencing technology,people can study gene expression and function at the overall level,and explore specific biological processes and molecular mechanisms in the occurrence and development of diseases.Although preliminary results have been achieved in the screening and prevention of renal cancer,there is still a lack of accurate and reliable biomarkers for prediction,diagnosis,treatment and prognosis of renal cancer [1].Therefore,exploring the expression and regulation of effective molecular targets in renal cancer and the related mechanisms that affect the metastasis of renal cancer are the key scientific issues to analyze the pathogenesis of renal cancer.The purpose of this study is to find an effective molecular target of renal cancer and to obtain the whole transcriptome expression profile data regulated by this target in renal cancer,so as to reveal the molecular mechanism of the development and metastasis of renal cancer,and to provide a powerful reference for solving the above scientific problems..The research content of this topic includes three parts: 1)Construction of ESRP2-overexpressing lentiviral vector and empty(control treatment)transfected renal cancer cells,and detection of cytological function changes to understand the role of ESRP2 in renal cancer cells 2)Obtain the whole transcriptome data of ESRP2 overexpression and control renal cancer cells,screen the differential genes and alternative splicing events regulated by ESRP2 through comparative analysis,and analyze the differential expression and splicing events of the transcriptome.GO and KEGG functional clustering of genes with variable splicing changes to further investigate biological processes and signaling pathways related to the occurrence and development of renal cancer to establish gene-pathway-disease associations;3)Explore the influence of ESRP2 through the transcription factor IRF3 The transcription and splicing of the renal cancer-related gene CAMK1 regulates the expression of CAMK1 to reveal the role and mechanism of ESRP2 in renal cancer metastasis through the regulation of gene expression by transcription factors.Method:1.The 786-0 cell was transfected with overexpressing ESRP2 lentivirus vector to construct 786-0 cell model with overexpressing ESRP2 gene.The influence of ESRP2 on proliferation,migration and apoptosis of 786-0 renal cell carcinoma cells were detected by CCK8,Transwell and flow cytometry.2.Full transcriptional sequencing was performed on 786-0 cell samples overexpressing ESRP2 or empty.Relevant different expressed genes and alternative splicing events were screened and analyzed,followed by go and KEGG analysis.3.Download and analyze the different expressed genes and alternative splicing events after ESRP2 overexpression from TCGA KIRC,conduct Venn analysis with the above RNA-seqdata,and analyze the go and KEGG functions of overlapping genes.Combined with JASPAR2020 database,predict the TF that may be combined with ESRP2 and analyze the combined motif information,and construct the ESRP2-TF-DEG regulation network.4.The overlapping different expressed genes and alternative splicing events between TCGA data and RNA-seqdata were verified by qRT-PCR.Meanwhile,the protein expression levels of IRF3 and CAMK1 after ESRP2 overexpression were detected by Western blot.5.The experiment was divided into NC,ESRP2 and ESRP2 + Si-IRF3 groups.In addition,the Protein expression level of CAMK1 was detected by Western blot and the migration ability of 786-0 cells was detected by Transwell.6.The nude mouse tumor model was established by subcutaneous injection of786-0 cells,and the effects of ESRP2 on the proliferation and progression of renal cell carcinoma cells were analyzed in vivo.In addition,the expression levels of IRF3 and CAMK1 in tumor tissues were detected by Western blot.Results:1.786-0 cells were transfected with ESRP2-overexpressing lentiviral vector and empty vector,respectively,to construct a 786-0 cell model overexpressing ESRP2 gene and empty vector,and the proliferation,apoptosis,invasion,metastasis,and proliferation of two groups of cells were affected.The detection of inflammation showed that ESRP2 overexpression could significantly promote the proliferation,invasion and metastasis of 786-0 cells,inhibit cell apoptosis,and the expressions of inflammatory factors TNF-α and IL-1β were significantly increased(p<0.05);2.Whole transcriptomic analysis of ESRP2-overexpressing and empty 786-0 cell samples identified 1180 differentially expressed genes,of which 372 were up-regulated and 808 were down-regulated.GO function analysis of the up-regulated genes revealed positive regulation of cell migration,positive regulation of smooth muscle cell proliferation,immune response,negative regulation of extracellular apoptosis in the absence of ligands,positive regulation of T cell proliferation,cellular lipopolysaccharide response and inflammation Signaling pathways such as reaction were enriched.Among them,the functions of SNAI2,CD274 and DLC1 genes in the pathways were reported in renal cancer and cancer-related literature.At the same time,GO function analysis of down-regulated genes found that cell adhesion-related pathways were enriched.,among which DDIT4,IGFBP3,CHST1,CAMK1,FDFT1 and LDLR have been reported to be associated with the occurrence of renal cancer and cancer;at the same time,transcriptome data identified a total of 1340 differentially alternative splicing events,and the level of alternative splicing after ESRP2 overexpression GO analysis of significantly altered genes identified RNA splicing,membrane reorganization,cellular component movement,gene expression,response to folic acid,positive regulation of myocyte differentiation,myocyte differentiation,endocytosis,DNA catabolism and nucleic acid endocytosis Signaling pathways such as excision and nuclear m RNA splicing through the spliceosome were enriched,among which INF2,CD44,CDKN3,ANXA6 and IRF3 deserve special attention.3.Download and analyze the transcriptome data of differentially expressed genes and differentially alternative splicing events of the 20 samples with the highest ESRP2 expression and the 20 samples with the lowest ESRP2 expression from TCGA KIRC(Kidney renal clear cell carcinoma),JASPAR2020 database prediction The TF that may be bound to ESRP2 and the analysis of the combined motif information found that the TCGA data and the RNA-seqdata had overlapping differentially expressed genes and alternative splicing events.A total of 286 identical differentially expressed genes were identified in the two sets of data,of which 47 were Up-regulated expression,239 down-regulated expressions,combined with the genes screened in the transcriptome data,further proved that CHST1,DDIT4,and IGFBP3 are related to the occurrence and development of renal cancer.The GO and KEGG function analysis of overlapping genes found that cell adhesion,internal At the same time,217 identical differential alternative splicing events were identified from TCGA data and RNA-seqdata,and the changes of alternative splicing events of ANXA6,INF2 and IRF3 in the two sets of data Consistently,alternative splicing abnormalities in these genes may affect the pathogenesis of renal cancer.4.There were overlapping differentially expressed genes and alternative splicing events between TCGA data and RNA SEQ data,which was verified by qRT-PCR.We found that the alternative splicing levels of IRF3、INF2、CD44、CDKN3、ANXA6、SCRIB 、 TCF3 genes were significantly affected by ESRP2.In addition,it can increase the expression of FDFT1、LDLR、DDIT4、CHST1,and reduce the expression of RND3、CAMK1.5.The study found that the protein expression of IRF3 increased and CAMK1 expression decreased after ESRP2 overexpression.Meanwhile,CAMK1 protein expression increased significantly after si-IRF3.Above these show that ESRP2 was positively correlated with IRF3 and positively correlated with camk1.In addition,ten expression of IRF3 was positively correlated with CAMK1.6.Transwell was used to detect the migration and invasion ability of renal cancer cells.we found that ESRP2 and IRF3 could promote the invasion and metastasis of renal cancer cells.However,CAMK1 inhibited the metastasis of renal cancer cells.7.ESRP2 can promote the growth of renal cell carcinoma and significantly promote the expression of IRF3 and inhibit the expression of camk1 in vivo by subcutaneous tumorigenesis.Combined with the regulatory relationship between ESRP2,IRF3 and CAMK1 in vivo and in vitro and their effects on the migration and invasion of renal cell carcinoma cells,we found that ESRP2 can promote the invasion and metastasis of renal cell carcinoma through IRF3 /CAMK1 axis.Conclusion:In this study,the differentially expressed genes and alternative splicing events were screened out by whole transcriptome sequencing of ESRP2-overexpressed786-0 cells,and the functional pathways enriched by the genes were analyzed,and then the key genes in the disease-related pathways of ESRP2 were constructed,Furthermore,according to the RNA binding function of ESRP2,the transcription factor IRF3 interacting with it was predicted,and the target gene CAMK1 regulated by IRF3 at the transcriptional level was found out.The regulatory relationship among ESRP2,IRF3 and CAMK1 and their effects on the migration and invasion of renal cell carcinoma cells were verified in vivo and in vitro.It was found that ESRP2 can promote the invasion and metastasis of renal cell carcinoma through IRF3 / CAM1 axis,which provides a reliable molecular target for the further study,diagnosis and treatment of molecular mechanism of renal cell carcinoma.