Based On TCGA Database Construction Of LncRNA Related CeRNA Regulatory Network In Cervical Squamous Cell Carcinoma Radiotherapy Resistance And MiR-204 Preliminary Verification Analysis

Objective: Radiotherapy resistance is the main reason leading to the failure of concurrent chemoradiotherapy for cervical cancer;however its molecular mechanism remains unclear.In recent years,the research of lncRNA ceRNA(lncRNA-miRNA-mRNA)regulation mechanism has become a hotspot in cancer,but there has been few reports on of radiotherapy resistance in cervical squamous cell carcinoma(CSCC).In this study,based on the TCGA database and to construct lncRNA related ceRNA regulatory network in CSCC radiotherapy resistance by using bioinformatics technology.We took the target gene miR-204 as the main object of study to verification and function analysis in CSCC tissues and cells with different radiotherapy sensitivities.Method: 1.According to the inclusion study criteria,RNAseq data and corresponding clinical information of 67 radiotherapy resistant samples and 23 radiotherapy sensitive samples in CSCC were screened from the TCGA.Using edger package analysis selected differentially expressed genes with | log FC |>2.0 and P <0.05.Then we use the Ensenbl,miRcode,Targetscan and Tarbase databases to implement comprehensive analysis and construct a ceRNA regulatory network by Cytoscape.In addition,we also explored the relationship between allRNAs in the ceRNA network and prognosis,the relationship between DElncRNA and clinical features,and the relationship between DElncRNA-DEmRNA and the enrichment analysis of DEmRNA in GO and KEGG in order to screen more reliable molecular targets,.2.We collected 32 cases of CSCC tissues with different radiotherapy sensitivities,used q RT-PCR to detect the expression of miR-204 and analyzed the significance of its expression level in different clinical factors.In addition,Si Ha(radiotherapy resistance)and Me180(radiotherapy sensitive)CSCC cell lines were selected for the detection of miR-204 expression level,and then the miR-204 stable transfected strain was constructed using a lentiviral vector,and miR-was explored through in vitro proliferation and apoptosis experiments.Whether 204 can regulate the sensitivity of radiotherapy is a preliminary study.Results: 1.A total of 565 mRNAs,237 lncRNAs and 16 miRNAs were identified based onRNA-seq expression profile data of different radiotherapy sensitivities in the TCGA database,of which 17 lncRNAs,3 miRNAs and 15 mRNAs were involved in the construction of ceRNA regulatory network.LINC00461,MIAT,ERVMER61-1,TLR8-AS1,FAM181A-AS1,and PSORS1C3 are associated with clinicopathological features and DEmRNA.The enrichment analysis of GO and KEGG suggested that the differential mRNA was significantly enriched in the three components of the membrane component,plasma membrane and rough endoplasmic reticulum in cell composition and involved in c AMP signaling pathway,Neuroactive ligand-receptor interaction and apoptosis-variety,ascorbic acid and alginate metabolism,retinol metabolism.2.q RT-PCR results showed that the expression of miR-204 in the radiotherapy resistant group tissues and cell lines was significantly lower than that in the radiotherapy sensitive group(P<0.05),and the low expression of miR-204 was related to the tumor size(P<0.05);However,PCR results showed that compared with me180 cells,miR-204 target gene TRPM3 showed low expression in Si Ha cells,which was inconsistent with our previous prediction results.In vitro experiments show that transfection of miR-204 in Si Ha cells combined with radiotherapy can increase the level of apoptosis and inhibit the proliferation of cell lines,thereby increasing the sensitivity of cells to radiotherapy.Conclusion: 1.This study was the first to successfully construct a ceRNA regulatory network related to radiotherapy resistance of cervical squamous cell carcinoma,in which we speculated that LINC00461,MIAT,ERVMER61-1 /miR-204 / LY6G6 D,TRPM3,LY6G6F;PSORS1C3,TLR8-AS1,FAM181A-AS1 / miR-204 / LRRC55 axis were likely to be a mechanism of radiotherapy resistance of CSCC,especially MIAT-miR-204-TRPM3 axis but further experiments are needed to confirm.2.The expression level of miR-204 in cervical squamous cell carcinoma-resistant tissues and cells is significantly lower than that of radiotherapy-sensitive tissues and cells,and the low expression of miR-204 is related to the larger tumor volume of cervical squamous cell carcinoma and is related to the pre-ceRNA The network results are consistent,which initially suggests the value of constructing a ceRNA regulatory network to screen candidate target molecules;however,the PCR results of TRPM3 have an opposite trend to the predicted results,indicating that in CSCC radiotherapy resistance,this gene is likely not a downstream target gene of miR-204,The specific mechanism still needs further exploration.3.The successful construction of miR-204 overexpression stable transfectants in Si Ha and Me180 cell lines,and the successful transfection of miR-204 overexpressed Si Ha confirmed that miR-204 can regulate the sensitivity of CSCC radiotherapy and continue to verify miR-204 Lay the foundation for the mechanism research of regulating the sensitivity of CSCC radiotherapy.