Screening And Identification Of Key LncRNAs Associate With Colorectal Cancer And The Functionary Mechanism Of MAMDC2-AS1 In Carcinogenesis And Development Of Colorectal Cancer

Objective:The morbidity of colorectal cancer has been increasing last decade,and colorectal cancer has become a severe public health problem.Though the methods of treating colorectal cancer have improved greatly these years,the prognosis of the patients who suffered advantaged colorectal are still unsatisfied.Therefore,it is important and meaningful to look for some biomarkers which could be used for early diagnosis or target therapy.Long non-coding RNA(lncRNA)plays an important role in the regulation of gene expression,its abnormal expression associates with the carcinogenesis and developments of various cancers,and thus lncRNA has potential value on early diagnosis and target therapy for some cancers.However,the functions and mechanisms of lncRNA in colorectal cancer still require in-depth research.So,our study will analysis the RNA-seq data in TCGA data base,to identify and screen some Key lncRNAs associate with carcinogenesis of colorectal cancer,the final aim is to explore some meaningful lncRNAs which have potential value on diagnosis and prognosis of colorectal cancer patients.Methods:Downloaded RNA-seq data from TCGA data base,and analyzed the differential expression genes,which included lncRNAs and coding RNA(mRNA).Firstly,we analyzed the GO function enrichment,KEGG pathway enrichment and PPI networks of the differential expression genes;Secondly,we screened the key lncRNAs which associate with the prognosis of colorectal cancer.After gaining the key lncRNAs,miRNA and mRNA from the bioinformatic analysis above,we validated its expression through the operation specimens of colorectal cancer patients,the expression level of the lncRNA,miRNA and mRNA of the 21 paired simples contained normal colorectal mucosa and colorectal cancer tissues were tested by RT-qPCR,and then we synthesized the siRNA and expression plasmid of the lncRNA,and transfected them into the colorectal cancer cell line HCT116 cell,and then transwell assay was used for observing the changes of migration and invasion of HCT116 induced by knocking down or increasing the expression of the IncRNA,meanwhile,the proportion of cell apoptosis of HCT116 cells was examined by flow cytometry.Results:We analyzed the RNA-seq data from 453 specimens of colorectal cance,and found that 119 lncRNAs expressed differently,there were co-expression relations between lncRNAs and coding RNAs.From the results of GO function enrichment and KEEGG pathway enrichment analysis,we found that the differential expression IncRNAs had complicated functions,and involved in various biological process of cells,most of them associated with the carcinogenesis and development of colorectal cancer.What was more,the expression level of 16 differential expression IncRNAs refered to prognosis of the colorectal cancers,3 of them:BVES-AS1,HAND2-AS1 and MNAMDC2-AS1 were the hub lunRNAs,because they associated with the prognosis of colorectal cancers,but also co-expressed with miRNA and mRNA strongly,the interaction networks of lncRNAs,miRNAs and mRNAs prompted that the "MAMDC2-AS1-miR-197-3p-MAPK4" might be the key axis involved in carcinogenesis of colorectal cancer,because MAMDC2-AS1 was over expression in colorectal cancer,and miR-197-3p acted as antioncogene but MAPK4 was considered to be oncogene.We validated the expression of MAMDC2-AS1,miR-197-3p and MAPK4 by RT-qPCR in the 21 pared clinical specimens,the results showed that MAMDC2-AS1 and MAPK4 were over expression,but miR-197-3p was low expression,the expression level among them excited significant correlations.Next,we synthetized MAMDC2-AS1 siRNA and expression plasmid,and then transfected them into colorectal cell line HCT16 cells respectively,the results of transwell showed that the deletion of MAMDC2-AS1 weakened the migration and invasion of HCT116 cells,and increased the proportion of apoptosis of HCT116 cells;by contrary,overexpression of MAMDC2-AS1 strengthened the migration and invasion of HCT116 cells,and decreased the proportion of apoptosis of HCT116 cells.Conclusions:The differential expression lncRNAs in colorectal cancer associate with the carcinogenesis and development of colorectal cancer,they involve in various biological function and mechanism of cancer cells,the lncRNA MAMDC2-AS1 gained from the bioinformatic analysis has potential value on diagnosis,prognosis and target therapy of colorectal cancer patients.