LncRNA Expression Features In CRC Tissues And Mechanism Of GAPLINC In Carcinogenesis And Development Of CRC

Abjective:The incidence of colorectal cancer(CRC)had increased consideratly these years,and the survival time was improved by the development of the integrate therapies,which depended on the progress of surgery mainly.While,the outcomes of some patients suffering advanced carcinoma were still unsatisfied.Therefore,it was important and meaningful to explore some biomarks of early diagnoses and targeted therapy for CRC patients.Long non-coding RNA(lncRNA)was the member of non-coding RNA family,and played an important role in some deseases,especially for the carcinogenesis and development of some tumors.There were some prelimary studies showed that the gastric adenocarcinoma predictive long intergenic non-coding RNA(GAPLINC)was over expressed in gastriointestinal cancer and performed as a carcinogenesis in the progress of tumor’s proliferation and invasion.Therefore,we would study the IncRNA expression profiles and features in CRC tissues and their potential value associtated with prognosis assessment using RNA-seq data from public data base,and further study the role of GAPLINC in the carcinogenesis and development of CRC,and discuss its potential value on the diagnose and target therapy of CRC patients.Methods:Analyzed the differential expression,functional and pathway enrichment,intergation network and lncRNA-mRNA co-expression of IncRNA using RNA-seq data from TCGA;meanwhile,analyzed prognosis changes of CRC patients induced by the differential expression level of lncRNA.To detect the expressed difference of lncRNA preliminarily,lncRNA PCR microarray was performed on the 3 clinical specimens of CRC tissues and their paired non-cancerous tissues,and established the targeted lncRNA for the further study,which was GAPLINC.The real time quantitive PCR(RT-qPCR)was performed on the 21 specimens to confirm the expressed difference between the carcinour tissue and non-cancerous tissue.Cultured the CRC cell line HCT116,studied the functions of GAPLINC by synthesizing and transfecting GAPLINC siRNA and expressed plasmid,to confirm the changes of the apoptosis,migration and invitation of HCT116 cells during GAPLINC being over expressed or being knocked down;The cell cytoplasm/nucleus isolation was carried on,to confirm the location of GAPLINC;Predicted the micorRNAs(miRNAs)bound to GAPLINC by bioinformatic software,and established the targeted miRNA miR-34a by RNA pull-down assays and the construction of reporter plasmids and luciferase assays,which could confirm the sites of GAPLINC 3,untranslated region(3’UTR)bind to miR-34a.Tested the expressed relations of GAPLINC and miR-34a by RT-qPCR,and then constructed GAPLINC plasmid and miR-34a mimics transfected HCT116 cells together,studied the apoptosis,migration and invasion of the CRC cells;Predicted the targeted gene of miR-34a which involved in the carcinogenesis and development of CRC by bioinformatic principle,and established the targeted gene mesenchymal epithelia transition factor(c-MET).For further studying the relations among GAPLINC,miR-34a and c-MET,the Western blot(WB)was performed on the HCT116 cells which were transfected GAPLINC siRNA or miR-34a mimics,and RT-qPCR was used for testing the expression of c-MET mRNA,miR-34a and GAPLINC in 21 pair clinical specimens.Results:Analyzed the TCGA RNA-seq data from 453 specimens of CRC patients,it could be found that there were 119 lncRNAs which expressed differentially;IncRNA and coding gene had the relation of co-expression;IncRNA played various roles in biological features of cells;there existed lncRNA-miRNA-mRNA regulatory network;16 of these IncRNAs had potential value on the prognosis of CRC patients.From the results of IncRNA arrays,it could be found that there existed great difference in the expression of IncRNA;the expressed level of IncRNA GAPLINC was stable and high in CRC tissues,and the expressed difference between cancerous tissue and paired-no cancer tissue in 21 clinical specimens was significant from the results of RT-qPCR.Cultured the HCT116 cells;constructed GAPLINC small interfered RNA(siRNA)and expressed plasmid pcDAN3.1,and then transfected into HCT116 cells,the results of transwell assays showed that up regulated the expression of GAPLINC could promote the migration and invasion,meanwhile inhibit apoptosis of HCT cells;contrarily,knocked down the expression of GAPLINC reduced the migration and invasion of HCT116 cells,increased the apoptosis of HCT116 cells.GAPLINC was mainly located in cytoplasm,based on the mechanism of competitive endogenous RNA(ceRNA)and the principle of bioinformatics,we predicted several miRNAs which were regulated by GAPLINC,RNA pull-down assays,reporter plasmids and luciferase assays established the targeted miRNA miR-34a,RT-qPCR carried on the 21 clinical specimens had approved that there exited significant correction in the expression of GAPINC and miR-34a;constructed the miR-34a mimics and transfected into HCT116 cells with GAPLINC plasmids,it could be found that miR-34a could inhabit the migration and invitation of HCT116 cells,and counteracted some function of GAPLINC.The target gene of miR-34a was also predicted by bioinformatic principles,and established c-MET as the target gene.The results of WB and RT-qPCR showed the three were negative corrections between miR-34a and c-MET,while there were positive corrections in GPLINC and c-MET.Conclusions:IncRNA expressed differently in CRC tissues,and played an important role in biological feature of CRC cells,had potential value in diagnosis,targeted therapy and prognosis of CRC patients.GAPLINC played an important role of regulation in the carcinogenesis and development of CRC,which promoted the migration and invasion of CRC cells,but inhibited their apoptosis.GAPLINC acted as oncogene,and its mechanism depended on binding miR-34a,and increasing the expression of c-MET,thus effected the carcinogenesis and development of CRC cells.GAPLINC had potential value on the diagnoses and target therapy of CRC.