Identification Of Colorectal Cancer-associated MicroRNA And The Relationship Between MicroRNA-20a And TRAIL Sensitivity Incolorectal Cancer

Objective: For patients with advanced colorectal cancer,tumor resection combined with the chemotherapy and biotherapy become the only strategy for treatment.However,these treatments are mostly ineffective because of the development of chemoresistance in cancer patients.TNF-related apoptosis-inducing ligand(TRAIL)has been considered as a potential therapeutic agent in cancer treatment due to its capability to induce apoptosis selectively in tumor cells,but not in normal cells.However,a significant proportion of cancer cells exhibit low sensitivity to TRAIL-induced apoptosis,which precludes its use in many cases.MicroRNAs(miRNAs)were found to be associated with the TRAIL sensitivity.Therefore,this study aimed to present insight into the role of miRNAs by screening the key miRNA in colorectal cancer-associated gene expression profile datasets and then to verify the role of the key miRNA in TRAIL sensitivity in colorectal cancer through a series of in vitro experiments.Methods: In this study,we downloaded the miRNA microarray dataset of GSE41655 and mRNA microarray dataset of GSE41657 from Gene Expression Omnibus(GEO)database.GSE41655 included 15 normal mucosae samples,39 low-grade adenomas samples,20 high-grade adenomas samples and 33 adenocarcinomas samples.GSE41657 contained 12 normal mucosae samples,21 low-grade adenomas samples,30 high-grade adenomas samples and 25 adenocarcinomas samples.After data preprocessing,the differentially expressed miRNAs(DEMs)and genes(DEGs)between adenocarcinomas group and normal control group were identified with the cutoff values of p-value < 0.05 and log|FC| > 0.58(mi RNA)or 0.4(m RNA).Then we predicted the target genes of DEMs and performed KEGG pathway enrichment analysis for the target genes.For DEGs,we predicted their regulatory miRNAs,and then performed GO and KEGG pathway enrichment analyses for these DEGs.The DEGs were then imported into STRING online database for protein-protein interaction(PPI)network analysis.Moreover,we predicted thetranscription factor(TF)of DEGs and constructed the TF-miRNA-PPI network.We also analyzed the target genes of DEMs and DEGs to screen the key miRNAs and mRNAs associated with colorectal cancer.Finally,based on the results of the analyses and the relevant literatures,miR-20 was considered to play important roles in colorectal cancer.We thus performed KEGG pathway enrichment analysis for the target genes of miR-20.In vitro verification experiment,we collected 30 pairs of colorectal cancer tissues and the adjacent non-tumor tissues were obtained from the patients who underwent tumor resection in The First Affiliated Hospital of Wenzhou Medical University from 06/2013 to 09/2015.Both tumor and noncancerous samples were confirmed histologically.In addition,80 serum samples from 40 colorectal cancer patients and 40 healthy controls were also collected from the same hospital before treatment.FHC which is a normal colorectal epithelial cell line and the colorectal cancer cell lines SW480,SW948,NCI-H508,HT-29 were originated from ATCC.Then,we carried out a series of experiments inlcuding qRT-PCR,transfection,luciferase reporter assay,cell viability assay,western blot analysis,measurement of mitochondrial membrane potential and statistical analysis to analyze the expression levels of miR-20 a in serum,tumor tissues and cell lines of patients with colorectal cancer,and the relationship between miR-20a-directed regulation of BID and the TRAIL sensitivity in colorectal cancer.Results: A total of 96 DEMs and 1959 DEGs were identified between colorectal cancer and control groups.After target gene prediction,miR-20a-5p(the 5' mature of miR-20a)was found to have the most target genes(2752).Pathway enrichment analysis of the target genes predicted from the top five miRNAs found that these taegets were significantly enriched in Pathways in cancer,Hippo signaling pathway and Fox O signaling pathway.The DEGs were significantly enriched in GO:0006412~translation,GO:0006091~generation of precursor metabolites and energy,and hsa03010:Ribosome.In the PPI network,GAPDH,HSP90AA1,EFTUD2,and RPS3 had higher degrees among the up-regulated DEGs,and UBC,CYCS and PTEN had higher degrees among the down-regulated DEGs.In the TF-miRNA-PPI network,PHLPP2 and PTEN were found to interact with miR-20 a.KEGG pathway enrichment analysis found that the target genes of miR-20-5p were enriched in hsa04144:Endocytosis,hsa04010:MAPK signaling pathway,hsa04350:TGF-beta signaling pathway and hsa04068:Fox O signaling pathway.Additionally,the BID gene that induced apoptosis during the TRAIL treatment was one oftargets of mi R-20.In vitro verification experiment,we demonstrated the significant upregulation of miR-20 a in colorectal cancer patients' serum,tumor tissues,and cell lines by quantitative RT-PCR analysis.Furthermore,we found the TRAIL-induced apoptosis was associated with the expression level of miR-20 a in colorectal cancer.The knockdown of miR-20 a by inhibitors increased the anti-tumor effect of TRAIL via caspase-8dependent pathway.The gene of BID,which is the pro-apoptotic member of Bcl-2 family,was found to be directly regulated by miR-20 a in SW480 cells.The knockdown of miR-20 a inhibited the translocation of tBID to the mitochondria,which induced the mitochondrial pathway of apoptosis.Interestingly,we found the knockdown of miR-20 a also reversed the resistance of TRAIL in established TRAIL-resistant SW480 cells by tBID-mitochondria pathway.Conclusion:(1)miR-20 a was predicted to have 2752 target genes(the most one).The target genes of miR-20 a,like RPS3,BID,PHLPP2 and PTEN had been suggested to be implicated with apoptosis.The target genes of miR-20 a were significantly enriched in MAPK signaling pathway,TGF-beta signaling pathway and FoxO signaling pathway,and these pathways were appsociated with apoptosis and tumorigenesis.The BID gene that induced apoptosis during the TRAIL treatment was one of targets of miR-20.(2)miR-20 a was overexpressed in colorectal cancer tissues,cell lines and serum,and may act as a tumor promoter in colorectal cancer.(3)anti-miR-20 a could regulate the apoptosis process induced by TRAIL treatment through targeting BID gene.The understanding of the miR-20 a might be a potential therapeutic target for colorectal cancer.