The Analysis And Functional Studies Of Colorectal Cancer Related Gene TGFBR1 3 UTR MicroRNAs’ Binding Site

Objective: Colorectal cancer(CRC)is one of gastrointestinal cancer seriously harm to human health.Nationwide,there were nearly one million new cases each year,the mortality rate ranked third in developed countries.In China,the morbidity and mortality of CRC increased significantly with younger tendency,in recent years.The incidence of CRC was a complicate process in which many genes and factors take part in and go through a lot of stage developments.In order to improve the sensitivity and specificity of clinical treatment of CRC,clarify the mechanisms of colorectal cancer,looking for CRC markers,optimize the treatment programs of CRC has always been focused.Currently,the reports of single nucleotide polymorphisms of tumor-related genes(Single Nucleotide Polymorph Sims,SNPs)and a epigenetic markers: tiny RNA(microRNAs,miRNAs),which provide new perspectives for the molecular mechanisms of CRC development,and it has become a hotspots of cancer research.Many SNPs which have been found in the human genome,were located in the 3 ’untranslated region(3’UTR).There have been many studies confirmed that these SNPs located in the 3’UTR may affect mRNA stability through affect binding proteins and miRNA,thus participate in translational regulation of target gene,and then participate in a variety of biological functions such as cellgrowth,differentiation,apoptosis,development of tumors.microRNAs are a kind of small non-coding fragments of RNA,with a great power of gene regulation,regulate translation of target gene through complementary binding to mRNA’s 3’UTR of target gene.Currently,many studies have shown that miRNA target sites SNPs associated with the risk of colorectal cancer,but the relationship between the SNPs and 3’UTR regulation of target gene is still need to be resolved.The purpose of this study is looking for the miRNA target in the 3’UTR of colorectal cancer’s related gene TGFBR1,investigating the mechanism of between SNPs and miRNAs,analyze their relationships with colorectal cancer,and provide an important experimental basis for colorectal cancer’s molecular mechanism and regulation of gene expression.Methods : Our laboratory collected the clinic data from 371 cases of colorectal cancer patients and 246 cases of healthy people in Luzhou Han population,the colorectal cancer patients were confirmed by pathological diagnosis,after that,we were divided the patients and healthy people into two groups,such as treatment and control groups,annalyzed the clinic data through the "case-control" method.And detected the single nucleotide polymorphism loci(rs334348,rs334349)in TGFBR1 genes’ 3’UTR by PubMed(http://www.ncbi.nlm.nih.gov/pubmed/),Hapmap(http://hapmap.Ncbi.Nlm.Nih.gov/),Snpper(http://snpper.chip.org/),SHEsis-online(http://analysis.Bi o-x.cn/myAnalysis.php),and found they were associated with colorectal cancer.Then on these base,(1)we Screened the miRNA target sites in rs334348,rs334349 with RegRNA,RNAhybrid,miRanda,targetScan soft wares,and we found the candidate miRNA which were specific bind to rs334348,rs334349.(2)At first,we cultured the colorectal cancer cell lines,such as HT29,LOVO,SW620,HCT116,LS174 T.Then detected the target mutation in colorectal cancer cell lines by gene sequencing and OTUs.It was a pre-test for the next experiment.(3)Verified the candidate miRNAs’ impact on the expression of TGFBR1 and it’s transcription,instantaneous transfected target miRNA into colorectal cancer cell lines,and then take advantage of Real-time q PCR and Western-blot to detected the expression of TGFBR1’s mRNA and protein in cell lines.(4)Statistical analysis,we used SPSS17.0 statistical software for data analysis.The Real-time qPCR results of colorectal cancer cell lines were analyzed by Independent Samples T test,approximate T test would be used when data missed variance,we considered the Real-time qPCR results were statistically significant when p<0.05.Results:(1)In our experiment,we found that and the miRNA miR-133 b and miR-204-5p could specific bind to the single nucleotide polymorphism loci rs334348,rs334349 in TGFBR1 genes3’UTR and this bind have a highly degree of credibility.(2)We detected the colorectal cancer cell lines which has the target SNP from other kinds of colorectal cancer cell lines,and we found HT29 has the target SNP.(3)After we transfected the candidate miRNA into HT29 cell lines,the Real-time qPCR results showed that,Compared to miRNA mimics / inhibitors control group,the mRNA expression of HT29 cells which transfected by hsa-miR-133 b mimics and hsa-miR-204-5p mimics weresignificantly decreased,on the other hand the mRNA expression of HT29 cells which transfected by hsa-miR-133 b inhibitors and hsa-miR-204-5p inhibitors was significantly increased.(4)Western-blot results showed that: Compared to miRNA mimics / inhibitors control group,the expression levels of TGFBR1 protein decreased in two groups which were transfected by hsa-miR-133 b mimics and hsa-miR-204-5p mimics,but the expression levels of TGFBR1 protein increased in the other two groups which were transfected by hsa-miR-133 b inhibitors and hsa-miR-204-5p inhibitors,showed the remarkable interference effect.Conclusions: we found that the miRNA miR-133 b and miR-204-5p could specific bind to the single nucleotide polymorphism loci(rs334348,rs334349)through bio-informatics analysis,which in colorectal cancer related gene TGFBR1’s 3’UTR.And our experiment verified miR-204-5p,miR-133b could increase the TGFBR1 gene’s mRNA degradation and reduce the expression of TGFBR1.