Association Study Between Polymorphisms In MiR-196a Genes And Its Binding Site And Esophageal Squamous Cell Carcinoma

Esophageal cancer is one of the most common malignancies and occurs mainly in Asiaand Africa, and its incidence has obvious differences between ethnic groups. Esophagealcancer is divided into esophageal adenocarcinoma and esophageal squamous cell carcinoma,and esophageal squamous cell carcinoma is the main type in china. China is a high incidencearea of esophageal cancer, however, the incidence of disease type and morbidity are diverse indifferent areas. The high-risk areas of esophageal cancer in China are mainly distributed inHenan, Hebei, Shanxi, Shanxi,Sichuan and other eastern regions, which in Henan provinceare mainly composed of familial transmissibility, whereas in the southwest region mainlyshows sporadic feature. Chongqing is one of the esophageal cancer high-risk area, which islocated in southwest of Sichuan basin. According to the Report in Chongqing, esophagealcancer mortality accounted for13.37%. At present, esophageal cancer is a highly lethaldisease, the5-year survival rate is only18-30%.Esophageal cancer is result from the interaction between gene and gene, or gene andenvironment. A large number of studies have showed that the genetic polymorphism ofpopulation can affect the function of related genes and play an important role in canersusceptibility. In recent years, the human genome project has made great achievements. Onthis basis, the association study between single nucleotide polymorphism and complex diseasehad made tremendous progress, including the association study of Candidate genes andGenome-Wide Association Study. Association study has also become hot in the post-genomicera. Therefore, based on the sporadic samples in southwest China and corresponding clinicaldates to explore the interaction of esophageal cancer genetic susceptibility and environmentalfactors, and it is helpful to understand the etiology and pathogenesis of sporadic esophagealcancer. In addition, it is very important for improving the prevention and diagnosis esophageal cancer by searching for esophageal cancer susceptibility genetic markers.MicroRNA (miRNAs) are a class of naturally occurring small non-coding RNAs onlyabout22-25nucleotides that control gene expression by targeting mRNAs for translationalrepression or cleavage. Recent studies suggests that miRNAs can regulate important tumorrelated genes. Therefore, it may play a key role in tumorigenesis and provides new basis fortumor diagnosis and treatment. There are three steps in miRNA processing. Firstly, miRNAsare encoded in the genome and are transcribed by RNA polymerase II (polII) as longprecursor transcripts, which are known as primary miRNAs (pri-miRNAs). And then theRNase-III enzyme Drosha and Dicer performs a second cleavage to generate miRNAs. Amember of the Argonaute (Ago) protein family serves as the direct interaction partner of themiRNA within the RISC. Mature miRNA can bind to the3’untranslated region (3’UTR) of itstarget gene mRNA result in either mRNA degradation or protein translation inhibition.miRNAs regulate the expression of target genes by binding the3’UTR using its seed regionwhich is conserve in mammalian. MiRNAs can regulate many tumor related genes and thusaffect the tumorigenesis and development of a variety of tumors. Several miRNA have beenreported in the development of esophageal cancer.With the completion of Human Genome Project and the1000genome Project, the studyof single nucleotide polymorphism promotes the development of the genetic susceptibility oncommon diseases such as tumor, which makes it become one of the hotspot in diseasegenetics research. So far, a large number of tumor related genetic loci has been found. Thereare many association study between miRNA related SNPs and complex disease because of theimportance of miRNA. Then we investigate the genetic susceptibility of esophageal cancerusing the SNPs located in miRNA genes or miRNA binding sites, and study the underlinemechanism of the positive SNPs.The major results are presented below:1. The association study between miRNA binding sites SNPs and esophageal squamouscell carcinoma.We choose seven SNPs by bioinformatic tools considering the genetic and environmentalfactors. We genotyped these SNPs by SNapShot assay in large number of samples.Association study found that SNP rs6573was association with esophageal cancer and the Callele increased the risk of ESCC. 2. SNP rs6573associated with ESCC metastasis by affecting the regulation of miR-196ain RAP1A.SNP is located in the potential miR-196a binding site by bioinformatic tools. SNP rs6573affected the activity of luciferase by luciferase reporter assay, and found that overexpressionof RAP1A existed in most ESCC tissues and the expression of RAP1A was higher in patientswith lymph node metastasis than those without. This SNP C allele may prevent miR-196afrom binding to RAP1A mRNA, resulting in altered regulation of RAP1A expression andincreased ESCC risk and metastasis. RAP1A could promote esophageal cancer cell migrationand invasion through MMP2. Our research suggested a novel potential marker for predictingthe susceptibility and malignancy of ESCC.3.The association study between SNP in pre-miR-196a and esophageal squamous cellcarcinoma.miR-196a is involved in many cancer genesis and is a marker in EA. But there is littleresearch in ESCC. A SNP rs11614913is located in the pre-miR-196a region by UCSCdatabase. We genotyped this SNP in458esophageal cancer cases and489controls and foundthat CC genotype increased the risk of ESCC.The expression of miR-196a with C allele was higher than that with T allele by RT-PCR.Therefore the expression of pre-miR-196a is no difference. By RNA-protein pull-down andMS assay we found that the C allele could bind to the NonO protein but not the T allele.These results indicated that the C allele could increase the miR-196a expression by binding tothe NonO protein and increased the risk of ESCC.In conclusion, this study found two SNPs were association with the ESCC in southwestChina. And this study provide a new way for the studying of genetic susceptibility ofesophageal cancer and a new potential marker for early diagnosis and personal therapy of thisdisease.