The Role And Its Mechanism Of MiRNA-29c And Its Genetic Variation In Development And Prognosis Of Gastric Cancer

Background:Gastric cancer (GC) is one of the most prevalent malignant gastrointestinal tumors in the world. Epidemiological study has showed that the number of GC patients is only next to lung cancer, breast cancer and colon cancer; approximately eight hundred thousand new GC cases occur worldwide, accounting more than 9 percent of the entire new cancer cases. Each year, more then six hundred patients died of gastric cancer, which also meant the second lethal cause of the cancer-related death. Most GC patients inhabit in Eastern Asia, Eastern Europe, Central Africa, and Southern Africa. As the world’s most populous nation, China occupy about 42%of the world’s new GC cases every year. In China, near three-quarters GC patients died after the first diagnose, and the 5-year survival rate is generally below 25%. With regard to the hazard, the recurrence and metastasis are normal causes of death in GC patients. Therefore, efficient identification of some powerful element of GC will be helpful in prevention and treatment as well as the low morbidity and low mortality.MicroRNA (miRNA) is a newly discovered small single-stranded non-coding RNA, which composed of 18 to 24 nucleotides. And miRNAs exist in eukaryotic organisms widely. The function of miRNA is always accomplished by binding to the 3’-untranslated region (3’-UTR) of its target mRNA. Usually, there are two kinds of results after the integration between miRNA and its target mRNA-the degradation or retardation. An increasing number of researches have clarified that miRNAs exist in all kinds of important biological processes, and the aberrant expression of miRNAs are associated with the occurrence and progression of diseases, including cancers. In our previous study, we have screened 5 patients’tumor and normal tissues for searching the significant differences of miRNAs expression. As a result, miR-1246, miR-29c, miR-148a, miR-107, and miR-103 revealed their variance between cancerous and non-cancerous tissues (P< 0.05). In detail, miR-1246, miR-107, and miR-103 were up-regulated in tumors, whereas miR-29c and miR-148a were down-regulated in normal tissues.DNA methylation was one of the important epigenetic regulation mechanisms. As an acquired character in mammalian genome, the DNA methylation mainly appeares in the promoter CpG islands of gene. And it is also involved in many important biological processes, such as up-growth, X chromosomal inactivation, DNA imprinting and so on. In tumorigenesis, the DNA methylation might lead to the aberrant expression of oncogene and suppressor genes. In recent researches, the CpG islands have also been indentified in some miRNAs promoter region, which controlled miRNAs’own transcription.Single nucleotide polymorphisms (SNPs) are one kind of genetic polymorphism, which could acquire as a hereditary factor from parents to children. The SNPs were considered as the factors of some diseases according to Collins idea "common disease, common variant". So, the SNPs were also a key factor influencing susceptibility to malignant neoplasm. In recent, the SNPs on precursor miRNAs (pre-miRNAs) and primary miRNAs (pri-miRNAs) have become the focus of studies. As reported, the SNPs on pre-and pri-miRNA might change the expression or the function of mature miRNA, which might finally lead to the susceptibility to cancers. Therefore, searching for the functional SNPs might provide the powerful theory basis for the tumor pathogenesis. Meanwhile, SNPs might be also treated as the important biomarker, using in individualized prevention, intervention, diagnosis and treatment for cancers. Objective:Based on our previous miRNA expression profile on GC, we selected the miR-29c, miR-148a, and miR-107 as the candidated miRNAs and aimed to identify the complex regulation mechanism between the miR-29c, miR-148a, and miR-107, which could explore the potential molecular mechanism of miR-29c in the development and prognosis of GC. Meanwhile, we investigated the associations between the promoter genetic variant of miR-29c and risk and survival of GC in Chinese populations. This study will provide the evidence that the epigenetics involved in the development, prognosis and survival of GC and the important references in GC prevention, intervention, and therapy.Methods and Results:Based on our previous miRNA expression profile, we adopted the MSP, real-time PCR, Western blot, Flow Cytometry, Transwell, Scratch, CCK8, Plate Clone Formation, etc. were adopted to identify whether the miR-29c participate in the overexpression of miR-107 and decrease of miR-148a in GC tissues. The results showed that in gastric cancer cell lines, miR-29c positive regulated the expression of miR-148a by targeting the DNMT-3B for triggering the demethylation status of miR-148a CpG islands. Meanwhile, the miR-29c could bind to XPO5 mRNA 3’UTR, reduce the expression of XP05 and control the XPO5 activity to transport pre-miRNAs from nucleus to cytoplasm. In other words, the miR-29c negative regulated miR-107 by targeting XPO5. We also found the function of miR-29c could affect the invasion and migrate of GC, and this function is related to miR-148a. In addition, we identified one functional SNP rs2724377 existing in miR-29c promoter region, which could specific bind to CREB factor and influence the expression of miR-29c. The luciferase results reveal that individuals with CT/CC genotypes possessing a higher expression of miR-29c than those with TT genotype. The following two-stage case-control study also confirms that this SNP was significantly associated with the genetic susceptibility to GC. The individuals carrying CT/CC genotypes had a decreased risk of GC compared with those with the TT genotyoe (adjusted OR= 0.82,95%CI= 0.73-0.93). However, there is no evidence that this SNP is associated with the GC survival.ConclusionOur study found that different alleles of functional MIR29C SNP rs2724377 have different affinities to CREB transcriptional factor, which could ultimately influence the expression of miR-29c. The miR-29c may regulate the miRNAs without CpG islands by targeting XPO53’UTR, and it may also regulate miRNAs with CpG islands by targeting DNMT-3B. These mechanisms of miR-29c could affect several importance biological pathways and participate in the proliferation, invasion and migration of GC. These results suggested that the miR-29c participate in the epigenetic regulation of GC development, and the promoter genetic variant rs2724377 contribute to susceptibility of GC, which may be a valuable potential biomarker in GC prevention, intervention, and clinical therapy.