The Regulatory Mechanism Of MiR-181a In Gastric Cancer Cell SGC-7901

BackgroundmiRNAs are a family of small (~21nucleotides [nt]), endogenous, important regulatorynoncoding RNAs, they participate in almost all of the major life activities of genetranscription and post-transcriptional processing, cell differentiation and ontogenesis,heredity and epigenetic inheritance. Mature miRNAs are transcribed as mono-orpolycistronic, long, primary precursor transcripts (pri-miRNAs), processed by the nuclearRNase III-like enzyme Drosha and Dicer, transported by Exportin-5, are cleaved into80-nt precursor hairpins, known as pre-miRNAs, and mature miRNA. One strand of theduplex is incorporated into the RNA-induced silencing complex, guides to the targetmRNA with sequence complementarity, leading either to mRNA cleavage or totranslational repression. In theory, any alteration occurs during the process of miRNAdevelopment and maturation, may be result in the change of the expression of maturemiRNA. At the transcription stage of the miRNA, its expression is mainly regulated by themutation of the promoter sequence, epigenetic changes of the promoters (aberrant levelsof methylation and histone acetylation), and the expression of transcription factors; at thepost transcriptional stage, the expression of mature miRNA is mainly associated with thesequence variation, the function of Drosha, Dicer and their co-factors, and transportprotein Exportin-5.More and more evidences had revealed miRNA was closely associated withtumorigenesis, miRNA express abnormally in many human tumor tissues. Elevatedexpression in tumor tissues, promote tumor growth, proliferation, and metastasis, termedoncomiRNAs; however, reduced expression in tumor tissues, inhibit tumor growth,proliferation, or metastasis, termed antioncomiRNAs. In previous experiments, weverified miR-181a was upregulated in gastric cancer tissues, inhibited the expression ofmiR-181a in SGC-7901gastric cancer cells by RNA interference, the ability of cell proliferation, migration and invasion decreased remarkably, yet increased apoptosis. Theseresults hinted miR-181a maybe was a new oncomiRNA in human gastric cancer. However,the regulatory mechanism of elevated miR-181a in gastric cancer cells was unknown.CCAAT/enhancer binding protein beta(C/EBP β), also named nuclear factorinterleukin-6(NF-IL6), is the second member of C/EBPs family. C/EBP β is a nucleartranscription factor, not only involved in normal physiological processes, but alsoassociated with the occurrence and development of many diseases, regulated transcriptionpositively and negatively. Through regulate the transcription of target genes, C/EBP βinvolved in cell proliferation and differentiation, tumorigenesis and apoptosis,inflammatory response and other important life actions. The expression of C/EBP β waselevated in HepG2and HepG3β hepatoma cell lines, ovarian cancer cell lines, breastcancer and skin cancer cell lines, and C/EBP β can enhance the transformation ability ofoncogene Ha-Ras, promote tumor growth; but there were reports considered C/EBP βmediate apoptosis of tumor cells, up-regulated C/EBP β can induce fibroblasts apoptosisby activate cell apoptosis factors p53and p21. These results indicated that C/EBP β candecide the fate of tumor cells, through the regulation of oncogenes and anticogenes.PurposeThis study combined TaqMan real-time PCR,5’RACE, dual-luciferase activity assay,chromatin immunoprecipitation (CHIP) and RNAi technologies, to identify thetranscription start site of miR-181a-1, position the core promoter region, predicttranscription factors, confirmed combine between the binding site and transcription factors,finally certified transcription factors can regulate the expression of pri-miR-181a andmature miR-181a.Methods1. Using TaqMan real-time PCR to detect the expression of mature miR-181a,pri-miR-181a-1and pri-miR-181a-2in gastric cancer, its adjacent non-neoplastic tissues,and gastric cancer cells.2. Identifying the transcription start site of miR-181a-1by5’RACE.3. Construction of plasmids: miR-181a-1pro1~10（﹢379~﹢616bp、﹢140~﹢616bp、 ﹣37~﹢616bp、﹣165~﹢616bp、﹣295~﹢616bp、﹣609~﹢616bp、﹣994~﹢616bp、﹣1419~﹢616bp、﹣1779~﹢616bp、﹣2022~﹢616bp），using dual-luciferase activityassay to position the core promoter region of miR-181a.4. Predicting the transcription factor of miR-181a-1, confirming the relationship betweenbinding site and transcription factors by CHIP.5. Transfection SGC-7901cells with C/EBP β siRNA, detect the expression ofpri-miR-181a-1and mature miR-181a, furtherly certified C/EBP β can regulate miR-181aResults1. In gastric cancer tissues and cells, the expression of mature miR-181a andpri-miR-181a-1were up-regulated, and there was a positive correlation between them, andthe expression in SGC-7901is the highest.2. The transcription start site is at miR-181a-15’677bp. Compared to the PGL3-Basic, therelative luciferase activity of miR-181a pro1~10were all increased, and﹣37~﹢616bpwas the highest, the core promoter region of miR-181a was at the﹣37~﹢140bpposition.3. We predicted that there were many binding sites of C/EBP β in the core promoter regionof miR-181a. The result of CHIP verified that C/EBP β can combine to the DNA.4. Transfected SGC-7901cells with C/EBP β, knocked down C/EBP β mRNAand proteinsuccessfully, the expression of pre-miR-181a-1and mature miR-181a were reducedmarkedly.Conclusion1. The mature miR-181a was processed by pri-miR-181a-1and pri-miR-181a-2, and theexpression of mature miR-181a is influenced mainly due to pri-miR-181a-1.2. C/EBP β can combine to the particular DNA sequence of the core promoter region ofmiR-181a, thereby regulate the expression of mature miR-181a at the transcription stage.