MicroRNA-152Down-regulated Lead To The DNA Methylation Induced Bladder Cancer

Background:Bladder cancer is one of the most common the genitourinary systemmalignancies in the world, which is a direct threat to the survival of patients with thedisease. In recent years, just more serious industrial pollution and treatment meansincreased incidence of bladder cancer, and the rate of diagnosed showed an increasingtrend. Some scholars statistical analysis, from1985to2005, the number of newlydiagnosed bladder cancer increased by nearly50%, has become one of the commonmalignancies studied the health effects of people’s lives. Currently, many aspects ofbladder cancer diagnosis, treatment Shangqie lack of uniform standards, making roomfor progress in its treatment still exists. Bladder cancer has about80%of superficialbladder cancer tumors, the current main methods include transurethral resection(TURBt) and postoperative intravesical chemotherapy or immunotherapy; Have beenreported in the literature, TURBt treatment of postoperative bladder cancer have a10%-65%of patients relapse within1year; over24%-84%of patients relapse within fiveyears; nearly20%of patients relapse after tumor malignant degree will increase;eventually10%of patients will develop invasive cancer or metastasis; Similarly, some well-differentiated tumor progression occurs tumor invasion in five years, andtransferred occurs. Invasion type of bladder cancer treatment to surgical excision, butthere is a big flaw surgery, namely: trauma, complications,5-year survival rate (54.5%-68%), and the type of bladder cancer with invasion recur, easy to transfer, etc,because this series is still making the diagnosis of bladder cancer is one of thedifficulties of urinary tract tumors. So far, still no bladder cancer-specific biomarkersand therapeutic targets related to the occurrence of bladder cancer and the formationmechanism is not fully understood, which makes it more difficult to treat bladdercancer. Therefore, further study the molecular mechanisms of pathogenesis of bladdercancer, the discovery of new specific biomarkers and therapeutic targets for treatment,has a very important scientific significance.Formation of bladder cancer by a variety of different roles of the combined effectof factors: including:①associated mutations occur;②oncogene expression oroverexpression increases;③systemic factors; respective roles④between thetumor and the surrounding matrix;⑤urothelial changes in the microenvironment ofthe cells;⑥outside carcinogens; Under the stimulus of carcinogens, normal bladdercells produce DNA damage, leading to bladder cancer and to promote the formationand development. In this process, as well as up-or down-regulated activation ofoncogenes or inactivation of tumor suppressor genes, plays a very important role.Currently, the focus of research focused on the progression of bladder cancer tumorsuppressor gene effect. Mutated tumor suppressor gene inactivation, the loss will leadto bladder cancer. Thus, the import of wild-type tumor suppressor gene to repair thedefect, making the function of tumor suppressor genes is restored, is one method oftreatment for bladder cancer. Regulator of tumor suppressor gene is one of the processof differentiation and proliferation plays an important role in normal cells play a blockabnormal cell proliferation and cell differentiation function. If the tumor suppressorgene is mutated or inactivated, can lead to cell cycle regulation equilibrium is broken,thus tend abnormal proliferation of cells, leading to tumorigenesis. In recent years, theabnormal expression of MicroRNAs in bladder cancer and the role in tumordevelopment is being gradually clarified, indicating MicroRNAs play a role in cancer-causing genes or tumor suppressor genes in the development of bladder cancer.MicroRNAs(miRNA), also known as microRNAs, small non-codingsingle-stranded RNA molecules of about21-23nucleotides in a composition. Isindependent of intracellular miRNAs encoding gene is an important part of the body’sgenetic information. MiRNAs transcription by RNA polymerase II to become theprimary miRNAs transcripts, followed by enzymes in the nucleus that is cut to formmiRNAs precursors, and after being transported to the cytoplasm to form the maturemiRNA processing in the form of complementary bases complementary to themRNAs homologous pair, thereby inducing degradation miRNA cutting, or modulatethe expression of tumor suppressor genes translate other form, which is thepost-transcriptional regulation of gene expression. There are a total body about1000kinds of miRNA, accounting for5%of the total number of genes in the humangenome to predict, although a small number, but they are the most regulated andnon-protein-coding gene protein coding portion of the gene. It is mainly through thetarget gene mRNA3’-UTR region (mRNA3’untranslated region) pair complementeach other, and thus play a silent or activation of specific target genes. More and morestudies evidence, miRNAs are generally present in a wide variety of plants andanimals, and viruses, in humans and other mammals have more than30%of the genesis regulated by miRNAs. The results show that, miRNAs in organism development,proliferation, differentiation or apoptosis, in particular the development of tumors andother aspects play an important role in regulating. With the deepening of the study,more and more miRNAs were found and reported. There are many scholars believethat the change may be a large number of miRNAs exist, even in the pathophysiologyof human development tumors played a key role, and some even raised exception isthe most important tumor occurrence and development of miRNAs reasons. Throughthe review of the published literature, we found that abnormal expression of miRNAspresent in many tumors, including: urinary tract cancer, breast cancer, lung cancer,lymphoma and leukemia. MiRNAs by regulating downstream target genes in tumordevelopment plays a vital function of oncogenic or tumor suppressor genes.In our previous study, through MicroRNA microarray technology to detect bladder cancer tissue abnormalities in miRNA expression in adjacent tissues as well,occurring in bladder cancer tissues, the expression of miR-152is significantly reducedfurther by Real-time PCR experiments tests confirmed: Compared with normal tissue,miR-152is one of the most down-regulated miRNAs bladder cancer significantly, sowe infer that miR-152may play a role in the development of tumor suppressor genesin bladder cancer. However, miR-152specific function in bladder cancer yet reported,therefore, necessary for the mechanism of miR-152in bladder cancer research.Objective:.1. Role of miR-152in the development of bladder cancer, identified miR-152iswhether the process of bladder cancer tumor suppressor gene plays an importantrole, whether due to the absence of miR-152led to the occurrence and thedevelopment of bladder cancer.2. Identification of miR-152regulation of downstream target genes, analyzed andproposed deletion of miR-152due cause bladder cancer occurrence anddevelopment pathway.Methods:.1. Cultured human bladder cancer cells and the expression of bladder epithelial cellsimmortalized detect miR-152’s expression.Through Real-time detection of bladder cancer cell line T24immortalized humanurothelial cells Sv-huc-1in miR-152expression.2. Observed miR-152in bladder cancer cells and immortalized human urothelial cellsinfluence biological behavior.Cell proliferation transfected miR-152mimics the T24cells transfected withmiR-152inhibitor to the Sv-huc-1, respectively, by cell counting, cell colonyformation and Real-time PCR to detect bladder cancer cell lines capacity.3. Identification of downstream regulation of miR-152target genes.Target gene prediction software TargetScans predicted miR-152in bladdercancer is a potential target gene DNMT1. DNMT1gene were constructed containingthe3’non-coding region sequence DNMT13’-UTR (DNMT1-WT) and mutant sequences DNMT13’-UTR (DNMT1-MU) of the luciferase reporter, the role ofmiR-152region detection. After transfection of miR-152, using Real-time PCR andWestern blot Decorating miR-152expression and potential target genes DNMT1protein expression changes further by Real-time PCR detection of downstream targetgenes DNMT1gene, and thus clarify miR-152and its possible mechanism of actionof the target gene in bladder cancer.Results:.1. Real-time PCR test results show that immortalized human urothelial cellsSv-huc-1in miR-152is highly expressed in bladder cancer cell line T24, ScaBer,5637is low expression (P <0.01).2．Cells computational experiments and colony formation assay results show that,miR-152is significantly inhibit bladder cancer cell lines T24, ScaBer,5637proliferation (P <0.01).3. TargetScans prediction software is a potential target gene of miR-152, DNMT1gene3’untranslated region (3’-UTR) contains complementary binding sites formiR-152. Luciferase reporter gene analysis showed that: the transfected miR-152and DNMT1-WT fluorescent reporter vector fluorescence intensity has beensignificantly inhibited (P <0.001), while the mutant DNMT1(DNMT1-MU) genefluorescent reportervector the fluorescence intensity substantially unaffected.4After transfection of miR-152simulation agent expression by Western blot andReal-time PCR detection of DNMT1to detect DNA methylation. We found that theexpression of DNMT1enzyme is inhibited. After transfected miR-152mimc, wefound that thatdownstream genes by Real-time PCR detection of DNA methylationregulation. We found that the expression is downregulated HoxA9. Thus wespeculate, HoxA9is affected by DNA methylation downstream target generegulation.Conclusion:. 1: MicroRNA-152in bladder cells, is highly expressed; in bladder cancer is lowexpression. In normal bladder cells, DNA methylation is the low level. With theoccurrence of cancer, DNA methylation degree has increased.2: change MiR-152expression, the role of DNA in the3’-UTR region, leading toDNA methylation, DNA or increasing the degree of methylation.3: In the process of development of bladder cancer, miR-152plays a role in tumorsuppressor genes.4: In bladder cancer, HoxA9is one of the downstream target genes regulated by DNAmethylation.