Clinical Significance And Experimental Research Of MiR-490-5p And Its Influence On The Cellular Biological Characteristics In Bladder Cancer

Bladder cancer, with the highest morbidity and mortality among the urinary system tumors, is one of the most common malignant neoplasms of the urinary system in China, which threatens people's life and health severely. The clinical treatment strategies for bladder cancer is mainly surgical excision, combined with infusion chemotherapy and immunotherapy treatments, which has unsatisfactory results.Find the incidence of bladder cancer gene to understand the molecular genetics of bladder cancer, and provide a theoretical basis for the diagnosis and treatment of bladder cancer is the research focus of the current.MicroRNA is a class of small noncoding single-stranded RNAs found in eukaryotes, about22nucleotides in length, which usually regulates gene expression at the post-transcriptional level. A large number of studies have shown that microRNA participate in tumor cell proliferation, differentiation, invasion and apoptosis. MiRNA are highly conservative, timing and tissue-specific, which indicate that they may be in a new level of gene expression regulation. MiRNA expression profiles can be indicators as a tumor diagnosis and prognosis, and its mechanism of depth is expected to provide new ideas and targets for bladder cancer diagnosis and treatment.miR-490-5p is a newly discovered abnormal expression microRNA in tumors, which has been found in a low level expression in a variety of malignancies including bladder cancer, so we hypothesized that it function as a tumor-supprissive gene.The miR-490-5p expression in bladder cancer and adjacent tissues, three types of bladder cancer cells and normal bladder epithelial cells will be detected by real-time PCR in our project, to explore its relationship with the clinical stage and histological grade. The study will make use of bioinformatics to predict and screen target genes of miR-490-5p, and validate them by Western blot.And then studymiR-490-5p's influence on the biological behavior of a bladder cancer cell. We hope to clarify the possible molecular mechanisms of miR-490-5p on bladder cancer, so as to provide new strateges on early diagnosis and treatment of bladder cancer.Part I The expression of miR-490-5p and correlation with clinical stage and pathological grade of bladder cancerObjective:To study the expression of miR-490-5p in bladder cancer and clinical significance.Methods:The expression of miR-490-5p was detected by Real-time PCR in20cases of bladder cancer, adjacent non-tumor tissues, three bladder cancer cell lines (T24, BIU-87and5637) and bladder epithelial immortalized cell (SV-HUC-1).The relationship of miR-490-5p with clinical stage and pathological grade was also studied.Results:The expression of miR-490-5p was down-regulated in20samples compared with paired adjacent non-tumor tissue. The relative expression level of miR-490-5p in bladder cancer was1.175±0.643, which was significantly lower than that in the adjacent non-tumor tissue with8.539±1.920(P<0.05).According to the clinical stage, the relative expression level of miR-490-5p was1.149±0.677in Ⅱ phase, and1.214±0.632in Ⅲ-Ⅳ phase, which had no significant difference(P>0.05).According to the pathological grade, the relative expression level of miR-490-5p in well-differentiated group was1.770±0.452, which was significantly higher than that in low-moderately differentiated group with0.688±0.206(P<0.05).The miR-490-5p expression in normal bladder cell line SV-HUC-1was significantly higher than that in the three bladder cancer cell lines (T24, BIU87and5637)(P<0.05), and it gradually decreased (5637> BIU87> of T24) with increasing degree of malignancy of bladder cancer cells.Conclusion:1. The expression of miR-490-5pin bladder cancer was significantly lower than that in adjacent non-tumor tissue, indicating it may be associated with the pathogenesis of bladder cancer.2. The expression of miR-490-5p has no significant correlation with clinical stage in bladder cancer.3. The expression of miR-490-5p has significant correlation with pathological grade, and it gradually decreases with increasing degree of pathological grade in bladder cancer.4. The expression of miR-490-5p in the three bladder cancer cell lines was significantly lower than that in normal bladder cell line SV-HUC-1, and it gradually decreased with increasing degree of malignancy of bladder cancer cells. The expression of miR-490-5p was lowest in T24, and it was selected for further experiment. Part Ⅱ The target gene screen and verification of miR-490-5p in bladder cancer tissues and cellsObjective:To predict and screen target genes of miR-490-5pby bioinformatics, and validate them by Western blot.Methods:By searching databases of miRBase, TargetScan and miRanda.three miR-490-5p potential target genes were selected:c-Fos, TET1and UVRAG. Western blot was used to detect and validate the protein expression levels of these target genes in six cases withlowest expression ofmiR-490-5p, adjacent non-tumor tissue and four bladder cells.Results:Compared with adjacent non-tumor tissue of bladder cancer, c-Fos protein level in bladder cancer significantly upregulated, UVRAG significantly reduced, while TET1with no significant difference between the two groups (P<0.05). Similarly, c-Fos protein level in bladder cancer was significantly upregulated, UVRAG significantly reduced, while TET1with no significant difference compared with normal bladder cells (P<0.05).Conclusion:1. miR-490-5p in bladder cancer plays a similar role of tumor suppressor gene, and its deletion or low expression will lead to bladder cancer cell growth and differentiation, suggesting that it may become another target for bladder cancer gene therapy.2. c-Fos and UVRAG are the candidate target genes of miR-490-5p. miR-490-5p might have exert its impact on bladder cancer through these two target genes. Part Ⅲ Effects of miR-490-5pon cell proliferation, invasion, and apoptosis of human bladder cancer T24cellObjective:To study the effects of miR-490-5p high expression on bilogical characters of bladder cancer T24cell.Methods:1. miR-490-5p expression was increased in T24cell by exogenous transfection, and the experiment were divided into three groups:the normal group, negative control group (transfected meaningless mimics) and group of transfected miR-490-5p mimics.2. MTT assay was employed to draw cell growth inhibition curvesand evaluate miR-490-5p's impact on the ability of T24cell proliferation.3.Transwell invasion assay was used to determine the miR-490-5p' sinfluence on the ability of T24cell invasion.4.Caspase-3was detected in each group to evaluate miR-490-5p's influence on the apoptosis of T24cell.5. c-Fos and UVRAG were detected by Western blot to study the mechanism of miR-490-5p in bladder cancer.Results:1. Real-time PCR results showed that the expression of miR-490-5p in transfected miR-490-5p mimics group was significantly higher than that in the negative control group (P<0.05).2. The rate of T24cell survivalin transfected miR-490-5p mimics group were significantly lower when compared with the negative control group at48h and72hafter transfection (P<0.05).3. Transwell invasion assayshowed that the number of T24invasion in transfected miR-490-5p mimics group were significantly lower than that in the other groups (P<0.05).4. The caspase-3level in each group were as follows:(14.761±1.303) U/mg in normal control group,(15.719±1.613)U/mg in negative control group (15.719±1.613)U/mg, and (43.615±4.399) U/mg in transfection of miR-490-5p mimics group. There was no significant differences between the normal group and the negative control group (P>0.05). The caspase-3levelin transfection of miR-490-5p mimics group was significantly higher than tha in the normal group and negative control group (P<0.05).5. compared with the normal group and negative control group, the c-Fos protein level in ransfected miR-490-5p mimics group decreased significantly, while UVRAG increased significantly(P<0.05).Conclusion:1. In vitro experiments, the bladder cancer cell successfully imported by exogenous miR-490-5p can significantly inhibit proliferation and invasion, and promote apoptosis of T24cells. 2. miR-490-5p exerted imapact on the proliferation, invasion and apoptosis of bladder cancer cells by regulation of c-Fos and UVRAG at post-transcriptional level.