The Biological Function And Regulatory Mechanism Of MicroRNA-582 In Bladder Cancer

Background and ObjectiveBladder cancer is one of the common malignant tumors of the urinary system,but also the highest incidence of urinary tract cancer in China.The incidences of bladder cancer are complex,including two main factors:heredity and environment.Smoking and long-term exposure to chemical drugs is the two major environmental risk factors.Among them,smoking is the most dangerous factor.Studies have shown that smoking can increase the incidence of bladder cancer 2-4 times,and the risk factor is proportional to the intensity of smoking and smoking time.At present,according to the different depth of infiltration,bladder cancer is treated with different treatment plan.The non-muscle invasive bladder cancer is treated by transurethral resection combined with intravesical instillation.But the recurrence rate is as high as 60-70%,and there is a tendency to increase the degree of malignancy.And for the muscle invasive bladder cancer,the current standard treatment is radical cystectomy.However,there is a large surgical trauma,postoperative complications,and a great impact on the life quality of patients undergoing this operation.Therefore,urological surgeons hope to clarify the molecular biological process of the occurrence and development of bladder cancer,and to find a new method for early diagnosis and treatment of bladder cancer.MicroRNA is a class of small molecule nonprotein-encoded RNA(about 22 bases in length).Although they are not directly encoding the protein synthesis,but can inhibit the translation by partial or complete paired binding to the target gene 3 'UTR,or cause it to degrade specifically,thereby acting as a regulator of gene expression after transcription.We have found that miR-582 was low in bladder urothelial carcinoma cells and explored the role of miR-582 in human bladder cancer cells and provide a new target for the diagnosis and treatment of bladder cancer by in vitro cell experiments.Materals,Methods and ResultsPart I miR-582 expression differences in human bladder carcinoma tissues and human normal bladder tissuesMethods:1.Collected radical cystectomy carcinoma carcinoma tissue and corresponding nomal epithelial tissue.2.Used the PCR assay to detect the expression of miR-582 in bladder carcinoma tissues and cells.Results:1.The expression of miR-582 in bladder cancer tissues was significantly lower than that in normal tissues.2.The expression of miR-582 in bladder urothelial carcinoma was stable and low expression,and its expression was closely related to the pathological grade and clinical stage.Part ? Study on the biological function of miR-582 in the pathogenesis of bladder carcinoma Methods:1.MiR-582 mimic was transiently transfected into bladder cancer cell T24,5637 cells by lipofectamine 2000,resulting in down-regulation of miR-582 expression.The control group(transfected with miR-NC group)and the experimental group(transfected with miR-582 mimic)were established.2.The transfection efficiency was evaluated by QPCR method.The ability of bladder cancer cell migration was evaluated by wound healing assay.The invasive ability of bladder cancer cells was evaluated by transwell assay.The stemness of bladder cancer cells was evaluated by flow cytometry.Western blot was used to assess CD44 and.SOX2 posttransfection.Results:1.The expression of miR-582 in T24,5637 cells was significantly up-regulated after transfection with miR-582 mimic,and the difference was statistically significant(P<0.05).2.In wound healing assay,the wound healing time in miR-582 mimic group was significantly prolonged after 20 hours of transfection,and the difference was statistically significant(P<0.05).In transwell assay,the number of transmembrane cells in the T24,5637 cells of the miR-NC mimic group was much larger than that of the miR-582 mimic group,and the difference was statistically significant(P<0.05).By flow cytometry,the stemness of bladder cancer T24,5637 cells of the miR-582 mimic group was significantly decreased.Part? FOXG1 negatively targeted by miR-582 expression and the function FOXG1 in vitroMethods:1.Four bioinformatics software online(Target scan,miRanda,miR Target2,PITA and PicTar)were used to predict downstream target genes of miR-582.It was confirmated by luciferase.2.Co-transfected with miR-582 mimics and FOXG1 plasmid in the T24,5637 cells and used transwell assay and Western blot to detect the ability of bladder cancer cell migration and the protein expression.3.Human bladder cancer T24,5637 cells were treated with si-FOXG1 transfection.The ability of bladder cancer cell migration was evaluated by wound healing assay.Results:1.The miRNA target gene prediction softwares and luciferase reporter showed that miR-582 can conbine with 3' FOXG1 UTR.2.Result from transwell assay and Western blot showed that transfected with miR-582 in the T24,5637 cells weakened the ability of bladder cancer cell migration and MMP2,MMP9 expression,but transfected with FOXG1 plasmid strengthened the ability and expression.3.In transwell assay,the number of transmembrane cells in the T24,5637 cells of the si-NC group was much larger than that of the si-FOXG1 group,and the difference was statistically significant(P<0.05).These results suggested that FOXG1 up-regulation can effectively inhibit the invasive ability of bladder cancer cells.ConclusionsMiR-582 was down-regulated in bladder cancer tissues,and its expression was closely related to the pathological grade and clinical stage of the tumor.The higher the pathological grade,the later the clinical stage of the patient,the lower the expression was.FOXG1 negatively targeted by miR-582 expression,in order to achieve the inhibition of bladder cancer cell migration,invasion,sternness,and play a role in tumor suppressor gene.This study could provide experimental basis for molecular targeted therapy of bladder cancer.