The Functions And Mechanisms Of MicroRNA-31 In Urothelial Bladder Cancer

Background and ObjectivesUrothelial bladder carcinoma(UBC),the most common genitourinary tract cancer in China,is characterized by complex biological behaviors and high risks of recurrence and progression.Chemoresistance is one of main reasons for treatment failure of UBC MicroRNAs(miRNAs)are a conserved class of small noncoding RNAs that regulate protein expression either by mRNA degradation or repression,via specific binding to complementary site within the 3'untranslated region(3'UTR).MiRNAs are often aberrantly expressed in various cancers and exert their functions as oncogenes or tumor suppressors.MiR-31 is a well-recognized miRNA that exhibits diverse properties depending on cancer types.However,the functional mechanisms of miR-31 in UBC have not been illustrated until present.Hence,in levels of tissues,cells and animals,we invesitigated the expression patterns and biological functions of miR-31 in UBC and analyzed the relevant mechanisms in modulation of UBC behaviors and chemosensitivity The present study will help understanding the mechanisms of UBC development and provide evidence for novel stratergies of improving chemotherapeutic effects in UBCMaterials and Methods1.Surgical samples were collected from patients with nonmuscle invasive bladder cancer(NMIBC)or muscle invasive bladder cancer(MIBC).Quantative real-time PCR was employed to detect the expression of miR-31 in tumor tissues and adjacent normal urothelium.2.By using lipofectamine 2000,T24 and 5637 cells were transfected with miR-31 mimics or scramble oligonucleotides(miR-NC)mimics.Cell proliferation,migration and invasion were observed and cell cycle was analyzed after miR-31 overexpression.3.Luciferase reporter vector of integrin ?5(ITGA5)3'UTR was constructed and then co-transfected with miR-31 mimics(or miR-NC mimics)in T24 cells.Luciferase activaties were measured to identify whether ITGA5 was the target gene of miR-31.After transfection of miR-31 mimics(or miR-NC mimics)in T24 and 5637 cells,western blot assay was performed to detect the protein expression of ITGA5.ITGA5-expressing plasmid was constructed,and cell proliferation was observed after co-transfection of miR-31 mimics(or miR-NC mimics)and ITGA5 vector(or empty vector)in UBC cells.4.UBC cells co-transfected with ITGA5 vector(or empty vector)and miR-31 mimics(or miR-NC mimics)were seeded onto fibronectin-coated plates and then treated with mitomycin-C(MMC).Toxcities of MMC and cell apoptosis were detected in all groups of cells.Western blot assay was employed for analyzing the regulatory effects of miR-31/ITGA5 on Akt and Erk pathways.5.Recombinant miR-31-expressing lentivirus was constructed.T24 cells were infected with lentivirus and stable cells expressing miR-31 were subcutaneously inoculated into nude mice to induce tumor xenografts.By observation of tumor growth and pathologic analysis of xenografts,effects of miR-31 on UBC development and MMC efficacy were investigated in vivo.Results1.Compared with normal urothelium,we observed significantly down-regulation of miR-31 in both NMIBC and MIBC lesions,among which MIBC displayed markedly lower levels of expression than NMIBC2.Overexpression of miR-31 suppressed proliferation,migration and invasion of UBC cells and blocked G1 to S cell cycle transition3.In T24 cells,co-transfection with miR-31 mimics significantly reduced the luciferase activity of the reporter vector carrying wild-type ITGA5 3'UTR.In contrast,the suppressive effect was abolished when the miRNA binding sequence in ITGA53'UTR was mutated.After ectopic expression of miR-31,endogenous ITGA5 protein was remarkably down-regulated in both T24 and 5637 cells.Co-trasfection of ITGA5 vector and miR-31 mimics reversed the inhibited cell proliferation induced by miR-31 expression4.Overexpression of miR-31 increased the toxicity of MMC in UBC cells and aggrevated MMC-induced cell apoptosis,while co-expression of miR-31 and ITGA5 reversed these chemosensitization effects.MiR-31-expressing UBC cells showed inhibition of phosphorylation of Akt and Erk,while UBC cells co-expressing miR-31 and ITGA5 were similar with cells of control groups5.In the xenograft model,overexpression of miR-31 remarkably inhibited UBC tumor growth in vivo,decreased the expression of ITGA5 and improved therapeutic effects of MMCConclusions1.MiR-31 is lowly expressed in UBC tissues and the down-regulation is more significant in MIBC tissues2.MiR-31 inhibits proliferation,cell cycle,migration and invasion of UBC cells and improves sensitivity to MMC3.In UBC cells,ITGA5 is the target gene of miR-31 and miR-31 could play a tumor-suppressive effect by down-regulating ITGA5.By inactivating Akt and ERK pathways,miR-31/ITGA5 axis can inhibit cell adhesion mediated drug resistance and increase chemosensitivity of UBC cells.