Tumor-suppressing Effects Of MicroRNA-612 In Bladder Cancer Cells By Targeting Malic Enzyme 1 Expression

Objective Bladder cancer is a common malignancy of the urinary system,and is clinically characterized by a high recurrence rate and rapid progression.Surgical treatment is still the main treatment strategy.The postoperative chemotherapy is a new effectively adjuvant therapy strategy for preventing the recurrence and metastasis of bladder cancer,but the chemotherapy resistance is the main reason for its failure.Clarifying the pathogenesis of bladder cancer at the molecular level can help to provide a new way to detect and treat bladder cancer in the early stage,and also help to analyze the prognosis of bladder cancer patients,so as to improve the treatment of bladder cancer patients.Accumulating evidence suggested that micro RNA(miRNA)plays important regulatory roles in the initiation and development of various cancers.Previous study showed that micro RNA-612(miR-612)is dysregulated in human bladder cancer tissues,however,the biological function and underlying mechanisms of miR-612 in human bladder cancer remain unknown.In the present study,we aimed to investigate the clinical significance of miR-612 and its target gene ME1 in human bladder cancer,and to determine its effects on oncogenic phenotypes of this disease.Methods Real-time PCR was used to detect the expression of miR-612 in bladder cancer tissues and adjacent normal tissues and the relationship with TNM staging and distant metastasis of bladder cancer as well as human bladder epithelial immortalized SV-HUC-1 cells and bladder cancer cells(5637?J82,UMUC3?T24).Mi R-612 mimics was transfected in to bladder cancer T24 cells by using lipofectamine3000 reagent,and the overexpression of miR-612 was measured by real-time PCR.Cell Counting Kit-8(CCK-8),colony formation assays,and transwell invasion assays were used to examine the ability of cell proliferation,colony formation,migration and invasion,respectively.And the inhibition effect of mir-612 overexpression on tumor formation was verified by experiments in nude mice.At the same time,Western blooting experiment was used to examine the effect of expression of mir-612 on the process of EMT(Epithelial-mesenchymal transition).The online software DIANAm T,miRanda,miRDB,miRWalk,PICTAR5,RNA22 and Target Scan were used to predict possible target gene of miR-612,and real-time PCR,western blot and luciferase reporter assays were further used to verify the target gene.The small interfering RNA(si RNA)was used to knockdown the expression of ME1 in T24 cells,and the biological behaviors and the molecular markers of EMT of bladder cancer cells were detected.Rescue experiment that transfection of mir-612 mimic into T24 cells overexpressed mir-612 and transfected with ME1 overexpression plasmid was performed to confirmed that ME1 was the target gene of miR-612.Results The level of miR-612 expression was significantly reduced in both bladder cancer tissues and cell lines,compared to noncancerous ones.The reduced miR-612expression was associated with advanced tumor,lymph node,metastasis(TNM)stages and distant metastasis of bladder cancer.Transfection of a miR-612 mimic suppressed bladder cancer cell growth,colony formation,migration,invasion,and tumor cell epithelial–mesenchymal transition(EMT).Bioinformatic analysis identified that miR-612 could target the expression of malic enzyme 1(ME1).Indeed,western blot and luciferase reporter assay results confirmed the data.Moreover,the ME1 expression levels were inversely associated with miR-612expression in bladder cancer tissue specimens.Knockdown of ME1 expression using ME1 si RNA mimicked the effect of ectopic miR-612 overexpression in bladder cancer cells in terms of tumor cell growth,migration,and invasion.In contrast,ME1 overexpression weakened the inhibitory effect of the miR-612 mimic in bladder cancer cellsConclusion miR-612 may function as a tumor suppressor in bladder cancer by targeting ME1 expression,and provide novel insightful information regarding miR-612 in bladder cancer development and progression so that it could be further studied as a novel tumor biomarker or therapeutic strategy for bladder cancer in the future.