MiR-204 acts as a tumor suppressor in human bladder cancer cell T24 by targeting antiapoptotic BCL2

Abstract

ObjectiveBladder cancer (BC) is one of the most common noncutaneous malignant cancers with high mortality and recurrence rates in the general population, particularly in men. We previously reported detecting dysregulated micro-RNAs (miRNAs) in human BC tissues. Using an miRNA targeting database, we found that miR-204, which is downregulated in BC, targets the B-cell lymphoma 2 gene (BCL2). We hypothesized that the overexpression of miR-204 induces apoptosis by repressing BCL2 expression and causing cancer cell death. Materials and methodsTo overexpress miR-204 in cancer cells, a vector that expresses miR-204 was transfected into human BC, embryonic kidney, and cervical cancer cells. The expression of miR-204 in the transfected and mock cells was monitored through miRNA quantitative polymerase chain reactions (Q-PCRs). Luciferase reporter assays were performed to verify the direct binding of miR-204 to targeted sites on the BCL2 transcripts. The BCL2 mRNA and protein expression levels in the miR-204-transfected cells were measured using Q-PCRs and Western blotting, respectively. The cell viability of the miR-204 transfected cells was detected through WST-1 assays. The induction of apoptosis was determined by the increased release of cytochrome c, cleavage caspase 3, caspase 3/7 activity, and DNA fragmentation. ResultsThe expression of miR-204 in the transfected cells was elevated to 6.2-fold, compared with the controls, and the function of matured miR-204 was confirmed through the luciferase reporter assays. The expressions of BCL2 mRNA and protein were reduced in the miR-204-transfected cells, which generated increased DNA fragmentation, caspase 3 cleavage, caspase 3/7 activity, and reduced cell viability. Cotransfection of the reporter vector that harbored the BCL2 3′-untranslated region to compete with endogenous transcripts partially rescued the apoptosis that was induced by the overexpressed miR-204. This finding further supported that miR-204 targeting BCL2 induces apoptosis in BC cells. ConclusionmiR-204 targets BCL2 in human BC cells. The overexpression of miR-204 in tumors could thus be a novel strategy for inducing cell death by targeting antiapoptotic BCL2.