Targeting of Mcl-1 Expression by MiRNA-3614-5p Promotes Cell Apoptosis of Human Prostate Cancer Cells

Abstract

MicroRNA (miRNA) acts as a critical regulator of growth in various human malignancies. However, the role of miRNA-3614 in the progression of human prostate cancer remains unknown. In this study, our results demonstrated that miRNA-3614-5p exerts a significant inhibitory effect on cell viability and colony formation and induces sub-G1 cell cycle arrest and apoptosis in human prostate cancer cells. Myeloid cell leukemia-1 (Mcl-1) acts as a master regulator of cell survival. Using the miRNA databases, miRNA-3614-5p was found to regulate Mcl-1 expression by targeting positions of the Mcl-1-3′ UTR. The reduction of Mcl-1 expression by miRNA-3614-5p was further confirmed using an immunoblotting assay. Pro-apoptotic caspase-3 and poly (ADP-ribose) polymerase (PARP) were significantly activated by miRNA-3614-5p to generate cleaved caspase-3 (active caspase-3) and cleaved PARP (active PARP), accompanied by the inhibited Mcl-1 expression. These findings were the first to demonstrate the anti-growth effects of miRNA-3614-5p through downregulating Mcl-1 expression in human prostate cancer cells.

Loss of MTA2‐mediated downregulation of PTK7 inhibits hepatocellular carcinoma metastasis progression by modulating the FAK‐MMP7 axis

“…HEK293T cells were employed for the production of lentiviral shLuc and shPTK7 according to the methods outlined in a previous study. 19 Cells with stable PTK7 knockdown were established through lentiviral infection for 2 weeks. These cell lines were then subjected to a selection process using puromycin (2 μg/mL) for 2 weeks.…”

“…Cell viability was determined using the MTT assay according to the protocol outlined in the previous study. 19 MTT reagents were added to both shLuc-and shLuc-PTK7 HCC cells and allowed to incubate for 10 min, resulting in the formation of blue formazan crystals. The absorbance at 570 nm, indicative of the presence of these formazan crystals, was measured using an enzyme-linked immunosorbent assay reader.…”

“…Cell colonies were stained with crystal violet solution for 30 min and subsequently counted according to the established procedures. 19…”

“…Cells were lysed and collected in lysis buffer (0.5 M NaCl, 50 mM Tris, 1.0 mM EDTA, 1 mM BME, and 1% NP40) (Calbiochem, CA, USA) supplemented with a proteinase inhibitor cocktail (Roche Molecular Biochemicals) as described previously. 19 After electrophoresis and transfer to a polyvinylidene difluoride (PVDF) membrane (0.45 μm pore size), membranes were blocked with nonfat dry milk (5% in Tris buffered saline with Tween 20 [TBST buffer]), incubated with specific antibodies at 4 C overnight. The membranes were then incubated with HRP-conjugated secondary antibody for 10 min.…”

“…Reverse: 5 0 -TGAGGCTACTAGAAATGTCCCTG-3 0 amplified using the GoTaq RT-qPCR System (Promega Corporation, Wisconsin, USA) according to the methods reported in a previous study. 19 The specific target genes amplified included MTA2, PTK7, MMP1, MMP2, MMP3, MMP7, MMP9, MMP13, MMP14, and MMP15. The primer sequences for these target genes are provided in Table 1.…”