The same biological function of miR-15a was found in Huh7 cells (Physique 4B). [16,17]. It has been predicted that miRNAs may regulate protein expression from as many as 10% to 30% of all human genes [18]. Normally, miRNAs play important roles in crucial biological processes such as organ development, cell differentiation and proliferation, apoptosis, and cancer cell invasion [19]. In past few years, the deregulated expression of miRNAs has been widely reported in many diseases including cancer [20,21]. At present, more target genes of miRNAs have been constantly validated in experiments and verified in clinical samples. MiRNAs have been proven to have a new important role of regulating tumorigenesis, and these have been demonstrated to play important roles in various aspects of cancer progression including tumor metastasis [22]. For example, miR-15a is usually significantly decreased in HCC cells and tissues, especially in metastatic liver malignancy cells; resulting in increased FOS and Met expression [23]. Moreover, the expression of miR-15a was significantly reduced in HCC tissues, and this expression was negatively correlated with tumor TNM stage. Furthermore, this downregulation causes lymph node metastasis with a low metastasis-free survival in patients [24]. These studies indicate the importance of conducting thorough investigations on miRNAs that are aberrantly expressed during HCC progression, especially miRNAs associated with HCC metastasis. In the present study, miR-15a was found to be significantly downregulated in HCC MHCC97H and Vandetanib HCl Huh7 sphere cultured cells, compared with their parent cells. By manipulating miR-15a levels in HCC cells, we validated that miR-15a promotes the mobility of HCC cells. has been predicted to be a target of miR-15a by bioinformatics analysis, which was validated by luciferase assay and western blot. The function of in HCC metastasis was verified by loss-of-function and gain-of-function assays. Our study demonstrates that miR-15a plays a role as a metastasis promoter by directly targeting cDNA expression plasmid lacking the 3-UTR was purchased from GenePharma Co., Ltd (Shanghai, China), and the vacant plasmid served as the unfavorable control. The siRNA sequence targeting human mRNA was designed and synthesized by GenePharma Co., Ltd (Shanghai, China). A scrambled siRNA was used as unfavorable control. Sequences of the siRNA were as follows: 5-UGUUAUUGCCAAGCACUUAAA-3 (sense); 5-UAAGUGCUUGGCAAUAACAGAA-3 (antisense). The overexpression plasmid or siRNA was transfected into MHCC97H and Huh7 cells using Lipofectamine 2000 (Invitrogen), according to manufacturers instructions. The concentration for the transfection of the cDNA plasmid or siRNA was 0.05 g/mL or 50 nmol/L, respectively. Overexpression or knockdown of miR-15a The overexpression or knockdown of miR-15a was accomplished by transfecting cells with miR-15a mimic or inhibitor purchased from GenePharma Co., Ltd. (Shanghai, China). The transfection concentrations were 50 nmol/L for the miR-15a mimic and 200 nmol/L for the miR-15a inhibitor, which were also adopted when the control mimic Vandetanib HCl or inhibitor was transfected. Western blot Total protein extraction from cultured cells was used in electrophoresis and western blot. Briefly, 20 micrograms of total protein were separated by standard SDS-PAGE and transferred onto PVDF membranes. The membranes were washed, blocked and incubated with specific primary antihuman antibodies (1:1,500), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:5,000). The Vandetanib HCl reactions were detected by enhanced chemiluminescence assay. The anti-cMyb antibody was obtained from Santa Mouse monoclonal to DPPA2 Cruz Biotechnology (Santa Cruz, CA, USA), and a concentration of 1 1:1,500 was used. An antibody Vandetanib HCl against anti–actin, as the control, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); and a concentration of 1 1:1,500 was used. Luciferase reporter assay The entire 3-UTR of human was amplified by PCR using human genomic DNA as the template, and the PCR products were inserted into the p-MIR-reporter plasmid GenePharma Co., Ltd. (Shanghai, China). The insertion was confirmed by sequencing. In order to test the binding specificity, Vandetanib HCl sequences that interact with the miR-15a seed sequence were mutated (from TGCTGCTA to GGTTACG); and the mutant 3-UTR was inserted into an equivalent luciferase reporter plasmid. For the luciferase reporter assay, HEK293T cells were seeded in 24-well plates, and each well was transfected with 0.2 g of luciferase reporter plasmid, 0.4 g of -galactosidase (-gal) expression plasmid and 40 pmol of miR-15a mimic, 120 pmol of miR-15a inhibitor or scrambled negative control RNAs using Lipofectamine 2000 (Invitrogen). The -gal plasmid was used as the transfection control. At 24 hours after transfection, cells were harvested and analyzed for luciferase activity using luciferase assay kits (Promega,.
