ABSTRACT
Objective To investigate the effect of miR-107 on the function of human non-small cell lung cancer cell line A549 and its possible target genes.Methods The experiment was divided into the liposome+miR-107 mimics overexpression group(OV-miR-107 group),liposome+miR-107 inhibiting mimics downregulation group(KD-miR-107 group)and liposome+ negative control mimics group(NC group).The cell transfections of siRNA were siRNA-1,siRNA-2 and siRNA-3 respectively.The cellular prolifer-ation capacity was detected by MTT assay.The cellular cycle was detected by flow cytometry.The dual luciferase reporter gene as-say was performed to detect the downstream target gene of miR-107.The real time quantitative reverse transcription PCR and Western blot were respectively employed to examine mRNA and protein expression levels of downstream target gene.Results miR-107 inhibited the proliferation of A549 cells in a time-and dose-dependent manner(P<0.05).miR-107 arrested more A549 cells at the G0G1phase,and the proportions of S phase and G2M phase were decreased(P<0.05).miR-107 combinded with the 246 bp-253 bp of CCNE1 3′-untranslated region,and decreased mRNA and protein expression of CCNE1(P<0.05);after down-regulating CC-NE1 in A549 cells,siRNA-2 inhibited cellular proliferation(P<0.05)and blocked the cells to stay at G0G1phase(P<0.05).Con-clusion miR-107 inhibits the proliferation of human non-small cell lung cancer cell line A549 and regulates the cellular cycle by tar-geting CCNE1.
ABSTRACT
Background Eicosapentaenoic acid(EPA)function as the critical lipid mediators involved in several biological events in human body and play important role in suppressing the genesis of vascular endothelial growth factor (VEGF),migration and proliferation of vascular endothelial cells.Many ocular diseases were proved to be associated with neovascularization.Objecfive The purpose of this study was to investigate the inhibitory effect of EPA on the proliferation of human umbilical vein endothelial cells (HUVEC) indueed by VEGF. Methods HUVEC strain was cultured and passaged,and difierent concentrations of EPA were added to the medium with and without VEGF.The cultured cells were identified by antiofactor Ⅷ polyclonal antibody.The suppressing role of different concentrations of EPA on the proliferation of VEGF-induced or-uninduced HUVEC was assessed by MTT method.The influence of difierent concentrations of EPA on the cellular cycle of VEGF-induced HUVEC was assayed using flow eytometry.The expression of Flk-1,a receptor of VEGF,in the HUVEC Was detected by immunohistochemistry. Results Cultured HUVEC showed the ftlsiform in shape and presented with the cobblestone-like arrangement with the positive response for Ⅷ factor-related antigen.Various concentrations of EPA showed obviously inhibitory effect on VEGF-induced or-unindueed HUVEC at a dose-dependent manner (F=23.072.P=0.000).The inhibitory ability of EPA on VEGF-induced HUVEC was stronger than VEGF-uninduced HUVEC(F=41.417,P=0.000).In 24,48 and 72 hours,the action of EPA on the proliferation of HUVEC was gradually enhanced with the prolong of time(F=1.495,P=0.236).Cell cycle analysis indicated that EPA arrested VEGF-induced HUVEC in G0/G1 phase.The ratio of HUVEC in G0/G1 phase in EPA group was(75.83±1.56)%,and that in control groups was(68.62±1.44)%,showing a significant difference between them(t=-5.88,P=0.00),and no apoptosis of HUVEC was found in both groups.Flk-1 was strongly expressed in the cellular nucleus and cytoplasm in control group.However,the positive expressing intensity of Flk-1 in the HUVEC weakened,and the positive cell number was evidently less in EPA group. Conclusion EPA can inhibit the proliferation of VEGF induced HUVEC through arresting the synthesis of DNA of HUVEC and downregulate the expression of Flk-1 in HUVEC.These results suggest that EPA might exert an antiangiogenic effect.