【摘要】 目的 本研究旨在明确miR-409-3p在肝癌细胞系中的表达及其意义，并探讨可能的分子机制。方法 利用实时荧光定量PCR的方法检测miR-409-3p在正常肝细胞LO2,以及HepG2、BEL-7402、SMMC-7721、MHCC-97H四种肝癌细胞中的表达差异。阳离子脂质体法将miR-409-3p mimics及microRNA mimics control瞬时转染至肝癌细胞株HepG2中，利用CCK8法、平板克隆、流式细胞术检测miR-409-3p对体外癌细胞增殖、细胞周期及凋亡的影响。Western Blot检测过表达miR-409-3p的HepG2细胞中c-Met蛋白的表达变化，荧光素酶报告系统鉴定靶向关系。计量资料两组间比较采用成组t检验，多组间比较采用单因素方差分析，进一步两两比较采用SNK法。结果 基于TCGA肝癌microRNA表达谱数据，肝癌组织中miR-409-3p的表达水平显著低于癌旁组织(t=7.752,P<0.05)。与在LO2中的表达水平相比，miR-409-3p在HepG2、SMMC-7721、MHCC-97H、BEL-7402中的表达水平均明显降低(F=31.043,P<0.05)。与miR-con组相比，转染miR-409-3p mimics后的HepG2细胞中miR-409-3p的表达水平上升(t=-8.836,P<0.05),说明干扰有效。CCK8实验结果显示，与miR-con组相比，转染miR-409-3p mimics后的HepG2细胞增殖能力在48、72、96 h明显减弱，差异均具有统计学意义(t值分别为2.876、3.359、3.707,P值均<0.05)。平板克隆形成实验显示，miR-409-3p mimics组的细胞克隆形成率明显低于miR-con组(t=2.846,P=0.047)。流式细胞术结果显示，与miR-con组相比，过表达miR-409-3p后导致HepG2细胞G2期细胞数增多，差异具有统计学意义(t=-3.763,P<0.05);而凋亡率无明显统计学差异(t=0.714,P=0.515)。荧光素酶报告系统鉴定结果显示c-Met为miR-409-3p的靶基因(t=4.970,P=0.007)。与miR-con组相比，转染miR-409-3p mimics的HepG2细胞中，c-Met蛋白表达水平下降(t=-8.509,P=0.001)。结论 miR-409-3p通过抑制c-Met蛋白表达，进而调控下游信号通路引起细胞周期G2期阻滞，从而抑制肝癌HepG2细胞的增殖。更多还原

【Abstract】 Objective To investigate the expression and significance of miR-409-3p in hepatoma carcinoma cell lines and possible molecular mechanism. Methods Quantitative real-time PCR was used to measure the expression of miR-409-3p in normal LO2 hepatocytes and four hepatoma cell line(HepG2, BEL-7402, SMMC-7721, and MHCC-97H). Hepatoma HepG2 cells were transiently transfected with miR-409-3p mimics and microRNA mimics control using the cationic liposome method, and then CCK8, plate colony formation assay, and flow cytometry were used to observe the effect of miR-409-3p on the proliferation, cell cycle, and apoptosis of hepatoma cells in vitro. Western blot was used to measure the change in the expression of c-Met protein in HepG2 cells with overexpression of miR-409-3p, and luciferase reporter gene assay was used to identify targeting relationship. The independent-samples t test was used for comparison between two groups, and a one-way analysis of variance was used for comparison between multiple groups, followed by the SNK test. Results Based on the TCGA microRNA expression profile data of liver cancer, the expression level of miR-409-3p in liver cancer tissue was significantly lower than that in adjacent tissue(t=7.752, P<0.05). Compared with the LO2 cells, the HepG2, SMMC-7721, MHCC-97H, and BEL-7402 cells had a significant reduction in the expression level of miR-409-3p(F=31.043, P<0.05). Compared with the miR-con group, the HepG2 cells transfected with miR-409-3p mimics had a significant increase in the expression level of miR-409-3p(t=-8.836, P<0.05), suggesting that the interference was effective. CCK8 assay showed that compared with the miR-con group, the HepG2 cells transfected with miR-409-3p mimics had a significant reduction in proliferative capacity at 48, 72, and 96 hours(t=2.876, 3.359, and 3.707, all P<0.05). Plate colony formation assay showed that the miR-409-3p mimics group had a significantly lower plating efficiency than the miR-con group(t=2.846, P=0.047). Flow cytometry showed that compared with the miR-con group, overexpression of miR-409-3p resulted in the increased number of HepG2 cells in G2 phase(t=-3.763, P<0.05), while there was no significant difference in apoptosis rate(t=0.714, P=0.515). Luciferase reporter gene assay showed that c-Met was a target gene of miR-409-3p(t=4.970, P=0.007). Compared with the miR-con group, the HepG2 cells transfected with miR-409-3p mimics had a significant reduction in the expression of c-Met protein(t=-8.509, P=0.001). Conclusion By inhibiting the protein expression of c-Met, miR-409-3p regulates downstream signaling pathways to induce cell cycle arrest in G2 phase and thus inhibits the proliferation of hepatoma HepG2 cells.更多还原