微小RNA-375调控自噬抑制肝癌细胞SMMC-7721增殖和转移

目的 探讨微小 RNA-375（miR-375）通过调控自噬相关蛋白（Atg）14 介导的自噬对肝癌细胞（SMMC-7721）增殖和转移能力的影响。方法 将SMMC-7721细胞分别用miR-375模拟物、抑制物以及Atg14的干扰RNA处理，并建立缺氧1 h复氧6 h的模型，分为miR-375 NC组、miR-375 mimics组、miR-375 inhibitor组以及siRNA NC组、Atg14 siRNA 组、miR-375+Atg14 siRNA 组。TargetScan 预测 miR-375 与 Atg14 基因相关性。荧光实时定量 PCR（qRT-PCR）检测miR-375 NC组、miR-375 mimics组、miR-375 inhibitor组miR-375、Atg14 mRNA相对表达量。免疫细胞化学染色检测3组细胞内N-钙黏素（N-Cadherin）和β-连环蛋白（β-catenin）表达分布情况。绿色荧光蛋白-红色荧光蛋白-轻链（mGFP-RFP-LC3）自噬双标腺病毒转染3组细胞，荧光显微镜下观察自噬体形成情况。平板克隆检测肝癌细胞增殖情况。Western blot 检测Atg14、泛素结合蛋白（P62）、轻链3Ⅰ（LC3Ⅰ）、轻链3Ⅱ（LC3Ⅱ）、酵母Atg6同源物（Beclin1）、N-Cadherin、β-catenin和波形蛋白（Vimentin）表达情况。结果 miR-375与Atg14基因相关性较高，qRT-PCR显示过表达miR-375能抑制Atg14 mRNA表达；反之能促进Atg14 mRNA表达。过表达miR-375能抑制肝癌细胞内N-Cadherin表达、提高β-catenin表达水平，抑制肝癌细胞增殖能力（P＜0.01）；而抑制miR-375表达能促进N-Cadherin表达、降低β-catenin表达水平，提高肝癌细胞增殖能力（P＜0.01）。过表达miR-375能抑制Atg14、LC3Ⅱ、Beclin1表达，促进P62表达（P＜0.01）；反之能促进Atg14、LC3Ⅱ、Beclin1表达，抑制P62表达（P＜0.01）。荧光显微镜下观察到过表达 miR-375 可抑制自噬体形成，而抑制 miR-375 表达能促进自噬体形成（P＜0.01）。干扰Atg14表达后可增强miR-375对肝癌细胞增殖、转移能力的抑制作用（P＜0.01）。结论 激活miR-375能抑制肝癌细胞增殖和转移，而抑制miR-375能促进肝癌细胞增殖和转移，其机制可能与miR-375通过靶向调控Atg14抑制自噬，从而抑制SMMC-7721肝癌细胞增殖和转移有关。

MicroRNA-375 inhibited proliferation and metastasis of hepatoma cell line SMMC-7721 by regulating autophagy

Objective To investigate the effect of microRNA-375 (miR-375) targeting autophagy associated gene 14(Atg14)-mediated autophagy on the proliferation and metastasis of hepatoma cells (SMMC-7721). Methods SMMC-7721cells were divided into miR-375 mimics, miR-375 inhibitor and Atg14 interfering RNA treatment groups. Models with hypoxia for 1 h and reoxygenation for 6 h were established, and were divided into miR-375 NC group, miR-375 mimics group, miR-375 inhibitor group, siRNA NC group, Atg14 siRNA group and miR-375+Atg14 siRNA group. Targetscan was used to predict the genetic association between miR-375 and the Atg14. Fluorescence real-time quantitative PCR (QRTPCR) was used to detect the relative expressions of miR-375 and ATG14 mRNA in miR-375 NC group, miR-375 mimics group and miR-375 inhibitor group. Immunocytochemical staining was used to detect the expressions of N-Cadherin and β-catenin in the above three groups. The three groups of cells were transfected with GFP-RFP-LC3 adenovirus, and the formation of autophagosomes was observed by the fluorescence microscope. Plate cloning was used to detect the proliferation of liver cancer cells. The expressions of Atg14, P62, LC3Ⅰ, LC3Ⅱ, Beclin1, N-Cadherin, β-catenin and Vimentin were detected by Western blot assay. Results miR-375 gene was highly associated with the Atg14 gene. qRT-PCR showed that overexpression of miR-375 inhibited Atg14 mRNA expression, and conversely, it promoted Atg14 mRNA expression.Overexpression of miR-375 could inhibit the expression of N-Cadherin, increase the expression of β-catenin, and inhibit the proliferation of hepatoma cells (P＜0.01). On the contrary, the inhibition of miR-375 expression promoted the expression ofN-Cadherin, decreased β-catenin expression and increased the proliferation of liver cancer cells (P＜0.01). Overexpression of miR-375 inhibited the expression levels of Atg14, LC3Ⅱ and Beclin1, and promoted the expression of P62 (P＜0.01).Conversely, it promoted the expressions of Atg14, LC3Ⅱ and Beclin1, and inhibited the expression of P62 (P＜0.01). By fluorescence microscope, the inhibition of autophagosome formation with overexpression of miR-375 was observed, while the inhibition of miR-375 expression promoted autophagosome formation (P＜0.01). Interfering with the expression of Atg14 enhanced the inhibitory effect of miR-375 on the proliferation and metastasis of hepatoma cells (P＜0.01). Conclusion The activation of miR-375 can inhibit the proliferation and metastasis of hepatoma cells, while the inhibition of miR-375 can promote the proliferation and metastasis of hepatoma cells. The mechanism may be related to miR-375 activating Atg14 to inhibit hepatocellular autophagy, and further influence the proliferation and metastasis of hepatoma cellsSMMC-7721.