miRNA-30a-3p下调Atg3介导的自噬抑制肝癌细胞侵袭和转移

MiRNA-30a-3p inhibit the invasion and metastasis of liver cancer cells through down-regulating Atg3-mediated autophagy pathway

目的探讨miRNA-30a-3p通过靶向作用自噬相关蛋白3（Atg3）介导的自噬通路，对肝癌细胞增殖、侵袭和转移的影响。方法利用免疫组化检测人肝癌肝组织中miRNA-30a-3p、Atg3含量及其相关性：体外培养肝癌细胞，模拟饥饿环境诱导自噬，LC3自噬双标腺病毒转染肝癌细胞以检测自噬体形成情况。蛋白印迹实验（Western blot）检测自噬相关蛋白[自噬相关蛋白3（Atg3）、泛素结合蛋白（p62）、自噬微管相关蛋白轻链3（LC3）]和上皮-间充质转化（epithelial mesenchymal transition，EMT）相关蛋白[钙黏附分子N（N—cadherin）、波形蛋白（vimentin）、snail蛋白、胞质紧密粘连蛋白1（ZO-1）]表达水平。CCK-8试剂盒检测肝癌细胞活性。结果促进肝癌细胞中miRNA-30a-3p表达，可降低Atg3、LC3表达、增加p62表达，抑制自噬体形成；反之，可增加Atg3、LC3表达、降低p62表达，促进自噬体形成。抑制A邸表达，可使EMT相关蛋白表达降低。抑制miRNA-30a-3p表达，肝癌活性细胞数量在各个时间点呈升高趋势（F1=10．314，P〈0．05）；而抑制miRNA-30a-3p表达的同时抑制Atg3表达，肝癌活性细胞数量在各个时间点呈下降趋势（F2=6．599，P〈0．05）。结论miRNA-30a-3p能抑制Atg3介导的自噬通路，降低细胞自噬活性，从而抑制肝癌细胞增殖、侵袭和转移。

Objective To investigate the effect of miRNA-30a-3p on the proliferation, invasion and metastasis of liver cancer cells by targeting Atg3-mediated autophagy pathway. Methods The immunohistochemical staining was used to detect content of miRNA-30a-3p and Atg3 and their correlation in human hepatocellular carcinoma. Liver cancer cells were cultured in vitro and hunger environment was used to induce autophagy. RFP-GFP-LC3 double-labeled adenovirus infected hepatoma cells were used to detect autophagosomes in hepatoma cells. The expressions of autophagy-related proteins （autophagocytosis associated protein （Atg3）, polyubiquitin-binding protein p62, autophagy microtubule-associated protein light chain 3 （ LC3 ） ） and EMT-related proteins （ N-cadherin, vimentin, snail, ZO-1 ） were detected by Western blot. Platelet cloning assay and transwell assay were carried out to detect the proliferation,invasion and metastasis of carcinoma cell. CCK-8 kit was used to detect hepatocarcinoma cells＇ viability. Results The expression of miRNA-30a-3p was down-regulated. The expression of Atg3, E-cadherin and N-cadherin in miRNA-30a-3p high-expressed hepatocellular carcinoma was lower than that in miRNA-30a-3p low-expressed hepatocellular carcinoma. Increasing the expression of miRNA-30a-3p in hepatocellular carcinoma cells can decrease the expression of Atg3 and LC3, increase the expression of p62 and inhibit the formation of autophagosomes ; otherwise, Atg3 and LC3 were increased, p62 was decreased and the formation of autophagosomes was promoted. Inhibition of Atg3 expression could decrease the expression of EMT-related proteins. When miRNA-30a-3p was inhibited, the cell viability of HCC was increased at each time point （F1 = 10. 314,P 〈 0. 05 ）. When miRNA-30a-3p and Atg3 were inhibitor together, the cell viability of HCC was decreased at each time point（ F2 = 6. 599,P 〈 0. 05 ）. Conclusion miRNA-30a-3p can inhibit Atg3- mediated autophagy pathway and reduce cell autophagy activity, thus inhibiting the proliferation, invasion and metastasis of hepatocarcinoma ceils.