摘要
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.
作者
杜晨阳
张建军
王振
宋虎
李世朋
张海明
郑虹
沈中阳
Du Chenyang;Zhang Jianjun;Wang Zhen;Song Hu;Li Shipeng;Zhang Haiming;Zheng Hong;Shen Zhongyang(First Central Clinical College of Tianjin Medical University, Tianjin 300192, China)
出处
《中华普通外科杂志》
CSCD
北大核心
2018年第4期334-337,共4页
Chinese Journal of General Surgery
基金
天津市器官移植临床医学研究中心基金资助项目(15XLCSY00070)
国家高技术研究发展计划基金资助项目(863)(2012AA021001)
卫生公益行业科研专项基金资助项目(201302009)
关键词
癌
肝细胞
微RNAS
自噬
肿瘤转移
Carcinoma, hepatocellular
MicoRNAs
Autophagy
Neoplasm metastasis