自噬对肝癌SMMC-7721细胞侵袭迁移能力的影响

Effect of Autophagy on Invasion and Migration of Hepatocellular Carcinoma SMMC-7721 Cells

关键词:  肝细胞癌  自噬  照射  侵袭  迁移  EMT

Key words:  hepatocellular carcinoma  autophagy  radiotherapy  invasion  migration  EMT

摘要:

为探讨自噬对照射过程中肝癌SMMC-7721细胞侵袭和迁移能力的影响及其潜在的作用机制,将肝癌SMMC-7721细胞分为6组,分别为阴性对照(NC)组、8 Gy X-线照射(IR)组、自噬激活剂雷帕霉素(Rapa)组、自噬抑制剂三甲基腺嘌呤(3-MA)组、照射+雷帕霉素(IR+Rapa)组和照射+三甲基腺嘌呤(IR+3-MA)组,利用划痕实验和Transwell实验检测自噬对肝癌SMMC-7721细胞迁移和侵袭的影响;用CCK8实验检测照射、雷帕霉素和3-MA对肝癌细胞增殖的影响;用蛋白免疫印迹(Western blot)检测N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)和LC3B蛋白表达情况。研究结果显示,照射可增强肝癌SMMC-7721细胞侵袭迁移的能力(P<0.05),同时,照射可抑制细胞的增殖能力(P<0.05);雷帕霉素激活自噬可增强细胞的侵袭迁移能力(P<0.05),3-MA抑制自噬可抑制细胞侵袭迁移和细胞的增殖能力(P<0.05)。照射可上调N-cadherin、Vimentin表达水平(P<0.05),激活或抑制自噬可分别增加或抑制N-cadherin、Vimentin蛋白表达水平(P<0.05)。表明X-线照射可激活自噬,促进细胞上皮-间充质转化(Epithelial-Mesenchymal Transition,EMT)的发生。激活自噬可以提高肝癌细胞上皮间质转化进而促进肝癌细胞侵袭迁移;反之,抑制自噬可阻碍肝癌细胞的侵袭迁移。

Abstract:

To investigate the effect of autophagy on the invasion and migration of Hepatocellular Carcinoma (HCC) SMMC-7721 cells during irradiation and its potential mechanism,the HCC SMMC-7721 cells were divided into 6 groups,namely negative control (NC) group,8 Gy X-ray irradiation (IR) group,autophagy activator rapamycin (Rapa) group,autophagy inhibitor trimethyl Adenine (3-MA) group,irradiation+rapamycin (IR+Rapa) group,irradiation+trimethyladenine (IR+3-MA) group.The effects of autophagy on the migration and invasion of HCC SMMC-7721 cells were detected by scratch assay and Transwell assay.CCK8 assay was used to detect the effects of irradiation,rapamycin and 3-MA on the proliferation of HCC cells.The protein expressions of N-cadherin,Vimentin and LC3B were detected by Western blot.The results showed that irradiation could enhance the ability of invasion and migration of SMMC-7721 cells (P<0.05),and at the same time,irradiation could inhibit the proliferation of cells (P<0.005).Rapamycin activated autophagy could promote the ability of cell invasion and metastasis (P<0.05),and 3-MA inhibited autophagy could inhibit the ability of cell invasion,migration and cell proliferation (P<0.05).Irradiation could up-regulate the protein expression levels of N-cadherin and Vimentin (P<0.05),and activation or inhibition of autophagy could increase or inhibit the protein expression levels of N-cadherin and Vimentin (P<0.05).It indicated that X-ray irradiation could activate autophagy and promote the occurrence of Epithelial-Mesenchymal Transition (EMT).Activation of autophagy could improve epithelial-mesenchymal transition of HCC cells and promote invasion and migration of HCC cells.On the contrary,inhibition of autophagy could hinder the invasion and migration of HCC cells.