目的：检测eEF-2K基因在肝细胞癌（HCC）组织内的表达并分析其表达水平对HCC细胞增殖、侵袭和凋亡的影响。方法：用RT-qPCR及Western blot方法检测了64例肝癌组织及癌旁肝组织、五种HCC细胞系LM3, Hep3B, Huh7, 97H, HepG2及人正常肝细胞系 LO2中eEF-2K基因在mRNA及蛋白水平的表达。用eEF-2K 特异性siRNA沉默LM3及Hep3B细胞性中eEF-2K基因后，经qPCR检测确认。随后用CCK-8，Transwell方法与流式细胞技术检测eEF-2K基因沉默对增殖、侵袭与凋亡的影响。结果：相较癌旁组织，HCC组织内eEF-2K mRNA与蛋白表达显著上调（P＜0.05）；与正常人肝细胞系LO2相比，肝癌细胞系LM3, Hep3B, 97H, HepG2, Huh7中的eEF-2K mRNA和蛋白表达分别升高（Ｐ＜0.05）。在LM3及Hep3B细胞系中用特异性siRNA沉默eEF-2K后，与非特异性siRNA对照组相比，细胞增殖活性和侵袭能力显著降低（P＜0.05），凋亡率显著升高（P＜0.05）。TCGA数据库分析结果表明eEF-2K高表达与肝癌预后不良相关（P＜0.05）。结论：eEF-2K蛋白在肝癌组织和细胞中高表达且与预后不良相关；沉默eEF-2K后肝癌细胞增殖和侵袭能力降低并诱发凋亡，提示eEF-2K可用作HCC潜在的预后标志物和治疗靶点。

Objective: To detect the expression level of eEF-2K in hepatocellular carcinoma (HCC) and the effect of its silencing on proliferation, invasion and apoptosis of HCC cells. Methods: RT-qPCR and Western blot were used to examine the expression of eEF-2K gene at mRNA and protein levels in HCC and their adjacent liver tissues resected from 64 HCC patients, as well as in five HCC lines (LM3, Hep3B, Huh7, 97H, HepG2) and normal human liver cell line LO2. LM3 and Hep3b cells were transfected with eEF-2K specific and non-specific siRNAs respectively . The silencing of eEF-2K gene after siRNA trasfection in LM3 and Hep3B cell lines was confirmed by RT-qPCR. The effect of silencing of eEF-2K gene on proliferation, invasion and apoptosis of HCC were detected by CCK-8 assay, Transwell assay and flow-cytometry. Result: The expression of eEF-2K was significantly up-regulated both at mRNA and protein levels in HCC tissues as well as in LM3, HEP3B, 97H, Hepg2, Huh7 lines compared to the expressions in adjacent tissues (P＜0.05) and in normal LO2 line (P＜0.05) . The proliferation rates of eEF-2K-silenced groups were decreased compared to control group (p < 0.05) , and the percentages of transmembrane cells in eEF-2K-silenced groups groups were decreased compared to control group (P＜0.05). Silencing of eEF-2K by siRNA also led to increased apoptosis in HCC (p < 0.05) ..The analysis of TCGA database showed that the high expression of eEF-2K was associated with poor prognosis of HCC (P＜0.05) . Conclusion: eEF-2K was highly expressed in HCC tissues and cell lines, which is associated with poor prognosis. Knock-down of eEF-2K gene by siRNA inhibited the proliferation and invasion ability, and induced apoptosis in HCC cells. This study provided evidence that eEF-2K is potential prognostic marker and therapeutic target for HCC.

内蒙古医科大学“致远”人才计划“治学”人才团队项目(ZY0130011)