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目的:探讨Six同源盒蛋白4(Six homeobox 4，Six4)蛋白在肝癌中表达及对肝癌细胞增殖、迁移和上皮-间充质转化的影响。方法:选取2018年6月到2019年6月新乡医学院第一附属医院收集的169例肝癌组织和对应的癌旁组织作为研究对象，采用免疫组织化学分析肝癌组织和癌旁组织中Six4蛋白表达水平；采用常间回文重复序列丛集-Cas9(CRISPR-Cas9)在人类肝癌细胞HepG2中建立Six4敲除细胞系，命名为对照组和SIX4 KO组。采用细胞计数试剂盒(CCK-8)和5-乙炔基-2’脱氧尿嘧啶核苷(EdU)染色分析两组细胞增殖；采用Transwell分析两组细胞迁移；采用蛋白质印迹法(Western blot)分析上皮间质标志物上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)表达水平，计量数据比较采用n t检验。n 结果:肝癌组织中Six4蛋白表达水平(169.59±18.59)高于癌旁组织(79.43±10.43)，差异有统计学意义(n t＝4.179，n P<0.05)。本研究成功构建Six4敲除细胞系。Six4 KO组细胞吸光值(1.21±0.19)低于对照组(2.15±0.28)，差异有统计学意义(n t＝3.441，n P<0.05)。对照组细胞EdU染色阳性率(76.45±8.90)%明显高于Six4 KO组细胞(31.63±7.43)%，差异有统计学意义(n t＝4.693，n P<0.05)。Six4 KO组细胞迁移数量(65.64±9.99)个低于对照组(119.48±12.08)个，差异有统计学意义(n t＝3.748，n P<0.05)。Six4 KO组细胞E-cadherin相对表达水平(1.49±0.21)高于对照组(0.33±0.11)，差异有统计学意义(n t＝5.184，n P<0.05)。Six4 KO组细胞间质标记物N-cadherin和Vimentin相对表达水平(0.58±0.11、0.68±0.23)低于对照组(1.74±0.28、1.89±0.26)，差异均有统计学意义(n t＝4.274、4.109，n P<0.05)。n 结论:SIX4蛋白在肝癌组织中呈高表达，参与肝癌细胞增殖、迁移和上皮-间充质转化等恶性细胞生物学行为。“,”Objective:To investigate the expression of Six homeobox 4(Six4) protein in hepatocellular carcinoma (HCC) and its effects on the proliferation, migration and epithelial mesenchymal transition of HCC cells.Methods:A total of 169 cases of HCC tissues and corresponding adjacent tissues from June 2018 to June 2019 were selected as the research objects, and the expression levels of Six4 protein in liver cancer tissues and adjacent tissues were analyzed by immunohistochemistry. Six4 knockout cell lines were established in HepG2 cells by clustered regularly interspaced short palindromic repeats-cas9 (CRISPR-Cas9), named as control group and Six4 KO group. The cell proliferation in the control group and Six4 KO group was analyzed by cell counting kit-8 (CCK-8) and 5-ethynyl-2′-deoxyuridine (EdU) staining. The cell migration in the two groups was analyzed by Transwell. The expression of N-cadherin, E-cadherin and vimentin was analyzed by Western blotting.Results:Compared with the adjacent tissues (79.43±10.43), the expression level of Six4 protein in HCC tissues (169.59±18.59) was significantly increased (n t=4.179, n P<0.05). Six4 knockout cell line was successfully constructed by CRISPR-Cas9. Compared with the control group (2.15±0.28), the cell absorbance of Six4 KO group (1.21±0.19) significantly decreased (n t=3.441, n P<0.05). The positive rate of EdU staining in the control group (76.45±8.90)% was significantly higher than that in the Six4 KO group (31.63±7.43)%, and the difference was statistically significant (n t=4.693, n P<0.05). Compared with the control group (119.48±12.08), the number of migration cells in the Six4 KO group (65.64±9.99) significantly decreased (n t=3.748, n P<0.05). Compared with the control group (0.33±0.11), the relative expression level of E-cadherin in the Six4 KO group was significantly increased (1.49±0.21), and the difference was statistically significant (n t=5.184, n P<0.05). Compared with the relative expression levels of N-cadherin and vimentin in the control group (1.74±0.28, 1.89±0.26), the relative expression levels of N-cadherin and vimentin in the Six4 KO group (0.58±0.11, 0.68±0.23) were significantly decreased (n t=4.274, n P<0.05;n t=4.109, n P<0.05).n Conclusion:Six4 protein is highly expressed in HCC tissues, which is involved in the proliferation, migration and epithelial mesenchymal transition of HCC cells.