稳定抑制PAK2蛋白表达的HUH—7细胞株的建立

朴莲淑　王福光　于庆功等

[摘要] 目的 为研究细胞周期相关因子PAK2在原发性肝癌中的作用，拟用shRNA干扰技术建立稳定抑制PAK2蛋白表达的HUH-7肝癌细胞系。 方法 用基因转染技术将人PAK2的3个shRNA片段分别克隆至慢病毒载体pHBLV-U6-ZsGreen-Puro，包装成病毒后利用脂质体将载体病毒转染至HUH-7细胞，利用G418筛选稳定表达shRNA的细胞。利用Real-time PCR和Western blotting方法分别鉴定转染细胞内PAK2的RNA及蛋白表达水平。 结果 sh1-PAK2 HUH-7、sh2-PAK2 HUH-7和sh3-PAK2病毒载体均可转染至HUH-7细胞系，且转染效率较高（>80%）。Real time-PCR结果显示，与对照组sh-cont比较，转染sh1-PAK2、sh2-PAK2和sh3-PAK可明显抑制HUH-7细胞内PAK2-mRNA的表达，差异均有统计学意义（均P < 0.05），其中sh2-PAK2和sh3-PAK2的干扰效果尤为明显，下调效率分别达68%和89%。Western blotting结果显示，转染sh3-PKA2细胞内PAK2的蛋白表达量较对照组sh-cont明显下调，其表达量为对照组sh-cont的14.4%。 结论 稳定抑制PAK2基因表达的肝癌细胞系shPAK2 HUH-7的建立为PAK2在肝癌细胞中的作用机制研究奠定细胞基础。

[关键词] PAK2；原发性肝癌；RNA干扰；HUH-7

[中图分类号] R735.7 [文献标识码] A [文章编号] 1673-7210（2015）09（c）-0014-04

[Abstract] Objective To study the role of cell cycle related factor PAK2 in primary hepatic carcinoma， shRNA interference technology was used to establish HUH-7 cell lines that could inhibit PAK2 protein expression stably. Methods Three shRNA fragments of human PAK2 were cloned into lentiviral vector pHBLV-U6-ZsGreen-Puro respectively by gene transfection technique， after they were transfected to virus， the carrier virus was transfected into HUH-7 cells by lipidosome， and G418 was used to screen cells that could express the shRNA stably. Real-time PCR and Western blotting were used to identify the expression levels of RNA and protein of PAK2 in transfection cells. Results All virus vectors of sh1-PAK2 HUH-7， sh2-PAK2 HUH-7 and sh3-PAK2 could be transfected into HUH-7 cell lines， and the transfection efficiency was high （>80%）. The results of Real time-PCR showed that， compared with control group sh-cont， transfection sh1-PAK2， sh2-PAK2 and sh3-PAK could inhibit the expression of PAK2-mRNA in HUH-7 cells， the differences were all statistically significant （all P < 0.05）， among which， the interference effectiveness of sh2-PAK2 and sh3-PAK2 was especially obvious， the down-regulated efficiency reached to 68% and 89% respectively. The results of Western blotting showed that， the protein expression level of PAK2 in sh3-PKA2 cells was lower than that of control group sh-cont， the expression level of which was 14.4% of control group sh-cont. Conclusion The establishment of cell line shRNA-PAK2 HUH-7 that can inhibit PAK2 genetic expression stably provides a cell model for mechanism study of PAK2 in hepatocellular carcinoma cells.

[Key words] PAK2； Primary hepatic carcinoma； RNAi； HUH-7

p21激活激酶（p21-activated kinases，PAKs）是一类丝氨酸/ 苏氨酸激酶，是小G蛋白Rho家族Rac和Cdc42重要的下游效应分子，可分为2个亚类：PAK2属于Ⅰ类范畴[1]。随着研究的不断深入，PAK2被证明作为Caspase酶的效应底物，在凋亡的过程中被激活，发现PAK2可以被Caspase裂解从而显示出催化活性[2]。近年来，有很多学者指出PAK2的高表达在乳腺癌、头颈部肿瘤、胃癌等多种肿瘤中，对其发生、发展中起促细胞增殖、抗凋亡作用，且在放射治疗的抵抗中起着重要作用，有望成为肿瘤靶向治疗的新靶点[3-7]。