RNA干扰黏着斑激酶抑制人肝癌细胞sk-hep-1生长的实验研究

RNA Interference Targeting Focal Adhesion Kinase Inhibited the Growth of Human Hepatocellular Carcinoma sk-hep-1

目的通过运用RNA干扰技术(RNA interference,RNAi),干扰人肝癌细胞系sk-hep-1的黏着斑激酶(FAK)的表达,初步探讨干扰效果。方法在体外,用转染试剂FugeneHD将靶向FAK基因的小发夹RNA质粒(pshFAK)转染至sk-hep-1细胞中,通过流式细胞术、RT-PCR和Western bolt分析靶向FAK基因的RNAi对细胞的影响。在体内,用阳离子脂质体包装质粒,通过尾静脉注射治疗裸鼠皮下肿瘤模型,通过分析肿瘤生长曲线,肿瘤体积及瘤重,肿瘤组织的FAK、CD31、PCNA的免疫组化以及TUNEL,分析体内治疗效果。结果 sk-hep-1细胞经pshFAK作用后,FAK的mRNA及蛋白质水平均降低(P<0.001),凋亡率增高。皮下肿瘤模型经治疗后,生长速度缓于对照组,肿瘤大小及瘤重降低(P<0.001)。肿瘤标本的免疫组化及TUNEL显示肿瘤标本的FAK蛋白表达下调,血管生成减少,增殖减少及凋亡增加(P<0.001)。结论靶向FAK基因的RNAi可以抑制FAK在人肝癌细胞sk-hep-1的表达,抑制肿瘤生长速度,促进其凋亡,FAK可以成为有潜力的肝癌治疗的靶点。

Objective To test the impact of silencing focal adhesion kinase（FAK） gene on human hepatocellular carcinoma cell line sk-hep-1 using RNA interference（RNAi） in vitro,and its therapeutic effect on human hepatocellular carcinoma xenograft in vivo.Methods The plasmids generating short hairpin RNA that target FAK gene were transfected into sk-hep-1 cell line through Fugene HD.The impact was analyzed using flow cytometry,RT-PCR and western bolt.Human hepatocellular carcinoma xenograft in nude mice was established and treated with injection of the plasmids that packaged by cationic liposome into the tail veins the mice.The volumes and weights of the tumors were measured.The tumor tissues were analysed with FAK,CD31 and PCNA immunohistochemistry and TUNEL.Results The RNAi targeting FAK significantly down regulated the expression of FAK in mRNA and the level of protein（P0.001）,and increased the cell apoptosis in vitro. The mice treated with pshFAK had slower tumor growth,resulting in smaller tumor size and lower tumor weight（P0.001） comparing to the controls.Decrease in FAK protein expression,tumor angiogenesis and proliferation,and increase in tumor apoptosis were found in the mice treated with pshFAK compared to the controls（P0.001）.Conclusion The shRNA targeting FAK suppresses the FAK expression of human hepatocellular carcinoma cells sk-hep-1,inhibits tumor growth,and promotes cell apoptosis in vitro and in vivo.FAK could become a candidate target for liver cancer treatment.