RNA干扰技术靶向SMYD3基因治疗肝癌的实验研究

目的 构建针对人SET-和MYND-结构域含有蛋白3（SMYD3）编码基因的短发夹状RNA（shRNA）干扰质粒,并研究其对肝癌细胞的作用。方法 应用逆转录聚合酶链反应（RT-PCR）方法检测SMYD3mRNA在肝癌细胞系HepG2、Hep3B、SMMC7721的表达。构建重组SMYD3shRNA表达质粒Pgenesil-1-s,并采用介导转染法导入HepG2细胞,蛋白免疫印迹法（Western blot）检测阻抑效应,噻唑蓝比色法（MTT）检测细胞生长增殖抑制率,流式细胞术检测细胞凋亡率。建立人肝癌细胞HepG2皮下移植瘤裸鼠模型,注射Pgenesil-1-s,动态观察肿瘤体积并于2周后处死裸鼠称取瘤重。结果肝癌细胞株HepG2、Hep3B、SMMC7721的SMYD3mRNA表达水平显著高于正常肝细胞株L-02;Pgenesil-1-s转染HepG2细胞后,SMYD3蛋白表达明显下调,而且细胞凋亡率显著增加,细胞生长明显受抑;经重组质粒治疗14d后,裸鼠皮下移植瘤生长缓慢,相较于对照组瘤体明显缩小、瘤重明显减轻（P〈0．01）。结论 肝癌细胞高表达SMYD3,shRNA干扰特异性抑制SMYD3表达,可以促进肝癌细胞凋亡并抑制裸鼠移植瘤的生长。

Experimental research of therapeutic effect on hepatocellular carcinoma of targeting SMYD3 gene inhibition by RNA interference

Objective To determine the potential of SMYD3 as a therapeutic target for hepatocellular carcinoma(HCC) by potent and highly sequence-specific RNA interference(RNAi) technique.Methods The mRNA of SMYD3 was detected by RT-PCR in different HCC cell lines,such as HepG2,Hep3B and SMMC7721.Recombinant SMYD3 shRNA plasmid Pgenesil-1-s was constructed and transfected into HepG2 cells,and Western blot was used to identify the down regulation of SMYD3 protein expression after transfection.MTT and flow cytometry analysis(FCM) were respectively applied to analysis cell proliferation and apoptosis.In vivo study was carried out by injecting recombinant SMYD3 shRNA plasmids into transplanted tumors of nude mice.Results The expression of SMYD3 mRNA was abundant in HCC cell lines HepG2,Hep3B,SMMC7721,whereas none in normal hepatic cell line L-02.RNA interference was able to suppress SMYD3 expression greatly and then inhibited cell growth effectively and induced apoptosis of HepG2 cells efficiently.After injection of recombinant SMYD3 shRNA plasmid,transplanted tumors grew slowly and reduced in size and weight when compared with those of control groups(P<0.01).Conclusions SMYD3 plays a major role in occurrence and progress of HCC.Inhibition of SMYD3 by RNAi can induce apoptosis in HepG2 cells and suppress tumor growth in nude mice.Therefore SMYD3 could be an ideal therapeutic target for HCC.