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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SMYD3 mRNA在肝癌细胞系HepG2、Hep3B、SMMC7721的表达。构建重组SMYD3 shRNA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(W estern b lot)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SMYD3 mRNA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14 d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。
Objective To construct short hairpin RNA (shRNA) interference plasmids targeting human SET- and MYND-domain containing protein 3 (SMYD3) genes and to study their effect on hepatoma cells. Methods The expression of SMYD3 mRNA in HepG2, Hep3B and SMMC7721 cells was detected by RT-PCR. Recombinant SMYD3 shRNA expression plasmid Pgenesil-1-s was constructed and transfected into HepG2 cells by Western Blot. Western blotting was used to detect the inhibitory effect. MTT assay was used to detect the proliferation and proliferation of HepG2 cells. Inhibition rate, flow cytometry apoptosis rate. The human hepatocellular carcinoma HepG2 transplanted tumor model in nude mice was established. Pgenesil-1-s was injected into the nude mice. The tumor volume was observed dynamically and the nude mice were sacrificed after 2 weeks. Results The expression of SMYD3 mRNA in HepG2, Hep3B and SMMC7721 cells was significantly higher than that in normal liver cell line L-02. The SMYD3 protein expression was significantly down-regulated and the apoptosis rate was significantly increased in HepG2 cells transfected with Pgenesil-1-s, After 14 days of recombinant plasmids treatment, the growth of subcutaneous xenografts in nude mice was slower than that in control group, and the tumor weight was significantly reduced (P <0.01). Conclusion High expression of SMYD3 in hepatocellular carcinoma cells and shRNA interference specifically inhibit the expression of SMYD3, which can promote the apoptosis of hepatoma cells and inhibit the growth of xenografts in nude mice.