shRNA干扰SMYD3对肝癌细胞c-Myc表达及凋亡的影响

Effects of shRNA-induced SMYD3 silence on c-Myc expression and apoptosis of HepG2 cells

目的:观察SMYD3(SET and MYND-domain containing3)基因沉默后c-Myc的表达及对HepG2细胞凋亡的影响.方法:构建针对SMYD3的shRNA干扰质粒Pgenesil-1-s1、Pgenesil-1-s2和阴性对照质粒Pgenesil-1-hk,同时设空白对照组,采用Lipofectamine2000脂质体介导转染法转染质粒.转染后24、48、72h,RT-PCR检测HepG2细胞SMYD3和c-Myc的表达情况.流式细胞术法检测各组细胞的凋亡.结果:SMYD3、c-Myc基因在HepG2细胞中强表达.RT-PCR显示Pgenesil-1-s1、Pgenesil-1-s2转染组与阴性对照质粒转染组Pgenesil-1-hk转染24、48、72h后相比,SMYD3基因表达均明显受到抑制(F=67.46,P<0.01;F=176.79,P<0.01;F=175.28,P<0.01),同时c-Myc表达下调(三组之间:F=11.58,P=0.009;F=126.41,P<0.01;F=261.25,P<0.01).Pgenesil-1-s1、Pgenesil-1-s2转染组细胞早期凋亡率与Pgenesil-1-hk转染组(LSD-t=-13.58,-12.62,均P<0.01)、空白组(LSD-t=-18.62,-17.67,均P<0.01)相比有显著性差异.结论:RNA干扰技术特异性沉默HepG2细胞SMYD3基因后,抑制了c-Myc的表达,促进了HepG2细胞的凋亡.

AIM： To observe the c-Myc gene expression and apoptosis of HepG2 cells after SET and MYND-domain containing 3 （SMYD3） silence induced by short hairpin RNA. METHODS： Three short hairpin RNA interference plasmids targeting SMYD3 were prepared as 3 groups： Pgenesil-1-s1 （with interfering effect）, Pgenesil-1-s2 （with interfering effect）, and Pgenesil-1-hk （negative control plasmid, without interfering effect）. Meanwhile, an empty controlgroup was also designed. Transfection was per- formed using the Lipofectmine2000 liposome. Reverse transcription-polymerase chain reaction （RT-PCR） was employed to detect the expression of SMYD3 and c-Myc gene 24, 48 and 72 h after transfection. Flow cytometry （FCM） was used to detect cell apoptosis in each group. RESULTS： SMYD3 and c-Myc gene were strongly expressed in HepG2 cells. The expression of SMYD3 gene was signifi cantly inhibited after 24-, 48- and 72-h transfection （F = 67.46, P 〈 0.01; F = 176.79, P 〈 0.01; F = 175.28, P 〈 0.01）. At the same time, c-Myc gene mRNA expression was down-regulated in the Pgenesil-1 transfected group as compared with that in the Pgenesil- 1-hk group （F = 11.58, P = 0.009; F = 126.41, P 〈 0.01; F = 261.25, P 〈 0.01）. Moreover, the early apoptosis rate was significantly higher in the Pgenesil-1-s1 or Pgenesil-1-s2 group than that in the Pgenesil-1-hk group （LSD-t = -13.58, -12.62; both P 〈 0.01） and the empty control group （LSD-t = -18.62, -17.67; P 〈 0.01）. CONCLUSION： Short hairpin RNA interference targeting SMYD3 may inhibit the expression of c-Myc gene in HepG2 cells, thus promoting the apoptosis of HepG2 cells.