小RNA干扰技术沉默血管内皮生长因子-C对人肝癌HepG2细胞增殖及凋亡的体外研究

Effect of silenced vascular endothelial growth factor-C through small RNA interference technique on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells in vitro

目的用小RNA干扰(siRNA)技术在体外研究其对人肝癌HepG2细胞的增殖及凋亡作用。方法将3组siRNA-VEGF-C质粒分别瞬时转染到HepG2细胞中,用qRT-PCR和免疫印迹法检测转染前后VEGF-C基因的表达,挑选干扰效率最强的一组表达载体用于后续实验;在后续实验将细胞分为3组:空白对照组,阴性对照组,siRNA-VEGF-C1组。通过噻唑蓝比色法(MTT)检测HepG2细胞增殖情况;Hoechst 33258染色法检测HepG2细胞核形态变化;流式细胞仪检测人肝癌HepG2细胞周期及凋亡的变化。结果各干扰组VEGF-C与空白对照组比较,基因和蛋白水平明显降低,siRNA-VEGF-C1抑制率最高达80.0%,用siRNA-VEGF-C1进行后续实验。siRNA-VEGF-C1组的生长曲线平坦,细胞生长率低于对照组。空白对照组、阴性对照组,siRNA-VEGFC1组的早期凋亡率分别是(0.25±0.09)%,(4.80±1.62)%,(20.10±3.41)%,siRNA-VEGF-C1组与空白对照组比较,差异有统计学意义(P<0.01)。空白对照组、阴性对照组,siRNA-VEGF-C1组的G0/G1期细胞数分别是(62.28±3.28)%,(62.20±4.81)%,(76.46±4.69)%,siRNAVEGF-C1组与空白对照组比较,差异有统计学意义(P<0.01)。结论针对人VEGF-C基因的siRNA表达载体能抑制VEGF-C的基因表达,并可抑制HepG2细胞增殖,诱导其凋亡。

Objective To investigate the effect of silenced vascular endothelial growth factor-C（ VEGF-C） through small interfering RNA（ siRNA） technique on the proliferation and apoptosis of human hepatocellular carcinoma HepG2 cells in vitro. Methods Three siRNA-VEGF-C plasmids were transiently transfected into HepG2 cells. The transcription and translation level of vascular endothelial growth factor-C（ VEGF-C） expression were detected by qRT-PCR and Western blot. The expression vector with the highest interference efficiency was selected for subsequent test. The subsequent experiment was divided into three groups： blank control group,negative control group,siRNA-VEGF-C group. The effect on the proliferation of HepG2 cells was detected by 3-（ 4,5-dimethyl-2-thiazolyl）-2,5 – diphenyl-2-H-tetrazolium bromide（ MTT） assay. The nucleus morphological changesof HepG2 cells was observed by Hoechst 33258 fluorescence staining. The changes of cycle and apoptosis of HepG2 cells were detected by Flow cytometry. Results The expressions of VEGF-C gene and protein in each interference group significantly reduced when compared with the blank control group. The inhibition rate of siRNA-VEGF-C1 was up to 80. 0%,therefore,siRNA-VEGF-C1 was used for subsequent experiments. The growth curve of siRNA-VEGF-C1 group was flat and the cell growth rate was lower than that of control group. The early apoptotic rates in the blank control group,negative control group and siRNA-VEGF-C group were（ 0. 25 ± 0. 09） %,（ 4. 80 ± 1. 62） %,（ 20. 10 ± 3. 41） %,respectively; and the number of G0/G1 cells in the blank control group,negative control group and siRNA-VEGF-C group were（ 62. 28 ± 3. 28） %,（ 62. 20 ± 4. 81） % and（ 76. 46 ± 4. 69） %,respectively.Compared with the blank control group,the differences of apoptotic rates and G_0/G_1 cells in siRNA-VEGF-C group were statistically significant（ all P 0. 01）. Conclusion VEGF-C gene silenced by siRNA can inhibit the proliferation and induce the apoptosis of human hepatocellular carcinoma HepG2 cells. The process might serve as a potential approach for cancer gene therapy.