摘要
目的应用siRNA干扰技术抑制人肝细胞癌系HepG2细胞MTH1基因表达,并研究MTH1基因干扰后对HepG2细胞凋亡和迁移能力的影响。方法设计并化学合成3对特异性MTH1-siRNA,采用阳离子脂质体法瞬间转染肝癌HepG2细胞,离体培养肝癌HepG2细胞,并设正常对照组、阴性对照组及MTH1-siRNA1、MTH1-siRNA2、MTH1-siRNA3转染组。采用蛋白印迹(Western blot)法检测转染MTH1-siRNA后HepG2细胞MTH1蛋白水平表达的变化,并筛选干扰效果最佳序列。运用流式细胞技术和划痕实验检测转染siRNA-MTH1后HepG2细胞凋亡、体外迁移能力的变化。结果 MTH1-siRNA3转染组干扰效果与正常组和阴性对照组相比效果具有统计学意义(P=0.002,P=0.005)。MTH1-siRNA3转染组细胞凋亡率高于正常组及阴性对照组,差异有统计学意义(P=0.012,P=0.013);MTH1-siRNA3转染组细胞的划痕24、36 h愈合率均低于正常组和阴性对照组,差异有统计学意义(P=0.001,P=0.000)。结论 MTH1在肝癌细胞的凋亡及迁移中起重要作用,干扰MTH1的表达能促进肝癌细胞凋亡并降低肝癌细胞的迁移能力。
Objective To investigate the inhibitory effect of small interfering RNA（siRNA） on the expression of MTH1 in human hepatocelluar carcinoma HepG2 cells, and to observe the effect of MTH1 silencing on the apoptosis and migration of human hepatocelluar carcinoma HepG2 cells. Methods Three pairs of specific siRNA targeting MTH1 were designed, synthesized, and then transiently transfected into HepG2 cells via cationic liposome transfection method. In vitro cultured HepG2 cells were divided into normal control group, negative control group and three transfection groups： MTHI-siRNA1, MTHI-siRNA2, MTHI-siRNA3. Then the protein levels of MTH1 were detected by Western blot, and we selected the best interfered MTH1- siRNA. Cell apoptosis was assessed by flow cytometry, and cell migratory ability was determined by wound healing assay. Results MTHI-siRNA3 had better interfering effect than control normal group and negative control group （（0.24±0.072） vs. （0.87±0.068） and （0.24±0.072） vs. （0.84±0.086）, P=0.002 and P=0.005）. Flow cytometry analysis showed that cell apoptosis rate was significantly higher in the MTHI-siRNA3 group than those in normal control group and negative control group （（21.81±3.58） % vs. （5.49±2.18）%, and （21.81± 3.58）% vs. （6.47±2.41）%, P=0.012 and P=0.013）. Wound healing assay suggested that the relative migration abilities of HepG2 cells in the MTHI-siRNA3 group at 24, 36h after transfection were significantly lower than those in normal control group and negative control group （（51.35±4.25）% vs. （70.33±3.58）% and （51.35± 4.25）% vs. （69.28±3.67）%, P=0.001, （82.16±2.39）% vs. （99.28±0.43）% and （82.16±2.39）% vs. （98.68±0.47）%, P=0.000）. Conclusion MTHI plays an important role in the apoptosis and migration of HepG2 cells. The inhibition of MTH1 expression could promote the apoptosis and reduce the migration of hepatocellular carcinoma HepG2 cells.
出处
《肿瘤防治研究》
CAS
CSCD
北大核心
2016年第12期1049-1054,共6页
Cancer Research on Prevention and Treatment
基金
江西省自然科学基金(20151BBG70176)
