目的探讨HBx基因通过调节miR-192的表达影响人肝癌细胞株HepG2周期进展的机制。方法流式细胞仪分析以下3组细胞的周期变化:HepG2/HBx细胞(HepG2细胞稳定转染HBx基因)、HepG2/pcDNA3.1细胞(HepG2细胞稳定转染空载体pcDNA3.1)以及HepG2细...目的探讨HBx基因通过调节miR-192的表达影响人肝癌细胞株HepG2周期进展的机制。方法流式细胞仪分析以下3组细胞的周期变化:HepG2/HBx细胞(HepG2细胞稳定转染HBx基因)、HepG2/pcDNA3.1细胞(HepG2细胞稳定转染空载体pcDNA3.1)以及HepG2细胞。3组细胞中miR-192的表达采用Taqman探针荧光定量PCR法检测。流式细胞术观察转染miR-192后HepG2细胞的周期分布变化,SYBR Green荧光定量PCR和Westernblot分别检测miR-192对HepG2细胞p53、CDKN1A mRNA和蛋白表达的影响。结果 3组细胞中,HepG2/HBx细胞G0/G1期细胞比例明显降低[(52.78±4.08)%vs(67.37±4.87)%,(65.08±5.15)%],S期和G2/M期比例明显升高[S期:(25.22±1.84)%vs(19.78±1.26)%,(18.84±1.68)%;G2/M期:(22.00±2.07)%vs(12.85±1.29)%,(16.08±1.44)%]。HepG2/HBx细胞miR-192表达显著下调[(49.1±5.9)%vs(98.0±8.9)%,(100.0±9.1)%]。转染miR-192引起HepG2细胞G0/G1期和G2/M期阻滞,同时p53、CDKN1A mRNA(p53:1.68±0.12 vs 0.90±0.06;CD-KN1A:2.36±0.12 vs 1.05±0.06)和蛋白(p53:3.07倍;CDKN1A:2.82倍)的表达水平亦显著上升。结论 miR-192通过促进p53和CDKN1A表达引起HepG2细胞周期阻滞,而HBx通过下调miR-192加速HepG2细胞周期进程。展开更多
To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHa and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatoc...To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHa and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the β-actin as the interior control, real-time polymerase chain reaction （Real-time qPCR） was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHα in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector （HepG2/pDNA3.1）. The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHα in the HepG2/HBx （0.021±0.007） was significantly lower than that of HepG2 （0.099±0.041） （P〈0.05） and HepG2/pDNA3.1 （0.121±0.005） （P〈0.05）. However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains （P〉0.05）. The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 （P〈0.05）. It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHα mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.展开更多
文摘目的探讨HBx基因通过调节miR-192的表达影响人肝癌细胞株HepG2周期进展的机制。方法流式细胞仪分析以下3组细胞的周期变化:HepG2/HBx细胞(HepG2细胞稳定转染HBx基因)、HepG2/pcDNA3.1细胞(HepG2细胞稳定转染空载体pcDNA3.1)以及HepG2细胞。3组细胞中miR-192的表达采用Taqman探针荧光定量PCR法检测。流式细胞术观察转染miR-192后HepG2细胞的周期分布变化,SYBR Green荧光定量PCR和Westernblot分别检测miR-192对HepG2细胞p53、CDKN1A mRNA和蛋白表达的影响。结果 3组细胞中,HepG2/HBx细胞G0/G1期细胞比例明显降低[(52.78±4.08)%vs(67.37±4.87)%,(65.08±5.15)%],S期和G2/M期比例明显升高[S期:(25.22±1.84)%vs(19.78±1.26)%,(18.84±1.68)%;G2/M期:(22.00±2.07)%vs(12.85±1.29)%,(16.08±1.44)%]。HepG2/HBx细胞miR-192表达显著下调[(49.1±5.9)%vs(98.0±8.9)%,(100.0±9.1)%]。转染miR-192引起HepG2细胞G0/G1期和G2/M期阻滞,同时p53、CDKN1A mRNA(p53:1.68±0.12 vs 0.90±0.06;CD-KN1A:2.36±0.12 vs 1.05±0.06)和蛋白(p53:3.07倍;CDKN1A:2.82倍)的表达水平亦显著上升。结论 miR-192通过促进p53和CDKN1A表达引起HepG2细胞周期阻滞,而HBx通过下调miR-192加速HepG2细胞周期进程。
基金supported by a grant from the National Natural Sciences Foundation of China (No.30570821)
文摘To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHa and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the β-actin as the interior control, real-time polymerase chain reaction （Real-time qPCR） was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHα in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector （HepG2/pDNA3.1）. The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHα in the HepG2/HBx （0.021±0.007） was significantly lower than that of HepG2 （0.099±0.041） （P〈0.05） and HepG2/pDNA3.1 （0.121±0.005） （P〈0.05）. However, the no significant differences existed in the expression of DNA repair enzyme hOGG1 among the three cell strains （P〉0.05）. The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 （P〈0.05）. It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHα mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.
