反义端粒酶RNA与1,25-(OH)_2D_3抑制肝癌细胞增殖的协同作用

Promotion of 1,25-dihydroxyvitamin D_3 inhibited cell proliferation through down regulation of telomerase activity by transfected anti-sense telomerase RNA into hepatocarcinoma cells

【目的】观察1,25-(OH2)D3对转染反义端粒酶RNA的肝癌细胞增殖的作用。【方法】经脂质体介导,将反义端粒酶RNA真核表达载体pBBS212-hTR导入维生素D3受体(VDR)阳性的肝癌细胞株SMMC7721细胞中培养。细胞克隆转移扩大培养,添加1,25-(OH)2D3分别作用于各实验组细胞后,采用四唑蓝比色实验(MTT)和平板克隆形成实验,检测细胞的存活和生长;TUNEL法检测细胞凋亡。【结果】转染组细胞和对照组细胞的端粒酶活性分别为0.686和1.685,说明反义端粒酶RNA可显著降低肝癌细胞端粒酶活性。10 nmol/L浓度的1,25-(OH2)D3对肝癌细胞的增殖具有明显的抑制作用;且较之于单独的1,25-(OH2)D3或转染反义端粒酶RNA对肝癌细胞增殖的抑制作用,在转染反义端粒酶RNA,降低肝癌细胞端粒酶活性后,1,25-(OH2)D3抑制肝癌细胞增殖的作用显著得到增强。各组细胞的凋亡率分别为3.4%、12.2%、8.8%、23.6%。经统计学处理,上述实验结果证实均存在显著的差异(P<0.01)。【结论】在转染反义端粒酶RNA、降低细胞端粒酶活性的基础上,1,25-(OH2)D3抑制肝癌细胞增殖、诱导细胞凋亡的作用进一步得到增强。

【Objective】This study is to observe the inhibition of cell proliferation by 1,25-dihydroxyvitamin D3 after telomerase activity is blocked by transfected anti-sense telomerase RNA into hepatocarcinoma cells.【Methods】pBBS212/hTR,anti-sense telomerase RNA gene expression vector,was introduced into SMMC7721 cells with vitamin D receptor（VDR） by using lipofectin mediation.After the transfected cells were screened by hygromycin and cloned,the effects of anti-sense telomerase RNA on telomerase activity of SMMC7721cells were observe by TRAP-PCR-ELISA,and then the transfected cells were treated by 1,25-dihydroxyvitamin D3.MTT assay,plate clone formation assay,and TUNEL were employed to detect the rate of growth,the phenotypes,and the apoptosis in cells,respetively.【Results】TRAP-PCR-ELISA showed that the transfected cells registered 0.686 and the control group 1.685 in telomerase activity,suggesting that anti-sense telomerase RNA had a significant inhibition of telomerase activity of SMMC7721cells.Anti-sense telomerase RNA or 1,25-dihydroxyvitamin D3 could inhibit cell growth and induce cell apoptosis,and that,the inhibition of cell growth and induction of cell apoptosis by 1,25-dihydroxyvitamin D3-induced was more significant enhancement after expression of telomerase activity in SMMC7 721 cells was blocked with transfected anti-sense elomerase RNA.【Conclusion】It is demonstrated that anti-sense telomerase RNA can decreased the telomerase activity of hepatocarcinoma cells,and that inhibition of telomerase activity could significant enhance the cell apoptosis and inhibit cell proliferation by 1,25-dihydroxyvitamin D3-induced in hepatocarcinoma cells.