联合干扰人端粒酶逆转录酶和端粒重复序列结合因子2基因的表达对乳腺癌MCF-7细胞生长的抑制作用

目的 探讨联合干扰人端粒酶逆转录酶(hTERT)基因和端粒重复序列结合因子2(TRF2)基因在乳腺癌基因治疗中的协同效应.方法 构建表达小分子干扰RNA(siRNA)-hTERT和siRNA-TRF2的重组腺病毒载体rAd-hTERT和rAd-TRF2,单独或联合转染人乳腺癌MCF-7细胞.采用Western blot法检测MCF-7细胞中hTERT和TRF2蛋白的表达,采用四甲基偶氮唑蓝(MTT)法检测MCF-7细胞的增殖抑制情况,采用流式细胞仪榆测MCF-7细胞的细胞周期变化,采用细胞平板克隆形成实验检测MCF-7细胞的克隆形成能力.结果 转染48 h后,PBS对照组、空载体对照组、阴性对照组、rAd-hTERT转染组、rAd-TRF2转染组以及rAd-hTERT和rAd-TRF2联合转染组乳腺癌MCF-7细胞中hTERT蛋白的相对表达量分别为1.00、0.94±0.02、0.95±0.04、0.18±0.04、0.95±0.01和0.18±0.04;TRF2蛋白的相对表达量分别为1.00、1.01±0.08、0.96±0.02、0.95±O.08、0.22±0.01 和0.26±0.02.单独转染rAd-hTERT或rAd-TRF2后,乳腺癌 MCF-7 细胞的增殖抑制率仅为54.6%和48.4%,有8.9%±1.2%和9.2%±2.3%的细胞进入分裂期,66.4%±1.5%和64.6%±1.9%的细胞阻滞于G_0+G_1期,细胞的克隆形成能力显著下降;联合转染rAd-hTERT和rAd-TRF2后,乳腺癌MCF-7细胞的增殖抑制率高达82.1%,进入分裂期的MCF-7细胞明显减少(为4.4%±1.2%),大量细胞阻滞于G_0+G_1期(为81.4%±1.3%),细胞的克隆形成能力下降更加明显.结论 联合干扰hTERT和TRF2 基因较单基因干扰可获得更有效的乳腺癌基因治疗效果.

Inhibitory effect of interference hTERT and TRF2 gene on the growth of breast cancer MCF-7 cells

Objective To explore the effect of combined gene therapy with interference hTERT and TRF2 gene on the treatment of breast cancer. Methods Recombinant adenovirus rAd-hTERT and rAd-TRF2 expressing siRNA-hTERT and siRNA-TRF2 was constructed, and the vectors were transfected into MCF-7 cells. Than the expressions of hTERT and TRF2 proteins were detected by Western blot, the inhibition of MCF-7 cell proliferation by MTT colorimetry, and the changes of MCF-7 cell cycle by flow cytometry and the colony forming ability of MCF-7 cells by clone form test. Results At 48 h after transfection,the relative expression amounts of hTERT protein of the PBS control group, rAd-blank group, rAd-HK control group, rAd-hTERT group, rAd-TRF2 group and rAd-hTERT and rAd-TRF2 group were 1.00, 0.94±0.02, 0.95±0.04, 0.18±0.04, 0.95±0.01 and 0.18±0.04, respectively. The relative expression amounts of TRF2 protein were 1.00, 1.01±0.08, 0.96±0.02, 0.95±0.08, 0.22±0.01 and 0.26±0.02, respectively. After transfection of rAd-hTERT or rAd-TRF2 into MCF-7 cells separately, the inhibition rate of cell proliferation was only 54.6% and 48.4%, there was 8.9%±1.2% or 9.2%±2.3% of MCF-7 cells into M phase, 66.4%±1.5% or 64.6%±1.9% of MCF-7 cells was arrested at G_0/G_1 phase,and the cell colony forming ability was decreased significantly (cell colony number from 100 in PBS control group down to 41.3±5.1 and 43.7±6.4). But after transfection by rAd-hTERT and rAd-TRF2 simultaneously, the inhibition rate of cell proliferation was about 82.1%, and M phase cells was significantly reduced to 4.4%±1.2%. Large numbers of cells were arrested at G_0/G_1 phase (81.4%±1.3% ), and the cell colony forming ability was more significantly decreased ( cell colony number there were only 29.2± 3.9). Conclusion More effective effect of tumor gene therapy can be achieved by combination of interference hTERT and TRF2 genes as compared with interference by either of the single gene alone.