miR-210反义慢病毒对乏氧人肝癌细胞放射敏感性的影响

为探讨miR-210表达下调对乏氧人肝癌SMMC-7721细胞放射敏感性的影响,利用分子生物学技术构建miR-210反义和随机寡核苷酸慢病毒载体,经慢病毒转染建立稳定转染miR-210反义和随机寡核苷酸的人肝癌SMMC-7721细胞株。以实时定量RT-PCR检测乏氧SMMC-7721细胞HIF-1αmRNA表达和miR-210表达,以四甲基偶氮唑盐(MTT)法检测细胞增殖活性,流式细胞术检测细胞周期和细胞凋亡,克隆存活实验检测细胞放射敏感性变化。结果表明:SMMC-7721细胞乏氧培养后,HIF-1α和miR-210表达随时间逐渐增加,稳定转染miR-210反义寡核苷酸能明显下调miR-210在乏氧细胞的表达;下调miR-210表达明显抑制乏氧SMMC-7721细胞体外增殖活性(P<0.05;P<0.01),诱导G1期阻滞(P<0.05),促进细胞凋亡(P<0.01),提高乏氧SMMC-7721细胞放射敏感性,放射增敏比约为1.21。结果提示,下调miR-210表达可抑制乏氧SMMC-7721细胞体外增殖,诱导凋亡,增强其放射敏感性。

Effects of mir-210 anti-sense lentivirus on radiosensitivity of hypoxic human hepatoma cells

To investigate the effect of miR-210 down-regulation on radio-sensitivity of hypoxic human hepatoma SMMC-7721 cells, lentivirus vector carrying miR-210 anti-sense or scrambled oligos was constructed by molecular biology techniques, and SMMC-7721 Cells with stable integration of the anti-sense miR-210 or scrambled sequence were generated through lentiviral-mediated gene transfer. Time courses of HIF-1a mRNA expression and miR-210 expression in SMMC-7721 cells exposed to 1% oxygen were analyzed by real-time RT-PCR. Cell viability was measured by MTT assay. Cell cycle and apoptosis were measured by flow cytometry assay. Radio-sensitivity of hypoxic SMMC-7721 cells was examined using a colony-forming assay. The results showed that hypoxia resulted in an increment of HIF- 1 a and miR-210 expression in SMMC-7721 cells. Stable integration of anti-sense miR-210 significantly decreased hypoxia-induced miR-210 expression. MiR-210 down-regulation decreased viability of hypoxic SMMC-7721 cells （P〈0.05 or P〈0.01）. SMMC-7721 cells with miR-210 down-regulation arrested in the G0/G1 phase of cell cycle after 72 h hypoxic culture （P〈0.05）. Apoptotic rate of cells with miR-210 down-regulation was significantly increased （P〈0.01） after 48 and 72 h hypoxic culture. MiR-210 down-regulation could enhance the radio-sensitivity of hypoxic SMMC-7721 cells with a radio-sensitization ratio of 1.21. These results suggest that miR-210 down-regulation can inhibit proliferation, induce apoptosis and enhance radio-sensitivity in hypoxic human hepatoma SMMC-7721 cells in vitro.