稳定转染HMGA1基因的小分子干扰RNA对卵巢上皮性癌细胞生物学特性的影响

目的 观察稳定转染HMGA1基因的小分子干扰RNA(siRNA)对卵巢上皮性癌(卵巢癌)细胞生物学特性的影响.方法 实验分为3组,反义序列载体(卵巢癌细胞系OVCAR细胞转染载有HMGA1基因siRNA序列的pSilence4.1-CMV-Hs质粒)、无关序列载体(OVCAR细胞转染载有HGMA1基因无关序列的pSilence4.1-CMV-Hn质粒)和空白对照组(OVCAR细胞仅转染脂质体),3组均经抗性筛选获得稳定转染的细胞系.采用RT-PCR技术、蛋白印迹法检测3组细胞中HMGA1mRNA和蛋白的表达;四甲基偶氮唑蓝比色法观察细胞增殖情况,绘制细胞生长曲线;体外侵袭实验观察3组细胞转染前后侵袭能力的变化;裸鼠接种3组细胞后观察其成瘤能力的变化.结果 (1)反义序列载体组转染前、后OVCAR细胞中HMGA1 mRNA表达水平分别为(86.3±2.7)%和(35.8±3.1)%,HMGA1蛋白表达水平分别为(68.6±2.8)%和(22.3±4.2)%,分别比较,差异均有统计学意义(P<0.05);无关序列载体组和空白对照组转染前、后HMGA1 mRNA和蛋白表达水平分别比较,差异均无统计学意义(P>0.05).(2)反义序列载体组OVCAR细胞增殖速度明显慢于无关序列载体组和空白对照组(P<0.05).(3)转染前、后的穿膜细胞数,反义序列载体组分别为(53±6)和(21±6)个,两者比较,差异有统计学意义(P<0.05);无关序列载体组和空白对照组转染前、后的穿膜细胞数分别比较,差异均无统计学意义(P>0.05).(4)接种转染后的OVCAR细胞后,反义序列载体组裸鼠的成瘤时间为(6.0±0.9)d,明显短于无关序列载体组的(12.3±3.9)d和空白对照组的(13.0±2.3)d(P<0.05).接种5周后,反义序列载体组裸鼠的肿瘤重量和体积分别为(0.8±0.3)g和(205±34)mm~3,分别与无关序列载体组分别为(2.1±0.4)g和(987±82)mm~3]和空白对照组分别为(2.3±0.3)g和(956±79)mm~3]比较,差异均有统计学意义(P<0.05).结论 HMGA1 siRNA可显著降低卵巢癌细胞中HMGA1基因的表达,并对卵巢癌细胞的生长有抑制作用.

Effects of small interfering RNA specific to stably transfected HMGA1 gene on biological characters of ovarian carcinoma cells

Objective To investigate the effects of biological characters of the human ovarian cell line (OVCAR) by stable transfection short hairpin RNA into the target HMGA1 gene. Methods Experiments were divided into two groups: transfected the OVCAR cells with pSilence4. 1-CMV-Hs plasmid as group A, while transfected OVCAR cells with pSilence4. 1-CMV-Hn plasmid as group B, in which stably transfected cells were gained by antibiotic screening. The comparative expressions of HMGA1 were detected by RT-PCR and western blot. Methyl thiazolyl tetrazolium (MTT) method was applied to measure cell proliferation and at the same time, the cell growth curve was also be mapped. Vitro invasion assay was used to observe the invasion ability of the cancer cells, and the tumor growth of the nude mice inoculated of tumor cells were compared with before and after transfection. Results In group A, the expression level of mRNA and protein HMGA1 gene in OVCAR cells were remarkably reduced before and after the stable transfected with HMGA1 siRNA, in which the percents of mRNA expression were (86.3±2.7) % vs. (35.8± 3.1) %, P<0.05], the expression of protein were (68.6±2.8) % vs. (22.3±4.2) %, P<0.05)]. The OVCAR cell growth in stable transfection status was more significantly decreased than that in non-transfection status (P<0.05). In group B, there were no statistical difference in the expression of HMGA1 siRNA, protein and the cell growth between before and after transfection states (P>0.05). The invasion cell numbers were reduced from before to after transfection state in group A (53±6)vs. (21±6), P< 0.05] ,while there was no significant difference in group B (51±6)vs. (47±8), P>0.05]. After inoculated transfected cells into nude mice, it took (6.0±0.9) days to grow the planed tumors in group A, which was much shorter than that (12.3±3.9) days in group B (P<0.05 ). After 5 weeks, the tumor weight and volume in group A was were significantly lower than those in group B (0.8±0.3)g vs. (2.1± 0.4)g, (205±34)mm~3 vs. (987±82) mm~3, all P <0.05]. Conclusion HMGA1 siRNA could remarkably reduce the expression of HMGA1 gene in ovarian cell and also inhabit the ovarian cell growth.