PLK1基因缄默在K562细胞凋亡中的作用

Effects of PLK1 gene silence on apoptosis of K562 cells

目的观察缄默PLK1基因的短发夹状RNA(short hairpin RNA,shRNA)对K562细胞内PLK1表达和细胞凋亡的影响,探讨PLK1在白血病发病中的作用,寻找更为有效的白血病治疗途径.方法设计合成针对PLK1基因1416～1436位点的shRNA片段,并将其克隆于绿色荧光蛋白的表达载体pEGFP-H1中,命名为pEGFP-H1/PLK1.通过电穿孔转染法将其导入K562细胞中,分为对照组、pEGFP-H1空载体转染组和pEGFP-H1/PLK1 shRNA重组质粒转染组,转染后24,48 h,分别通过RT-PCR和Western blot检测各组细胞PLK1基因和蛋白水平的变化,以MTT方法测各组细胞的增殖活性,比色法检测各组细胞的半胱天冬酶3(caspase-3)蛋白酶活性,并通过流式细胞仪检测各组细胞的凋亡和G2/M期转变情况.结果PLK1 mRNA相对水平(与内参的灰度比值):对照组为1.25±0.07,空载体转染24,48 h组分别为1.21±0.08和1.23±0.09,shRNA重组质粒转染24,48 h组分别为0.52±0.04和0.25±0.02,shRNA重组质粒转染组较其他两组明显降低(P＜0.01).各组细胞PLK1的蛋白水平变化趋势与PLK1 mRNA表达相似.相应地,对照组、空载体转染48 h组、shRNA重组质粒转染24,48 h组的凋亡率分别为(8.3±0.6)%、(8.7±0.7)%、(49.7±3.8)%和(82.3±6.9)%,后两者与前两组之间差异有统计学意义(P＜0.05).此外,与对照组和空载体转染组相比,shRNA重组质粒转染组的细胞增殖能力明显下降(P＜0.05),且位于G2/M期的细胞较对照组和空载体转染组显著增加.以上结果均于转染后48 h时较明显(P＜0.05).结论构建的shRNA能明显抑制转染细胞PLK1的表达和增殖,并可促进转染细胞的凋亡,并使停留于G2/M期的细胞显著增加.

Objective To investigate the effects of PLK1 gene silence by short hairpin RNA （ sh RNA） on PLK1 expression amt apoptosis in K562 cells, and explore the role of PLK1 in the pathogenesis of leukemia. Methods The shRNA fragment targeting at 1416-1436 bp of PLK1 mRNA was synthesized and cloned into pEGFP-H1 vector, named as pEGFP-H1/PLK1. The empty control, pEGFP-H1 and pEGFP-H1/ PLK1 were transfected into K562 cells respectively via electroporation. 24 h or 48 h after transfection , gene and protein expression of PLK1 in the cells were assayed by RT-PCR and Western blot analysis respectively, cells viability by MTT assay, caspase-3 activity by colorimetry, cell cycle and apoptosis by FACS. Results 24 and 48 h after transfection, PLK1 expression in K562 cells was 1.25 ± 0.07 for control group, 0.52 ± 0.04 and 0.25 ±0.02 for pEGFP-H1/PLK1 group, and 1.24 ± 0.08 and 1.23 ± 0.09 for pEGFP-H1 group respectively. The uheration status of PLK1 protein levels were similar to that of PLK mRNA levels. The apoptosis rate was （ 8.3 ± 0.6） % in control group, （ 8.7 ± 0.7 ） % in pEGFP-H1 group and （49.7 ± 3.8 ） % and （82.3 ± 6.9）% in pEGFP-H1/PKLK1 group at 24 and 48 h, respectively. 1n addition, cell fraction at G2/ M phase was increased obviously compared with control and pEGFP-H1-transfected group. Conclusion The constructed shRNA can remarkably inhibit PLK1 expression and transfected K562 cell proliferation, increase apoptosis and block cell-cycle, suggesting that PLK1 play important roles in apoptosis and cell-cycle control of leukemia cells.