PLK1基因缄默在K562细胞凋亡中的作用

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/PLK1shRNA重组质粒转染组,转染后24,48h,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PLK1mRNA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24,48h组分别为1.21±0.08和1.23±0.09,shRNA重组质粒转染24,48h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PLK1mRNA表达相似。相应地,对照组、空载体转染48h组、shRNA重组质粒转染24,48h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48h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。

Effects of PLK1 gene silence on apoptosis of K562 cells

OBJECTIVE: To investigate the effects of PLK1 gene silence by short hairpin RNA (shRNA) on PLK1 expression and 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 ulteration 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. In addition, cell fraction at G(2)/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.