PKCα siRNAs有效序列的筛选及其对肺癌A549细胞的作用

Effective sequence screening of siRNAs targeted protein kinase C α and their effects on A549 cell line

目的探讨人蛋白激酶Cα(PKCα)小干扰RNA(siRNA)对肺癌A549细胞生长增殖及凋亡的影响。方法设计并化学合成6条PKCα siRNAs,转染A549细胞,通过改良MTT法及RT-PCR筛选;对3条效果较好的PKCα siRNAs,进一步采用克隆形成抑制实验、Hoechst 33258染色、PI单染和AnnexinⅤ/PI双染以及Western blot检测。结果6条PKCα siRNAs均显著下调了PKCα mRNA水平;除No.5siRNA外,其他5条siRNA均显著地抑制了A549细胞增殖(P<0.05)。3条效果较好的PKCα siRNAs转染组PKCα蛋白的表达显著下调,克隆形成抑制率、亚二倍体百分率和早期凋亡细胞比例均显著高于对照组(P<0.05),并出现了核固缩、边聚和裂解等凋亡形态学变化。结论本实验发现3条能显著抑制A549细胞增殖并诱导其凋亡的PKCα siRNAs。

Objective To investigate the effects of siRNAs targeted protein kinase Cct （PKCa） on the proliferation and apoptosis of A549 cell line. Methods Six PKCα siRNAs were designed and chemical synthesized. Candidate PKCct siRNA was transfected into A549 cells, PKCα mRNA level was determined by semi-quantitative reverse transcription-polymerase chain reaction （RT-PCR）. Then, the effects of more effective PKCα siRNAs on A549 were further investigated by using clone formation assay, Hoechst 33258 staining, propidium iodide （PI） staining, Annexin V-FITC and PI dual staining and western blot. Results Six candidate PKCct siRNAs were designed and named as No. 1, No. 2, No. 3, No. 4, No. 5 and No. 6 PKCα siRNA. Compared with controls, PKCα mRNA levels were all significantly downregulated and the cell proliferation was inhibited in A549 cells treated with candidate PKCα siRNAs except No. 5 （ P 〈 0.05 ）. The No. 3, No. 6 and No. 1 PKCa siRNAs, which inhibited A549 cell growth and PKCα expression more effectively, were identified among 6 candidate PKCα siRNAs. Compared with controls, the clone formation was significantly inhibited （ P 〈 0. 05 ） , percentages of hypodiploid cells and early apoptosis rates were significantly increased （P 〈0. 01）, and the expression of PKCα protein was significantly decreased in A549 cells treated with No. 3, No. 6 or No. 1 PKCα siRNA（P 〈0. 01 ）. In addition, the typical morphologic changes of apoptosis were found in PKCα siRNA-treated A549 cells. Conclusion Three PKCα siRNAs in six candidate PKCα siRNAs can effectively downregulate PKCα protein expression, induce apoptosis and inhibit proliferation of A549 cells.