shRNA沉默Bmi-1质粒构建及对鳞癌细胞ECA109增殖的影响

Construction of shRNA-Bmi-1 Plasmid and Its Effect on Proliferation of ECA109 Cells

目的研究shRNA沉默Bmi-1基因对鳞癌细胞ECA109增殖变化的影响,探讨Bmi-1基因在鳞癌细胞增殖中的作用。方法采用RT-PCR和Western blot法分别检测Bmi-1在Fibroblast细胞、Hacat细胞、A431细胞、ECA109细胞中的表达。构建重组质粒GPU6/GFP/Neo-sh Bmi-1,脂质体转染ECA109细胞后,检测细胞中Bmi-1表达变化。MTT法检测转染前后细胞抑制率,流式细胞仪检测转染前后细胞周期的变化。结果与Fibroblast细胞和Hacat细胞相比,Bmi-1在鳞癌细胞系A431、ECA109中含量明显升高。重组载体GPU6/GFP/Neo-sh Bmi-1构建成功,转染ECA109细胞后Bmi-1表达明显降低(P<0.05)。与对照组相比,转染后细胞增殖明显受到抑制(P<0.05);G1期细胞明显增加(P<0.05),S期细胞明显减少(P<0.05)。结论 Bmi-1与细胞恶性增殖相关。在ECA109细胞中沉默Bmi-1,可改变细胞周期,从而抑制细胞增殖。

Objective To explore the role of Bmi-1 gene in the proliferation of squamous carcinoma cells and whether the silencing Bmi-1 can inhibit the growth of squamous cell carcinomas cells. Methods The expressions of Bmi-1 in primary cultured Fibroblasts,karatinocyte cell line Hacat,squamous carcinoma cell line A431,and ECA109 were detected by reverse transcription polymerase chain reaction（ RT-PCR） and Western blot analysis. Recombinant plasmid inserted with Bmi-1 gene short hairpin RNA（ shRNA） expression vector PGPU6 / GFP / Neo-sh Bmi-1 was constructed and transfected into ECA109 cells with control set. After transfection for 48 and 72 hours,the mRNA and protein levels of Bmi-1 were examined with RT-PCR and Western blot analysis,respectively. The proliferation of the ECA109 cells was evaluated by MTT method and flow cytometry.Results Bmi-1 was highly expressed in A431 and ECA109 cells than in Fibroblast cells and Hacat cells. The mRNA and protein expressions of Bmi-1 were significantly silenced in ECA109 cells after recombinant expression vector PGPU6 / GFP / Neo-sh Bmi-1 transfection（ P 0. 05）. Compared with the control groups,the proliferation of ECA109 transfected with PGPU6 / GFP / Neo-sh Bmi-1 was significantly inhibited（ P 0. 05）,and cells inG1 phase increased while in S phase decreased（ P 0. 05）. Conclusions Bmi-1 is involved in the proliferation of squamous carcinoma cells. After the silencing of Bmi-1 expression,the proliferation ECA109 cells is suppressed due to the altered cell cycle.