国际肿瘤学杂志

国际肿瘤学杂志 ›› 2017, Vol. 44 ›› Issue (7): 481-484.doi: 10.3760/cma.j.issn.1673-422X.2017.07.001

• 论著 •    下一篇

外源性dsRNA对肾透明细胞癌细胞中p21表达的影响

黄耿，姜卫东，毛青，桂定文   

  1. 435000 鄂东医疗集团黄石市中心医院（湖北理工学院附属医院）泌尿外科 肾脏疾病发生与干预湖北省重点实验室
  • 收稿日期:2017-01-03 出版日期:2017-07-08 发布日期:2017-06-20
  • 通讯作者: 桂定文，Email: drguidingwen@163.com E-mail:drguidingwen@163.com

Effect of exogenous dsRNA on expression of p21 in renal clear cell carcinoma cells

Huang Geng, Jiang Weidong, Mao Qing, Gui Dingwen   

  1. Department of Urology, Huangshi Central Hospital of Edong Healthcare Group (Affiliated Hospital of Hubei Polytechnic University); Hubei Key Laboratory of Kidney Disease Pathogenesis and Intervention, Huangshi 435000, China
  • Received:2017-01-03 Online:2017-07-08 Published:2017-06-20
  • Contact: Gui Dingwen E-mail:drguidingwen@163.com

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摘要： 目的 探讨dsP21-555转染对肾透明细胞癌细胞株ACHN和786-O中抑癌基因p21表达的影响。方法  肾透明细胞癌细胞分别使用脂质体Lipofectamine 3000转染dsControl和dsP21-555。荧光实时定量PCR（RT-qPCR）及Western blotting分别检测分析p21 mRNA及蛋白的表达情况。流式细胞术（FCM）检测细胞周期分布，使用细胞活力实验（MTS法）和集落培养实验分析细胞活力和增殖能力。结果  在ACHN和786-O细胞株中，dsP21-555组p21 mRNA的表达（2.86±0.33、1.96±0.35）相比dsControl组（1.05±0.34、1.01±0.14），分别升高至2.72倍(t=7.640，P<0.001)和1.95倍(t=5.058，P=0.002)。Western blotting实验证明两种细胞株中P21蛋白表达水平的上升与p21 mRNA上调相一致。FCM结果显示，转染dsP21-555后，细胞周期被阻滞在G0-G1期（57.08%±5.66%∶46.06%±4.60%，t=3.023，P=0.023；61.58%±6.23%∶42.25±6.08%，t=4.444，P=0.004）。MTS结果显示，转染dsP21-555后两种细胞的活力较dsControl组明显下降,其吸光度分别为0.85±0.20∶1.27±0.13，t=3.410，P=0.014；1.04±0.25∶1.55±0.10，t=3.758，P=0.009。集落培养实验显示，两细胞株dsControl组形成的集落数分别为110.91±26.21、129.89±22.87，dsP21555组形成的集落数分别为59.37±14.23(t=3.456，P=0.014)、71.26±21.38(t=3.745，P=0.010)，表明dsP21-555组细胞增殖能力明显降低。结论  dsP21-555可明显上调肾透明细胞癌细胞中p21基因的表达，并显著抑制癌细胞的生长，提示dsP21-555可能成为一种新型的基因治疗工具。

关键词: 肾肿瘤, 癌基因蛋白质p21(ras)细胞增殖, dsP21-555

Abstract: ObjectiveTo investigate the effect of dsP21555 transfection on the expression of tumor suppressor gene p21 in renal clear cell carcinoma cell lines ACHN and 786O. MethodsRenal clear cell carcinoma cells were transfected with dsControl and dsP21555 with Lipofectamine 3000 respectively. Realtime quantitative PCR (RTqPCR) and Western blotting were used to detect the expression of p21 mRNA and protein. Cell cycle distribution was detected by flow cytometry (FCM). Cell viability and proliferation were analyzed by cell viability assay (MTS method) and colony culture assay. ResultsIn ACHN and 786O cells, the expressions of p21 mRNA in dsP21555 group (2.86±0.33, 1.96±0.35) were significantly higher than those in dsControl group (1.05±0.34, 1.01±0.14), which were increased to 2.72 times (t=7.640, P<0.001) and 1.95 times (t=5.058, P=0.002). Western blotting showed that the expressions of P21 protein were upregulated in both renal cell lines, which was consistent with p21 mRNA upregulation. The result of FCM showed that the cell cycle was blocked in G0G1 phase (57.08%±5.66% vs. 46.06%±4.60%, t=3.023, P=0.023; 61.58%±6.23% vs. 42.25%±6.08%, t=4.444, P=0.004) after transfection of dsP21555 in renal clear cell carcinoma cells. MTS result showed that the vitality of both cell lines after transfection of dsP21555 decreased compared with dsControl group， their absorbance values were 0.85±0.20 vs.1.27±0.13, t=3.410, P=0.014; 1.04±0.25 vs.1.55±0.10, t=3.758, P=0.009. Colony culture experiments showed that the numbers of colonies formed by ACHN and 786O in the dsControl group were 110.91±26.21 and 129.99±22.87 respectively, and the numbers of colonies formed in the dsP21555 group were 59.37±14.23 (t=3.456, P=0.014) and 71.26±21.38 (t=3.745, P=0.010), indicating that the proliferation of cells in the dsP21555 group was significantly reduced. ConclusiondsP21555 can upregulate the expression of p21 gene in renal clear cell carcinoma cells and inhibit the growth of carcinoma cells, suggesting that dsP21555 may become a new gene therapy tool.

Key words: Kidney neoplasms, Oncogene protein p21 (ras), Cell proliferation, dsP21-555