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  • 陈俊明,吴登爽,王志向,吴震杰,刘冰,鲍一,叶华茂,杨庆,曲乐,王林辉.RNA干扰p38基因对肾癌786-O细胞生物学特性的影响及对舒尼替尼的增敏作用[J].第二军医大学学报,2016,37(7):799-804    [点击复制]
  • CHEN Jun-ming,WU Deng-shuang,WANG Zhi-xiang,WU Zhen-jie,LIU Bing,BAO Yi,YE Hua-mao,YANG Qing,QU Le,WANG Lin-hui.Effect of p38 siRNA on biological behavior and sensitivities to sunitinib of human renal carcinoma cell line 786-O[J].Acad J Sec Mil Med Univ,2016,37(7):799-804   [点击复制]
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RNA干扰p38基因对肾癌786-O细胞生物学特性的影响及对舒尼替尼的增敏作用
陈俊明1,2△,吴登爽1△,王志向1,3,吴震杰1,刘冰1,鲍一1,叶华茂4,杨庆4,曲乐1,5,王林辉1*
0
(1. 第二军医大学长征医院泌尿外科, 上海 200003;
2. 武警河南总队医院泌尿外科, 郑州 450052;
3. 解放军458 医院泌尿外科, 广州 510602;
4. 第二军医大学长海医院泌尿外科, 上海 200433;
5. 南京大学医学院附属金陵医院、南京军区南京总医院泌尿外科, 南京 210002
共同第一作者
*通信作者)
摘要:
目的 探讨RNA干扰p38基因对人肾癌细胞株786-O增殖、侵袭、细胞周期和细胞对舒尼替尼敏感性的影响。方法 构建针对p38的siRNA531和siRNA659两条siRNA,分别将其转染至肾癌786-O细胞株,即为siRNA531组和siRNA659组,同时设置转染无义siRNA的阴性对照组和仅加转染试剂的空白对照组。应用RT-PCR技术检测786-O细胞p38 mRNA的表达,蛋白质印迹法检测p38蛋白的表达。CCK-8法检测细胞的增殖情况和对舒尼替尼的敏感性,流式细胞术检测细胞的周期改变情况,Transwell实验检测细胞的侵袭能力。结果 RT-PCR及蛋白质印迹法检测发现siRNA转染后786-O细胞p38 mRNA及蛋白的表达均降低。与空白对照组和阴性对照组相比,siRNA531组和siRNA659组786-O细胞在转染后3~5 d时的增殖率均降低(P<0.05,P<0.01),细胞对舒尼替尼的敏感性增加,两组对舒尼替尼的IC50值均低于阴性对照组[(3.2±0.3)、(1.4±0.1) μmol/mL vs (5.4±0.2) μmol/mL,P<0.05]。siRNA531组、siRNA659组G1期细胞数量明显多于对照组,且两组786-O细胞出现G0/G1阻滞。转染24 h后,两组的穿膜细胞数分别为56.43±6.02、34.00±8.12,与阴性对照组(76.27±5.08)相比,两组细胞的侵袭能力均下降(P<0. 01)。结论 通过转染p38特异性siRNA可以成功沉默肾癌细胞株786-O的p38基因的表达,抑制肾癌细胞株786-O的增殖、侵袭能力,增加其对舒尼替尼的敏感性,为后续研究肾癌治疗及靶向耐药奠定基础。
关键词:  肾肿瘤  肾细胞癌  p38  靶向治疗  增敏  siRNA干扰
DOI:10.16781/j.0258-879x.2016.07.0799
投稿时间:2016-01-05修订日期:2016-04-11
基金项目:国家自然科学基金(81272817,81172447),上海市自然科学基金(11ZR1447800).
Effect of p38 siRNA on biological behavior and sensitivities to sunitinib of human renal carcinoma cell line 786-O
CHEN Jun-ming1,2△,WU Deng-shuang1△,WANG Zhi-xiang1,3,WU Zhen-jie1,LIU Bing1,BAO Yi1,YE Hua-mao4,YANG Qing4,QU Le1,5,WANG Lin-hui1*
(1. Department of Urology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China;
2. Department of Urology, Hospital of Chinese People's Armed Police Forces Henan Headquarters, Zhengzhou 450052, Henan, China;
3. Department of Urology, No. 458 Hospital of PLA, Guangzhou 510602, Guangdong, China;
4. Department of Urology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China;
5. Department of Urology, Jinling Hospital, Nanjing University School of Clinical Medicine, General Hospital, PLA Nanjing Military Area Command, Nanjing 210002, Jiangsu, China
* Corresponding author)
Abstract:
Objective To investigate the effects of p38 gene sliencing on the proliferation, invasion, cell cycle and sensitivity to sunitinib of human renal carcinoma cell line 786-O. Methods We designed two sequence-specific small interfering RNA(siRNA531 and siRNA659) targeting p38 gene and transfected them into renal carcinoma cell line 786-O. 786-O cells transfected with nonsense siRNA served as negative control and those cultured with transfection medium served as blank control. The change of p38 gene expression was observed by RT-PCR and the expression of p38 protein was detected by Western blotting analysis.The proliferation, sensitivities to sunitinib, invasion capabilities, and the cell cycle of 786-O cells were examined by CCK-8 assay, transwell chamber test and flow cytometry, respectively. Results RT-PCR and Western blotting analysis revealed that p38 expression in p38 siRNA group was significantly decreased compared with the controls. The cell proliferation rates were also significantly decreased 3-5 days after siRNA531 or siRNA659 transfection compared with the controls (P<0. 05, P<0. 01), and cells in the siRNA531 and siRNA659 groups become more sensitive to sunitinib compared with negative control group, with two IC50 values being significantly lower than that of the negative control group ([3.2±0.3],[1.4±0.1] μmol/mL vs[5.4±0.2] μmol/mL; P<0.05). In addition, analysis of cell cycle demonstrated a marked G0/G1 arrest of the 786-O cells transfected with siRNA531 or siRNA659. We also noticed that 24 h after transfection, the cell invasion capabilities was significantly decreased in siRNA531 and siRNA659 compared with negative control (numbers of cells permeating septum:56.43±6.02, 34.00±8.12 vs 76.27±5.08; P<0. 01). Conclusion We have successfully suppressed p38 gene expression by specific siRNA, which can inhibit the proliferation and invasion of human renal carcinoma cell line 786-O and increase its sensitivity to sunitinib, paving a way for future treatment and targeted drug resistance of renal cell carcinoma.
Key words:  kidney neoplasms  renal cell carcinoma  p38  targeted therapy  enhanced sensitivity  siRNA interference