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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p38mRNA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p38mRNA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3~5d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24h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。
Objective To investigate the effects of p38 RNA interference on proliferation, invasion, cell cycle and cell sensitivity to sunitinib in human renal cell carcinoma 786-O cells. Methods Two siRNAs targeting p38, siRNA651 and siRNA659 were constructed and transfected into 786-O cell line of renal cell carcinoma respectively, which were siRNA531 group and siRNA659 group. At the same time, negative control group transfected with non- Reagents blank control group. The expression of p38 mRNA in 786-O cells was detected by RT-PCR and the expression of p38 protein was detected by Western blotting. The cell proliferation and sensitivity to sunitinib were detected by CCK-8 assay. Cell cycle changes were detected by flow cytometry. Transwell assay was used to detect cell invasiveness. Results RT-PCR and Western blotting showed that the expression of p38 mRNA and protein in 786-O cells decreased after siRNA transfection. Compared with the blank control group and the negative control group, the proliferation rates of 786-O cells in siRNA531 group and siRNA659 group decreased 3 ~ 5 days after transfection (P <0.05, P <0.01) The IC50 of sunitinib was lower than that of negative control group [(3.2 ± 0.3), (1.4 ± 0.1) μmol / mL vs (5.4 ± 0.2) μmol / mL, P <0.05]. The number of cells in G1 phase in siRNA531 group and siRNA659 group was significantly more than that in control group, and G0 / G1 block appeared in 786-O cells in both groups. After transfection for 24h, the number of transmembrane cells in both groups were 56.43 ± 6.02 and 34.00 ± 8.12, respectively. Compared with the negative control group (76.27 ± 5.08), the invasive ability of both groups decreased (P <0.01). Conclusion The p38 gene silencing of 786-O cell line can be successfully silenced by transfecting p38 specific siRNA, which can inhibit the proliferation and invasion of 786-O cell line and increase its sensitivity to sunitinib Follow-up study of renal cell carcinoma treatment and targeted drug resistance laid the foundation.