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shRNA沉默DNA-PKcs表达对肝癌耐药细胞BEL7402/5-FU耐药性的影响 被引量：7

Effects of DNA-PKcs gene silence on drug resistance of multidrug-resistant hepatocellular carcinoma cell line BEL7402/5-FU
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摘要 目的观察shDNA-PKcs沉默DNA-PKcs基因对肝癌耐药细胞Bel7402/5-FU耐药性的影响。方法采用双酶切、测序分析鉴定shDNA-PKcs干扰质粒;用阳离子脂质体法将shDNA-PKcs质粒转染BEL7402/5-FU细胞,根据荧光细胞数计算转染率,Real-time PCR及Western blot检测沉默效率;MTT法检测细胞药物敏感性;Real-time PCR和Western blot检测P-gp mRNA和蛋白表达。结果双酶切鉴定结果显示质粒约为6300bp,测序结果显示插入序列为shDNA-PKcs序列。荧光细胞计数显示质粒的转染率为62.2%。Real time PCR及Western blot检测结果显示DNA-PKcs mRNA及蛋白水平的沉默效率分别为82.6%、71.8%;MTT检测结果显示shDNA-PKcs实验组的ADM和5-FU的IC50值比对照组ADM、5-FU低(P<0.05),DDP和MMC的IC50值与对照组比无统计学意义(P>0.05)。Real time PCR和Western blot检测结果显示实验组P-gp mRNA、蛋白表达水平均比对照组低(P<0.01)。结论 shDNA-PKcs干扰质粒能够有效抑制BEL7402/5-FU细胞中DNA-PKcs表达,增加细胞对化疗药物的敏感性,其机制可能与P-gp蛋白的下调有关。 Objective To investigate the effects of shDNA-PKcs silencing DNA-PKcs on drug resistance of the multidrug-resistant hepatocellular carcinoma cell line BEL7402/5-FU.Methods shDNA-PKcs was identified by double digestion technique,sequence analysis and then transiently transfected into BEL7402/5-FU cells via cathodolyte liposome transfection method.The transfection efficiency was evaluated by calculating the fluorescent cells and the silence efficiency was analyzed using Real-time PCR and Western blotting assay.The drug sensitivity was tested by MTT.The mRNA and protein expressions of P-gp were detected by Real-time PCR and Western blotting assay,respectively.Results The results of double enzyme digestion showed that the plasmid was 6 300 bp,The sequencing results indicated that the insertion sequence was shDNA-PKcs.The transfection efficiency was 62.2%.Real-time PCR and Western blotting results showed the efficiency of RNA and protein interference for DNA-PKcs were 82.6% and 71.8%,respectively.MTT revealed the IC50 of ADM and 5-FU in shDNA-PKcs group were lower than those in the control group（P 〈 0.05）.However,there was no significant difference of the IC50 of DDP and MMC between the shDNA-PKcs group and the control group（P 〉0.05）.The mRNA and protein expressions of P-gp in the shDNA-PKcs group were significantly lower than those in the control group（P 〈 0.01）.Conclusion The shDNA-PKcs interference plasmid could effectively inhibit the DNA-PKcs expression in BEL7402/5-FU cells and increase the cell sensitivity to chemotherapeutic drugs.The mechanisms might be associated with the decreased P-gp protein expression.
作者 梁大敏 束波 杨加伟 生欣 范芳
机构地区 遵义医学院生物化学教研室
出处 《遵义医学院学报》 2014年第3期304-307,共4页 Journal of Zunyi Medical University
基金 贵州省社发攻关项目(NO:[2009]3066)
关键词 DNA依赖性蛋白激酶亚基 RNA干扰 BEL7402/5-FU 多药耐药 P-糖蛋白 DNA-PKcs RNA interference BEL7402/5-FU multidrug resistance P-gp
分类号 R735.7 [医药卫生—肿瘤]
  • 相关文献

参考文献12

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共引文献4

同被引文献51

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遵义医学院学报
2014年 第3期

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