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微RNA-21与BIM蛋白在逆转肺腺癌细胞对EGFR-TKIs耐药中的相互拮抗作用
         
Mutual antagonism of microRNA-21 and BIM protein in the reversal of multidrug resistance to EGFR-TKIs in lung adenocaricinoma cells

摘    要
目的: 探索微RNA-21(microRNA-21,miR-21)以及与Bcl-2相互作用的细胞死亡调节子(Bcl-2 interacting mediator of cell death,BIM)蛋白在人肺腺癌细胞发生表皮生长因子受体(epidermal growth factor receptor,EGFR)-酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)获得性耐药过程中的作用及调控关系。
方法: 将重组质粒pcDNA3.1-BIM和空质粒pcDNA3.1分别瞬时转染至吉非替尼耐药的肺腺癌细胞株PC9R中,采用实时荧光定量PCR法检测各组细胞中miR-21的表达水平,细胞计数试剂盒-8(cell counting kit-8,CCK-8)法检测PC9R细胞对吉非替尼的敏感性变化。同时,采用慢病毒感染的方法干扰PC9R细胞株中miR-21的表达,然后采用实时荧光定量PCR法和蛋白质印迹法检测细胞中BIM基因的表达水平,CCK-8法检测PC9R细胞对吉非替尼的敏感性变化。另外,在干扰miR-21表达的PC9R细胞中转染pcDNA3.1-BIM重组质粒,然后采用CCK-8法检测PC9R细胞对吉非替尼的敏感性变化。
结果: 重组质粒pcDNA3.1-BIM转染后,PC9R细胞中BIM的表达水平明显提高(P<0.01),miR-21的表达水平也相应升高(P<0.01)。慢病毒干扰miR-21表达后,PC9R细胞中miR-21的表达水平明显降低(P<0.05),BIM的表达水平也相应降低(P<0.05)。下调miR-21水平和上调BIM表达均能提高PC9R细胞对吉非替尼的敏感性(P值均<0.05),而在下调miR-21表达的同时上调BIM表达,更加提高了细胞对吉非替尼的敏感性(P<0.05)。
结论: miR-21和BIM基因在逆转吉非替尼耐药过程中可能起关键作用,并且二者存在相互拮抗的作用。
标    签 癌,非小细胞肺   抗药性,肿瘤   微RNAs   基因表达调控   基因,BIM   吉非替尼   Carcinoma, non-small cell lung   Drug resistance, neoplasm   MicroRNAs   Gene expression regulation   Gene, BIM   Gefitinib  
 
Abstract
Objective: To investigate the effect and interaction of microRNA-21(miR-21) and Bcl-2 interacting mediator of cell death (BIM) on acquired drug resistance to epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in human lung adenocarcinoma cells.
Methods: The empty plasmid pcDNA3.1 and recombinant plasmid pcDNA3.1-BIM were transiently transfected into gefitinib-resistant human lung adenocarcinoma PC9R cells, respectively, then the expression of miR-21 was determined by real-time fluorescent quantitative-PCR (RFQ-PCR), and the proliferation of PC9R cells treated with gefitinib was detected by cell counting kit-8(CCK-8) assay. Meanwhile, the expression of miR-21 in PC9R cells was interfered by lentivirus infection method, then BIM expression and cell proliferation were detected by RFQ-PCR, Western blotting and CCK-8 assay, respectively. In addition, PC9R cells were transfected with recombinant plasmid pcDNA3.1-BIM and infected with lentivirus of miR-21 interference, then the sensitivity of PC9R cells to gefitinib was detected by CCK-8 assay.
Results: After transfection with pcDNA3.1-BIM plasmids, the expression levels of BIM and miR-21 in PC9R cells were increased (P < 0.01). After infection of miR-21 expression interference lentivirus, the expression level of miR-21 and BIM in PC9R cells were down-regulated (both P < 0.05). Both knockdown of miR-21 and over-expression of BIM could improve the sensitivity of PC9R cells to gefitinib (both P < 0.05). Moreover, miR-21 knockdown combined with BIM over-expression could further improve the sensitivity of PC9R cells to gefitinib as compared with only miR-21 knockdown group (P < 0.05).
Conclusion: MiR-21 and BIM may play key roles in the reversal of gefitinib-resistance to EGFR-TKIs, and they have a mutually antagonistic effect.

中图分类号 R734.2   DOI 10.3781/j.issn.1000-7431.2015.11.033

 
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所属栏目 基础研究

基金项目 国家自然科学基金资助项目(编号:81172101)

收稿日期 2015/1/16

修改稿日期 2015/3/25

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引用该论文: WANG Qi,LI Jia-yu,LI Xue-fei,ZHAO Chao,CAI Wei-jing,CHENG Ning-ning,ZHAO Ming-chuan,ZHANG Li-min,PAN Hui,ZHOU Cai-cun. Mutual antagonism of microRNA-21 and BIM protein in the reversal of multidrug resistance to EGFR-TKIs in lung adenocaricinoma cells[J]. Tumor, 2015, 35(6): 613~620
王琪,李嘉瑜,李雪飞,赵超,蔡微菁,成宁宁,赵明川,张丽敏,潘慧,周彩存. 微RNA-21与BIM蛋白在逆转肺腺癌细胞对EGFR-TKIs耐药中的相互拮抗作用[J]. 肿瘤, 2015, 35(6): 613~620


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