miR-181a/b靶向抑制多种抗凋亡蛋白表达对肺癌细胞顺铂耐药的影响
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miR-181a/b modulates cisplatin resistance of human lung cancer cell by targeting multiple anti-apoptosis genes
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    目的:研究miR-181a/b在肺癌细胞顺铂耐药形成中的作用-方法:运用miRNA实时定量PCR方法检测miR-181a/b在肺癌顺铂耐药细胞A549/CDDP与母代A549细胞中的表达差异,运用Western blot检测A549/CDDP细胞与母代细胞抗凋亡蛋白BCL2-MCL1和XIAP的表达差异,分别构建BCL2-MCL1和XIAP的3′UTR荧光素酶报告质粒验证miR-181a/b的靶基因,在耐药细胞中瞬时转染miR-181a/b模拟物以检测上调miR-181a/b对A549/CDDP细胞中BCL2-MCL1和XIAP蛋白表达及耐药性的影响,并运用流式细胞术检测转染后耐药细胞对CDDP(Cisplatin,顺铂)诱导凋亡的影响-结果:在A549/CDDP细胞中miR-181a/b均呈低表达,而抗凋亡蛋白BCL2-MCL1和XIAP则呈高表达,荧光素酶报告实验证实BCL2-MCL1和XIAP是直接受miR-181a/b调控的靶基因,在耐药细胞中上调miR-181a/b显著抑制BCL2-MCL1和XIAP蛋白表达水平,显著增加耐药细胞对顺铂的敏感性,并显著增加耐药细胞对顺铂诱导的凋亡-结论:miR-181a/b靶向抑制多种抗凋亡蛋白表达可显著增加A549/CDDP细胞对顺铂的敏感性-

    Abstract:

    Objective:To investigate the possible role of miR-181a/b in the development of cisplatin resistance in human lung cancer cell line A549/CDDP. Methods:Using quantitative real-time PCR analysis for miRNA and Western blot to detect the expression of miR-181a/b and anti-apoptotic protein BCL2,MCL1 and XIAP in cisplatin resistant human lung cancer cell line A549/cisplatin (CDDP) and its parental A549 cell,respectively. The luciferase reporter plasmids carried 3'-untranslated region of BCL2,MCL1 and XIAP were constructed to testify the target genes of miR-181a/b,respectively. Using transient transfection of miR-181a/b to mimic the up-regulation of miR-181a/b in A549/CDDP cells and observe the effect of miR-181a/b on the expression level of BCL2,MCL1,XIAP and cisplatin resistance phenotype. Using flow cytometry to detect CDDP-induced apoptosis of the A549/CDDP cells after the transfection. Results:miR-181a/b was down-regulated in cisplatin-resistant human lung cancer cell line A549/CDDP,the down-regulation of miR-181a/b was concurrent with the over-expression of its targeted anti-apoptotic genes BCL2,MCL1 and XIAP in A549/CDDP cells,compared to the parental A549 cell line. The luciferase activity of BCL2,MCL1 and XIAP 3'-untranslated region-based reporters construct in A549/CDDP cells suggested that BCL2,MCL1 and XIAP were the common target genes of the miR-181a/b. Over-expression of miR-181a/b sensitized A549/CDDP cells to CDDP. Overexpression of miR-181a/b also inhibited the expression of BCL2,MCL1 and XIAP and sensitized A549/CDDP cells to CDDP-induced apoptosis. Conclusion:miR-181a/b could enhance the sensitivity of cisplatin in human lung cancer cell line A549/CDDP,at least in part,by modulation of apoptosis via targeting multiple anti-apoptosis genes.

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房 栋,徐 静,朱 伟,周 鑫,张红光,束永前. miR-181a/b靶向抑制多种抗凋亡蛋白表达对肺癌细胞顺铂耐药的影响[J].南京医科大学学报(自然科学版),2012,(12):1690-1695

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  • 收稿日期:2012-04-29
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  • 在线发布日期: 2013-01-09
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