miR-135a/b逆转肺癌A549/CDDP细胞对顺铂耐药性及相关机制研究
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国家自然科学基金(81171908);江苏省普通高校研究生科研创新计划项目(JX10231801);江苏高校优势学科建设工程资助


Mechanism of miR-135a/b on regulating cisplatin resistance of human lung cancer cell line A549/CDDP
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    目的:研究miR-135a/b对肺癌耐顺铂细胞株A549/CDDP顺铂耐药的影响。方法:运用实时荧光定量PCR检测miR-135a/b在A549和A549/CDDP 细胞株中的差异表达;MTT法检测转染后A549及A549/CDDP细胞对CDDP的敏感性;构建MCL1-3′-UTR荧光素酶报告质粒验证miR-135a/b的靶基因;Western blot检测转染前后细胞MCL1蛋白的表达差异;流式细胞术检测转染后耐药细胞对顺铂诱导凋亡的影响。结果:miR-135a/b在A549/CDDP细胞中表达量降低;在耐药株中上调miR-135a/b后显著增加细胞对顺铂的敏感性;荧光素酶实验证实MCL1是miR-135a/b的靶基因;抗凋亡蛋白MCL1在A549/CDDP细胞中呈高表达,上调miR-135a/b明显抑制耐药细胞中MCL1蛋白的表达;miR-135a/b显著增加A549/CDDP细胞对顺铂诱导的凋亡。结论:miR-135a/b通过靶向调控MCL1蛋白表达增加NSCLC细胞对顺铂的敏感性和凋亡。

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    Objective:To investigate whether miR-135a/b could modulate the drug resistance of the human lung cancer cell line A549/CDDP to cisplatin(CDDP),and explore the mechanism of miR-135a/b on the CDDP sensitivity of A549/CDDP cells. Methods:MiR-135a/b expression was measured by quantitative real-time PCR. Transient transfection was used in A549 and A549/CDDP cell lines. Cell viability was detected by MTT assay. MCL1 3’-untranslated region-based luciferase reporter plasmids was constructed to testify the target gene of miR-135a/b.Protein expressions were measured by western blot. Flow cytometry was used to detect CDDP-induced apoptosis. Results:We found that miR-135a/b were downregulated while MCL1 was upregulated in A549/CDDP cells,compared with the parental A549 cells. In vitro drug sensitivity assay demonstrated that the over-expression of miR-135a/b sensitized A549/CDDP cells to cisplatin. The luciferase activity of MCL1 3’-untranslated region-based reporter constructed in A549/CDDP cells suggested that MCL1 was the direct target gene of miR-135a/b. Enforced miR-135a/b expression reduced MCL1 protein level and sensitized A549/CDDP cells to CDDP-induced apoptosis. Conclusion:This study demonstrated that hsa-miR-135a/b could play a role in the development of CDDP resistance in lung cancer cell line at least in part by modulation of apoptosis via targeting MCL1.

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周 丽,邱天竹,陈雯姣,朱 伟,束永前,刘 平. miR-135a/b逆转肺癌A549/CDDP细胞对顺铂耐药性及相关机制研究[J].南京医科大学学报(自然科学版),2013,(4):449-455

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  • 收稿日期:2012-11-24
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  • 在线发布日期: 2013-04-29
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