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长链非编码RNA NORAD/微小RNA-202-5p通过靶向P-糖蛋白调节A549/DDP对顺铂的耐药性。

LncRNA NORAD/miR-202-5p regulates the drug resistance of A549/DDP to cisplatin by targeting P-gp.

作者信息

Shen Jian-Guo, Xu Shi-Ning, Yin Li-Guo

机构信息

Department of Emergency Medicine, Jinan Central Hospital, Ji'nan, Shandong, P.R. China.

出版信息

Gen Physiol Biophys. 2020 Sep;39(5):481-489. doi: 10.4149/gpb_2020027.

DOI:10.4149/gpb_2020027
PMID:33084601
Abstract

In this study, we established the DDP-resistant NSCLC cell line A549/DDP to detect the effect of NORAD on cisplatin resistance of A549/DDP cells. NORAD was highly expressed in A549/DDP cells compared with A549 cells. The MTT data showed that knockdown of NORAD enhanced the inhibition rate of cisplatin on the A549/DDP cells and decreased IC50 value. The colony formation and MTT assay suggested that cisplatin inhibited cell proliferation, and knockdown of NORAD enhanced the inhibitory effect of cisplatin on A549/DDP cells. Besides, we found that NORAD silence reduced the P-gp expression but not BCRP, LRP and MRP. Moreover, NORAD could directly bind to miR-202-5p, and ABCB1 was a target of miR-202-5p. The MTT assay found that miR-202-5p inhibitor reversed the effects of NORAD silence on cisplatin resistance of A549/DDP cells. Then, the Western blot data showed that knockdown of NORAD reduced P-gp expression, and miR-202-5p inhibitor enhanced P-gp expression. ABCB1 overexpression reversed the inhibitory effect of NORAD knockdown on A549/DDP cells. Moreover, NORAD could directly bind to miR-202-5p, and ABCB1 was a target of miR-202-5p. Inhibition of miR-202-5p and overexpression of ABCB1 eliminated the effects of NORAD silence on cisplatin resistance of A549/DDP cells. Overexpression of miR-202-5p suppressed P-gp expression in A549/DDP cells. Collectively, our data showed that NORAD could enhance the DDP resistance of A549/DDP cells and potentially increased P-gp expression by sponging the miR-202-5p.

摘要

在本研究中，我们建立了顺铂耐药的非小细胞肺癌细胞系A549/DDP，以检测NORAD对A549/DDP细胞顺铂耐药性的影响。与A549细胞相比，NORAD在A549/DDP细胞中高表达。MTT数据显示，敲低NORAD可提高顺铂对A549/DDP细胞的抑制率并降低IC50值。集落形成和MTT分析表明，顺铂抑制细胞增殖，而敲低NORAD可增强顺铂对A549/DDP细胞的抑制作用。此外，我们发现NORAD沉默降低了P-糖蛋白的表达，但对乳腺癌耐药蛋白（BCRP）、肺耐药蛋白（LRP）和多药耐药相关蛋白（MRP）无影响。此外，NORAD可直接与miR-202-5p结合，而ABCB1是miR-202-5p的靶标。MTT分析发现，miR-202-5p抑制剂可逆转NORAD沉默对A549/DDP细胞顺铂耐药性的影响。然后，蛋白质免疫印迹数据显示，敲低NORAD可降低P-糖蛋白表达，而miR-202-5p抑制剂可增强P-糖蛋白表达。ABCB1过表达可逆转NORAD敲低对A549/DDP细胞的抑制作用。此外，NORAD可直接与miR-202-5p结合，而ABCB1是miR-202-5p的靶标。抑制miR-202-5p和过表达ABCB1可消除NORAD沉默对A549/DDP细胞顺铂耐药性的影响。过表达miR-202-5p可抑制A549/DDP细胞中P-糖蛋白的表达。总的来说，我们的数据表明，NORAD可增强A549/DDP细胞的顺铂耐药性，并可能通过结合miR-202-5p来增加P-糖蛋白的表达。

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