MiR-7-5p-mediated downregulation of PARP1 impacts DNA homologous recombination repair and resistance to doxorubicin in small cell lung cancer
MiR-7-5p-mediated downregulation of PARP1 impacts DNA homologous recombination repair and resistance to doxorubicin in small cell lung cancer
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DOI：
10.1186/s12885-019-5798-7
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发表时间：
2019-06-18
期刊：
影响因子：
3.8
通讯作者：
Zhang, Qiuyu
中科院分区：
文献类型：
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作者：
Lai, Jinzhi;Yang, Hainan;Zhang, Qiuyu
背景化疗耐药是小细胞肺癌（SCLC）治疗面临的主要挑战之一。在SCLC治疗期间，多种机制被认为参与化学抗性，但不幸的是，这些机制尚未得到很好的阐明。在此，我们研究了小细胞肺癌细胞对阿霉素（Dox）的抗性中miRNA的作用。方法miRNA微阵列分析显示，与亲本细胞（H69）相比，在阿霉素抗性小细胞肺癌细胞（H69 AR）中，包括miR-7- 5 p在内的几种miRNA特异性降低。通过qRT-PCR证实miR-7- 5 p在Dox抗性细胞（H69 AR和H446 AR细胞）及其亲本细胞中的表达水平。生物信息学分析表明，聚ADP核糖聚合酶1（PARP 1）是miR-7- 5 p的直接靶点。miR-7- 5 p在PARP 1 3 UTR中的结合位点通过荧光素酶报告基因和蛋白质印迹测定来验证。为了研究miR-7- 5 p在SCLC细胞对阿霉素的化学抗性中的作用，将miR-7- 5 p的模拟物或抑制剂转染到SCLC细胞中，并通过同源重组（HR）报告基因分析miR-7- 5 p对HR修复的影响。结果miR-7- 5 p模拟物转染H446 AR细胞后，H69 AR和H446 AR细胞中miR-7 - 5 p的表达水平明显低于H69和H446细胞（4倍）。聚ADP核糖聚合酶1（PARP 1）是miR-7- 5 p的直接靶点，miR-7- 5 p可下调PARP 1的表达。miR-7- 5 p通过抑制HR修复因子（Rad 51和BRCA 1）的表达来阻碍Dox诱导的HR修复，从而导致SCLC细胞对阿霉素（doxorubicin.ConclusionsOur findings）的再敏感，这表明miR-7 - 5 p的表达改变可能是克服SCLC治疗中的化疗耐药性的一种有前景的策略。
BackgroundChemo-resistance is one of the major challenges in the therapy of small cell lung cancer (SCLC). Multiple mechanisms are thought to be involved in chemo-resistance during SCLC treatment, but unfortunately, these mechanisms have not been well elucidated. Herein, we investigated the role of miRNA in the resistance of SCLC cells to doxorubicin (Dox).MethodsMiRNA microarray analysis revealed that several miRNAs, including miR-7-5p, were specifically decreased in Dox-resistant SCLC cells (H69AR) compared to parental cells (H69). The expression level of miR-7-5p was confirmed by qRT-PCR in Dox-resistant cells (H69AR and H446AR cells) and their parental cells. Bioinformatic analysis indicated that poly ADP-ribose polymerase 1 (PARP1) is a direct target of miR-7-5p. The binding sites of miR-7-5p in the PARP1 3 UTR were verified by luciferase reporter and Western blot assays. To investigate the role of miR-7-5p in the chemo-resistance of SCLC cells to doxorubicin, mimic or inhibitor of miR-7-5p was transfected into SCLC cells, and the effect of miR-7-5p on homologous recombination (HR) repair was analyzed by HR reporter assays. Furthermore, the expression of HR repair factors (Rad51 and BRCA1) induced by doxorubicin was detected by Western blot and immunofluorescent staining in H446AR cells transfected with miR-7-5p mimic.ResultsThe expression level of miR-7-5p was remarkably reduced (4-fold) in Dox-resistant SCLC cells (H69AR and H446AR cells) compared with that in parental cells (H69 and H446 cells). Poly ADP-ribose polymerase 1 (PARP1) is a direct target of miR-7-5p, and PARP1 expression was downregulated by miR-7-5p. MiR-7-5p impeded Dox-induced HR repair by inhibiting the expression of HR repair factors (Rad51 and BRCA1) that resulted in resensitizing SCLC cells to doxorubicin.ConclusionsOur findings provide evidence that miR-7-5p targets PARP1 to exert its suppressive effects on HR repair, indicating that the alteration of the expression of miR-7-5p may be a promising strategy for overcoming chemo-resistance in SCLC therapy.