LncRNA ANRIL promotes HR repair through regulating PARP1 expression by sponging miR-7-5p in lung cancer.
LncRNA ANRIL promotes HR repair through regulating PARP1 expression by sponging miR-7-5p in lung cancer.
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DOI：
10.1186/s12885-023-10593-z
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发表时间：
2023-02-08
期刊：
影响因子：
3.8
通讯作者：
中科院分区：
文献类型：
--
作者：
放射治疗是肺癌的重要治疗方法，主要通过触发DNA双链断裂来诱导细胞死亡。阻断DNA损伤修复可增加肿瘤细胞的放射敏感性。最近的研究发现，长链非编码rna是DNA损伤修复的关键调控因子。lncRNA ANRIL先前被证明参与同源重组（homologous recombination， HR）修复，但其具体机制尚未完全阐明。利用miRanda软件预测ANRIL下游相互作用的mirna。采用荧光定量PCR检测ANRIL及候选mirna的表达水平。克隆形成实验和细胞活力测定检测电离辐射后的细胞活力。细胞凋亡法检测电离辐射8 h后细胞的凋亡情况。Western blot分析和免疫荧光分析验证了miR-7-5p下游靶分子PARP1和HR通路关键分子的蛋白表达水平。采用荧光报告基因实验验证ANRIL与miR-7-5p以及miR-7-5p与PARP1之间的相互作用。生物信息学分析和qPCR验证提示miR-7-5p可能是ANRIL的下游分子。低表达ANRIL后miR-7-5p表达上调，过表达ANRIL后miR-7-5p表达下调。同时，ANRIL与电离辐射前后miR-7-5p表达变化呈负相关。荧光素酶报告基因检测证实了ANRIL与miR-7-5p结合位点的存在，发现转染miR-7-5p抑制剂可降低ANRIL- kd细胞的辐射敏感性。通过网站预测筛选miR-7-5p与HR修复相关的下游靶分子PARP1。随后，通过Western blot和荧光素酶报告基因检测证实，miR-7-5p可以下调PARP1的表达，并且在PARP1 mRNA的3'UTR上存在miR-7-5p的结合位点。这表明ANRIL可能作为竞争性内源性RNA结合miR-7-5p并上调PARP1的表达。采用Western blot和免疫荧光染色检测电离辐射后ANRIL- kd细胞中HR修复因子的表达变化，发现ANRIL的下调可抑制PARP1、BRCA1和Rad51的表达，阻碍辐射诱导的HR修复，最终导致ANRIL- kd细胞对电离辐射的再敏化。我们的研究结果提供了证据，表明ANRIL靶向miR-7-5p/PARP1轴发挥其对HR修复的调节作用，表明改变ANRIL表达可能是克服辐射抗性的一种有希望的策略。在线版本包含补充材料，可在10.1186/s12885-023-10593-z获得。
Radiotherapy is an important treatment for lung cancer, mainly by triggering DNA double-strand breaks to induce cell death. Blocking DNA damage repair can increase the radiosensitivity of tumor cells. Recent studies have identified long noncoding RNAs as key regulators in DNA damage repair. The lncRNA ANRIL was previously shown to be involved in homologous recombination (HR) repair, but its specific mechanism has not been fully elucidated. The downstream interacting miRNAs of ANRIL were predicted according to miRanda software. Fluorescence quantitative PCR was used to detect the expression levels of ANRIL and candidate miRNAs. Clone formation experiment and cell viability assays detect cell viability after ionizing radiation. Apoptosis assay was used to detect the apoptosis of cells after 8 h of ionizing radiation. Western blot analysis and immunofluorescence assays verified the protein expression levels of the downstream target molecule PARP1 of miR-7-5p and key molecules in the HR pathway. Fluorescent reporter gene experiments were used to verify the interaction between ANRIL and miR-7-5p and between miR-7-5p and PARP1. Bioinformatics analysis and qPCR validation suggested that miR-7-5p might be a downstream molecule of ANRIL. The expression of miR-7-5p was up-regulated after knockdown of ANRIL, and the expression of miR-7-5p was down-regulated after overexpression of ANRIL. Meanwhile, there was a negative correlation between ANRIL and miR-7-5p expression changes before and after ionizing radiation. The luciferase reporter gene assay confirmed the existence of ANRIL binding site with miR-7-5p, and found that transfection of miR-7-5p inhibitor can reduce the radiation sensitivity of ANRIL-KD cells. A downstream target molecule of miR-7-5p related to HR repair, PARP1, was screened through website prediction. Subsequently, it was confirmed by Western blot and luciferase reporter assays that miR-7-5p could down-regulate the expression of PARP1, and there was a miR-7-5p binding site on the 3'UTR of PARP1 mRNA. This suggests that ANRIL may act as a competitive endogenous RNA to bind miR-7-5p and upregulate the expression of PARP1. Western blot and immunofluorescence staining were used to detect the expression changes of HR repair factors in ANRIL-KD cells after ionizing radiation, and it was found that knockdown of ANRIL can inhibit the expression of PARP1, BRCA1 and Rad51, hinder radiation-induced HR repair, and eventually result in resensitizing ANRIL-KD cells to ionizing radiation. Our findings provide evidence that ANRIL targets the miR-7-5p/PARP1 axis to exert its regulatory effect on HR repair, suggesting that altering ANRIL expression may be a promising strategy to overcome radiation resistance. The online version contains supplementary material available at 10.1186/s12885-023-10593-z.
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影响因子：
5.6
作者：
Sarkar D;Oghabian A;Bodiyabadu PK;Joseph WR;Leung EY;Finlay GJ;Baguley BC;Askarian-Amiri ME
通讯作者：
Askarian-Amiri ME
影响因子：
5.6
作者：
Tomita K;Nagasawa T;Kuwahara Y;Torii S;Igarashi K;Roudkenar MH;Roushandeh AM;Kurimasa A;Sato T
通讯作者：
Sato T
影响因子：
8.8
作者：
Cruz-Garcia, Andres;Lopez-Saavedra, Ana;Huertas, Pablo
通讯作者：
Huertas, Pablo
DOI：
10.1038/nrm.2017.53
发表时间：
2017-10
期刊：
Nature reviews. Molecular cell biology
影响因子：
--
作者：
Ray Chaudhuri A;Nussenzweig A
通讯作者：
Nussenzweig A
影响因子：
3.8
作者：
Lai, Jinzhi;Yang, Hainan;Zhang, Qiuyu
通讯作者：
Zhang, Qiuyu