LncRNA ANRIL promotes HR repair through regulating PARP1 expression by sponging miR-7-5p in lung cancer

Abstract

Background 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. Methods 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. Results 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. Conclusions 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 Long Non-Coding RNA ANRIL in Cancers

“…Since ANRIL has been described to be a key regulator in DNA damage repair (HDR), it is well suited to play a role in this process. In agreement, ANRIL positively acts on HDR by modulating the miR-7-5p/PARP1/RAD51 and miR-145-5p/MMP1 pathways in LC and TNBC respectively [ 100 , 228 ].…”

“…In the context of CRC, ceRNA activity of ANRIL has been proposed to promote resistance to 5-Fluorouracil by inhibiting the ABCC1/let-7a pathway [ 247 ]. Finally, ANRIL may favor cisplatin resistance in LC and OS by modulating the miR-328/ABCG2/MDR1 and miR-125a-5p/STAT3 signaling pathways, respectively [ 125 , 228 ].…”

Arbutus unedo L. Fractions Exhibit Chemotherapeutic Properties for the Treatment of Gastrointestinal Stromal Tumors

“…However, recent findings in the literature seem to support our hypothesis of an epigenetic mechanism of action. Indeed, a study has identified PARP-1 as a target of miR-7-5p in a model of lung cancer cells, demonstrating that sponging miR-7-5p promotes the homologous repair (HR) path through upregulating PARP1 expression [21]. In another study, Garmutin-A (GA), an NP isolated from Garcinia multiflora, induced apoptosis via the upregulation of miR-17-5p, triggering PARP-1 downregulation, in leukemic CB3 cells [22].…”

Long noncoding RNA ANRIL alleviates hypoxia‐induced pulmonary microvascular endothelial cell damage

“…20 ANRIL has been discovered to enhance homologous recombination (HR) repair, which is achieved by regulating the expression of poly(ADP-ribose) polymerase 1 (PARP1) through its interaction with miR-7-5p, effectively acting as a microRNA sponge. 21 Until now, there is no evidence regarding the regulation of miRNA by the ANRIL in HAPE. We observed that ANRIL exhibited a negative regulatory effect on miR-181c-5p in hypoxic PMVECs.…”

“…The report shows that the inhibition of ANRIL has been shown to hinder the cisplatin resistance in lung cancer, which is achieved through up‐regulation expression of miR‐98 20 . ANRIL has been discovered to enhance homologous recombination (HR) repair, which is achieved by regulating the expression of poly(ADP‐ribose) polymerase 1 (PARP1) through its interaction with miR‐7‐5p, effectively acting as a microRNA sponge 21 . Until now, there is no evidence regarding the regulation of miRNA by the ANRIL in HAPE.…”