长链非编码 RNA PANDAR 通过 ATR/CHK1 促进 NSCLC 中的辐射和顺铂诱导的 DNA 损伤修复。

Long non-coding RNA PANDAR promoted radiation and cisplatin-induced DNA damage repair through ATR/CHK1 in NSCLC.

机构信息

Department of Respiratory and Critical Care Medicine, Changhai Hospital, The First Affiliated Hospital of Naval Medical University, Shanghai, China.

Department of Respiratory and Critical Care Medicine, The Second Naval Hospital of Southern Theater Command, Sanya, China.

出版信息

J Gene Med. 2023 Dec;25(12):e3565. doi: 10.1002/jgm.3565. Epub 2023 Jul 17.

DOI:10.1002/jgm.3565

PMID:37460393

Abstract

BACKGROUND

DNA-damaging agents, including radiation and platinum-based chemotherapy, are indispensable treatments for non-small cell lung cancer (NSCLC) patients. However, cancer cells tend to be resistant to both radiation and chemotherapy, thus resulting in treatment failure or recurrence. The purpose of this study was to explore the effect and mechanism of long non-coding RNA (lncRNA) PANDAR (promoter of CDKN1A antisense DNA damage-activated RNA) on NSCLC sensitivity to radiation and chemotherapy.

METHODS

Cell counting kit (CCK-8), colony formation and flow cytometry were respectively performed to determine the cell cycle and apoptosis of NSCLC cells treated with γ-ray radiation and cisplatin. The extent of DNA damage was evaluated using a comet assay and immunofluorescence staining against γH2AX. In addition, we explored the role of PANDAR in DNA damage response pathways through western blot analysis. Finally, a nude mouse subcutaneous xenograft model was established to assess the sensitivity to radiation and chemotherapy in vivo.

RESULTS

In cell experiments, PANDAR knockdown can increase the sensitivity of NSCLC cells to radiation and cisplatin. The CCK-8 results showed that cell viability was significantly increased in the overexpression group after radiation and cisplatin treatments. The overexpression group also showed more colonies, less apoptosis and DNA damage, and G2/M phase arrest was aggravated to provide the time necessary for DNA repair. Contrary to PANDAR overexpression, the trends were reversed in the PANDAR knockdown group. Furthermore, PANDAR knockdown inhibited radiation and cisplatin-activated phosphorylation levels of ATR and CHK1 in NSCLC cells. Finally, our in vivo model showed that targeting PANDAR significantly sensitized NSCLC to radiation and cisplatin.

CONCLUSION

Our study showed that PANDAR knockdown promoted sensitivity to radiation and cisplatin in NSCLC by regulating the ATR/CHK1 pathway, thus providing a novel understanding as well as a therapeutic target for NSCLC treatment. In NSCLC cells, lncRNA PANDAR negatively regulates sensitivity to radiation and cisplatin. PANDAR can promote the repair of radiation and cisplatin-induced DNA damage and activation of the G2/M checkpoint through the ATR/CHK1 pathway. PANDAR knockdown results in defects in DNA damage repair accompanied by more cell apoptosis.

摘要

背景

包括辐射和铂类化疗在内的 DNA 损伤剂是治疗非小细胞肺癌（NSCLC）患者不可或缺的手段。然而，癌细胞往往对辐射和化疗具有耐药性，从而导致治疗失败或复发。本研究旨在探讨长链非编码 RNA（lncRNA）PANDAR（CDKN1A 反义 DNA 损伤激活 RNA 的启动子）对 NSCLC 对辐射和化疗敏感性的影响及其机制。

方法

采用细胞计数试剂盒（CCK-8）、集落形成实验和流式细胞术分别检测γ射线辐射和顺铂处理的 NSCLC 细胞的细胞周期和细胞凋亡。彗星实验和γH2AX 免疫荧光染色评估 DNA 损伤程度。此外，通过 Western blot 分析探讨 PANDAR 在 DNA 损伤反应途径中的作用。最后，建立裸鼠皮下移植瘤模型，评估体内对辐射和化疗的敏感性。

结果

在细胞实验中，PANDAR 敲低可增加 NSCLC 细胞对辐射和顺铂的敏感性。CCK-8 结果表明，辐射和顺铂处理后，过表达组细胞活力明显增加。过表达组集落更多，凋亡更少，DNA 损伤更少，G2/M 期阻滞加重，为 DNA 修复提供必要的时间。与 PANDAR 过表达相反，PANDAR 敲低组的趋势则相反。此外，PANDAR 敲低抑制了 NSCLC 细胞中 ATR 和 CHK1 的辐射和顺铂激活磷酸化水平。最后，我们的体内模型表明，靶向 PANDAR 可显著增强 NSCLC 对辐射和顺铂的敏感性。

结论

本研究表明，PANDAR 敲低通过调节 ATR/CHK1 通路促进 NSCLC 对辐射和顺铂的敏感性，为 NSCLC 治疗提供了新的认识和治疗靶点。在 NSCLC 细胞中，lncRNA PANDAR 负调控对辐射和顺铂的敏感性。PANDAR 可通过 ATR/CHK1 通路促进辐射和顺铂诱导的 DNA 损伤修复和 G2/M 检查点激活。PANDAR 敲低导致 DNA 损伤修复缺陷，伴随更多细胞凋亡。