目的:放疗治疗非小细胞肺癌(NSCLC)疗效显著。然而,抗辐射仍然是取得良好成果的主要障碍。本研究旨在确定放射增敏NSCLC的潜在靶点并阐明其潜在机制。
方法:使用基于慢病毒的感染和CRISPR/Cas9技术来调节microRNA-384(miR-384)的表达。细胞克隆形成测定和异种移植肿瘤模型用于分析NSCLC细胞中的放射敏感性。使用荧光激活的细胞分选来评估细胞周期和细胞死亡。免疫荧光染色,彗星测定,和同源重组或非同源末端连接I-SceI/GFP报告基因测定用于研究DNA损伤和修复。Western印迹和定量实时聚合酶链反应用于鉴定miR-384的靶标。进行染色质免疫沉淀和聚合酶链反应以评估miR-384的上游调节因子。
结果:MiR-384在非小细胞肺癌中表达下调。miR-384的过表达增加了NSCLC细胞在体外和体内的放射敏感性,而miR-384的敲除导致放射抗性。通过减少G2/M细胞周期阻滞上调miR-384放射致敏NSCLC细胞,抑制DNA损伤修复,并因此增加细胞死亡;miR-384耗竭具有相反的作用。进一步调查显示ATM机,Ku70和Ku80是miR-384的直接靶标。此外,miR-384被NF-κB抑制。
结论:MiR-384是一种被NF-κB抑制的电离辐射应答基因。MiR-384通过靶向ATM增强NSCLC细胞的放射敏感性,Ku80和Ku70,会损害DNA损伤修复。因此,miR-384可能作为NSCLC的一种新型放射增敏剂。
OBJECTIVE: Radiotherapy has achieved remarkable effects in treating non-small cell lung cancer (NSCLC). However, radioresistance remains the major obstacle to achieving good outcomes. This study aims at identifying potential targets for radiosensitizing NSCLC and elucidating the underlying mechanisms.
METHODS: Lentivirus-based infection and CRISPR/Cas9 technology were used to modulate the expression of microRNA-384 (miR-384). Cell clonogenic formation assays and a xenograft tumor model were used to analyze radiosensitivity in NSCLC cells. Fluorescence-activated cell sorting was used to assess the cell cycle and cell death. Immunofluorescence staining, Comet assays, and homologous recombination or non-homologous end-joining I-SceI/GFP reporter assays were used to study DNA damage and repair. Western blotting and quantitative real-time polymerase chain reaction were used to identify the targets of miR-384. Chromatin immunoprecipitation and polymerase chain reaction were performed to evaluate upstream regulators of miR-384.
RESULTS: MiR-384 was downregulated in NSCLC. Overexpression of miR-384 increased the radiosensitivity of NSCLC cells in vitro and in vivo, whereas knockout of miR-384 led to radioresistance. Upregulation of miR-384 radiosensitized NSCLC cells by decreasing G2/M cell cycle arrest, inhibiting DNA damage repair, and consequently increasing cell death; miR-384 depletion had the opposite effects. Further investigation revealed that ATM, Ku70, and Ku80 were direct targets of miR-384. Moreover, miR-384 was repressed by NF-κB.
CONCLUSIONS: MiR-384 is an ionizing radiation-responsive gene repressed by NF-κB. MiR-384 enhances the radiosensitivity of NSCLC cells via targeting ATM, Ku80, and Ku70, which impairs DNA damage repair. Therefore, miR-384 may serve as a novel radiosensitizer for NSCLC.