Background: Radiotherapy resistance has become one of the major causes of radiotherapy failure among patients with non-small cell lung cancer (NSCLC), but its underlying mechanism remains unclear. In recent years, the influence of mitochondrial autophagy on the radiotherapy resistance in treated tumor cells and its regulatory mechanism has become a hotspot in research, which is also the subject of our group research effort. The primary objective of our study is to investigate the mitophagy-associated pathway and the regulatory mechanisms underlying radiotherapy resistance in NSCLC.

Methods: We developed biologically stable radiotherapy-resistant NSCLC cell models A549/X and H520/X and verified the radioresistance of these cells. Subsequently, through high-throughput transcriptomic sequencing analysis and experimental verification, we found that the Forkhead box O 3a (FOXO3a) gene and the PINK1/Parkin mitochondrial autophagy pathway in NSCLC radiotherapy-resistant cell lines were consistent and upregulated more reactively than those of parent cells. The effect of gene expression status of the FOXO3a-PINK1/Parkin pathway on the survival outcomes of NSCLC was analyzed in The Cancer Genome Atlas (TCGA) database. Next, we inoculated nude mouse xenografts with small interfering RNA to interfere with the FOXO3a gene and short hairpin RNA to construct radiotherapy-resistant stable strains of NSCLC with stable knockdown of FOXO3a gene. Subsequently, the association and regulation of FOXO3a gene expression levels with radioresistance and mitochondrial autophagy PINK1/Parkin pathway at the cellular and animal levels were determined.

Results: The expression level of FOXO3a gene in NSCLC radioresistant cells was significantly positively correlated with the level of mitophagy and the expression level of PINK1/Parkin pathway. Higher expression levels of genes in the FOXO3a-PINK1/Parkin pathway had a negative effect on survival outcomes in NSCLC and were positively correlated with the radioresistance of cells.

Conclusions: FOXO3a regulates NSCLC radioresistance by modulating the mitochondrial autophagy PINK1/Parkin pathway, which may serve as a new molecular intervention target and therapeutic entry point for intervening and improving the radioresistance of patients with NSCLC in clinical practice.

Keywords: FOXO3a; Non-small cell lung cancer (NSCLC); PINK1/Parkin pathway; mitochondrial autophagy; radiotherapy resistance.

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背景: 放射治疗抵抗已成为非小细胞肺癌（NSCLC）患者放射治疗失败的主要原因之一，但其潜在机制尚不清楚。近年来，线粒体自噬对治疗肿瘤细胞的放射治疗抵抗及其调节机制的影响已经成为研究热点，这也是我们小组的研究课题之一。本研究的主要目的是探讨与放射治疗抵抗相关的线粒体自噬通路及调控机制在NSCLC中的作用。

方法: 我们建立了生物学稳定的放射治疗耐药NSCLC细胞模型A549/X和H520/X，并验证了这些细胞的放射抵抗性。随后，通过高通量转录组测序分析及实验验证发现，在NSCLC放射耐药细胞系中FOXO3a基因以及PINK1/Parkin线粒体自噬途径与亲本细胞相比表现出一致性和更明显的上调反应。接下来，我们利用小干扰RNA（siRNA）和短发夹RNA（shRNA）分别干扰FOXO3a基因，在裸鼠成瘤模型中构建稳定敲低FOXO3a基因的放射治疗耐药性NSCLC稳转株系，并进一步确定了FOXO3a基因表达水平与细胞及动物层面放射抵抗性和线粒体自噬PINK1/Parkin途径的相关性和调节关系。此外，我们在癌症基因组图谱（TCGA）数据库中分析了FOXO3a-PINK1/Parkin通路的基因表达状态对NSCLC预后的影响。

结果: 在NSCLC放射耐药细胞中，FOXO3a基因表达水平与线粒体自噬水平及PINK1/Parkin途径表达水平显著正相关。FOXO3a-PINK1/Parkin通路的较高基因表达水平对NSCLC预后有负面影响，并且与细胞放射抵抗性呈正相关。

结论: FOXO3a通过调节线粒体自噬PINK1/Parkin途径影响NSCLC放射耐药，这可能成为临床干预和改善NSCLC患者放射治疗抵抗性的新的分子靶标及治疗方法。

关键词: FOXO3a；非小细胞肺癌（NSCLC）；PINK1/Parkin途径；线粒体自噬；放射治疗抵抗性。