敲低APLNR基因对NK细胞杀伤肺腺癌细胞敏感性影响及其机制研究

Study on the Effect and Mechanism of Knockdown of APLNR Gene on the Sensitivity of NK Cells to Kill Lung Adenocarcinoma Cells

摘要: 目的：本研究旨在探究敲低肺腺癌细胞的APLNR基因对自然杀伤(NK)细胞杀伤肺腺癌细胞敏感性的影响及其分子机制，评估APLNR在肺腺癌进展及免疫微环境重塑中的临床意义。通过细胞体外共培养实验，明确敲低APLNR基因后，NK细胞对肺腺癌细胞生物学行为的影响，揭示APLNR敲低调控NK细胞敏感性的信号通路，为开发靶向APLNR联合免疫治疗策略提供实验依据，推动肺腺癌个体化治疗的发展。方法：慢病毒转染敲低人肺腺癌A549细胞和NCI-H1299细胞APLNR基因表达，同时设置转染NC组为对照组，使用含有嘌呤霉素的培养基筛选获得稳定敲低APLNR基因的细胞株。通过Western Blot实验检测APLNR的蛋白表达水平，确保敲低效果。采用CCK-8方法检测不同效靶比NK-92MI细胞与肺腺癌A549细胞和NCI-H1299细胞共培养后NK-92MI细胞对肺腺癌A549细胞和NCI-H1299细胞杀伤力的变化；利用Transwell小室侵袭实验检测敲低APLNR基因后NK-92MI细胞对肺腺癌A549细胞和NCI-H1299细胞侵袭迁移能力的影响；KEGG差异基因富集分析得到敲低APLNR基因与p53信号通路相关；Western Blot法进一步检测敲低APLNR基因对p53信号通路相关蛋白表达水平的影响。结果：CCK-8结果显示，按照不同效靶比将NK-92MI细胞和A549细胞及H1299细胞共培养4小时后，其杀伤力随效靶比的增加明显提高。且在NK细胞与肺腺癌细胞效靶比为5:1与10:1时差异具有统计学意义(P < 0.05)。Transwell小室侵袭实验结果显示，共培养24小时后，敲低组肺腺癌A549细胞和H1299细胞穿过小室的细胞数显著减少(P < 0.01)，表明敲低APLNR基因后，肺腺癌细胞侵袭迁移能力明显降低。KEGG差异基因富集分析显示，APLNR基因在p53信号通路中显著富集(P < 0.001)，Western Blot实验进一步表明敲低APLNR基因后，p53、Bax蛋白表达量明显升高(P < 0.001)，Bcl-2蛋白表达量降低(P < 0.001)。结论：本研究表明，敲低肺腺癌细胞中APLNR基因可显著增强NK92-MI细胞对肺腺癌A549细胞和H1299细胞的杀伤作用，并显著降低肺腺癌细胞的侵袭能力，且增强NK细胞对肺腺癌A549细胞和H1299细胞侵袭迁移的抑制作用。同时通过p53信号通路增强NK细胞介导的细胞毒性作用。APLNR或可作为肺腺癌联合免疫治疗的新靶点，靶向抑制APLNR联合NK细胞过继免疫疗法可能成为改善肺腺癌患者预后的潜在策略。

Abstract: Objective: This study investigates the effects of APLNR gene knockdown in lung adenocarcinoma cells on NK cell-mediated cytotoxicity and its molecular mechanisms, while evaluating the clinical relevance of APLNR in tumor progression and immune microenvironment remodeling. Through in vitro co-culture experiments, we elucidate the effects of APLNR depletion on NK cell-regulated biological processes in cancer cells and identify the associated signaling pathways. These findings provide crucial experimental support for developing novel combination therapies targeting APLNR and immunotherapy, ultimately contributing to the advancement of personalized treatment strategies for lung adenocarcinoma. Methods: Lentivirus transfection was used to knock down the expression of the APLNR gene in human lung adenocarcinoma A549 cells and NCI-H1299 cells. Meanwhile, the transfected NC group was also set as a control group. Stable APLNR-knockdown cell lines were obtained by selection with puromycin-containing culture medium. The protein expression level of APLNR was detected by Western Blot to ensure the knockdown effect. The cytotoxicity of NK-92MI cells against A549 and NCI-H1299 cells was assessed by co-culturing NK-92MI cells with A549 and NCI-H1299 cells at different effector-to-target ratios using the CCK-8 method. The impact of APLNR knockdown on the invasive and migratory abilities of NK-92MI cells against A549 and NCI-H1299 cells was evaluated by Transwell invasion assays. KEGG differential gene enrichment analysis revealed that APLNR knockdown was associated with the p53 signaling pathway. Western Blot was further employed to detect the effects of APLNR knockdown on the expression levels of proteins related to the p53 signaling pathway Results: The results of the CCK-8 assay showed that after co-culturing NK-92MI cells with A549 and H1299 cells for 4 hours at different effector-to-target (E:T) ratios, the cytotoxicity of NK-92MI cells significantly increased with the increase of the E:T ratio. Moreover, statistically significant differences were observed when the E:T ratios were 5:1 and 10:1 (P < 0.05). The Transwell invasion assay results indicated that after 24 hours of co-culture, the number of A549 and H1299 cells that migrated through the chamber in the knockdown group was significantly reduced (P < 0.01), suggesting that the invasive and migratory abilities of lung adenocarcinoma cells were markedly decreased after APLNR gene knockdown. KEGG differential gene enrichment analysis revealed that the APLNR gene was significantly enriched in the p53 signaling pathway (P < 0.001). Western Blot analysis further demonstrated that after APLNR gene knockdown, the expression levels of p53 and Bax proteins were significantly increased (P < 0.001), while the expression level of Bcl-2 protein was decreased (P < 0.001). Conclusion: This study demonstrates that knocking down the APLNR gene in lung adenocarcinoma cells significantly enhances the cytotoxic effect of NK-92MI cells against A549 and H1299 cells, and markedly reduces the invasive capacity of lung adenocarcinoma cells. Additionally, it significantly enhances the inhibitory effect of NK cells on the invasion and migration of A549 and H1299 cells. This effect is achieved by enhancing NK cell-mediated cytotoxicity through the p53 signaling pathway. APLNR may serve as a novel target for combined immunotherapy in lung adenocarcinoma, and targeting APLNR in combination with NK cell adoptive immunotherapy could potentially be a promising strategy to improve the prognosis of patients with lung adenocarcinoma.