吲哚美辛通过NOS3影响线粒体呼吸链对非小细胞肺癌的机制

Research of indomethacin affecting mitochondrial respiratory chain via NOS3 in non-small cell lung cancer

目的探讨吲哚美辛抑制非小细胞肺癌的相关机制。方法CCK-8检测NCI-H520、A549肺癌细胞增殖情况;克隆形成实验检测肺癌细胞增殖以及成瘤能力;分子靶向对接预测吲哚美辛相互作用蛋白;GEPIA公共数据库分析肿瘤与癌旁组织、肿瘤切除术后远期生存率与NOS3 mRNA水平关系;免疫组化检测NOS3表达水平;通过LinkedOmics数据库分析NOS3富集的基因;使用KEGG数据库分析与NOS3调控的信号通路;MitoTracker Red CMXRos探针检测线粒体ROS生成水平;电镜下观察线粒体形态;Western blot检测ATP5A、UQCRC2、SDH8、NDUFB8的表达水平。结果20μmol·L^(-1)吲哚美辛可抑制肺癌细胞增殖以及成瘤能力;吲哚美辛与NOS3蛋白存在作用位点;NOS3抑制剂L-NMMA可逆转吲哚美辛抑癌作用。通过生物信息学分析发现,肺癌组织中NOS3表达较低,NOS3基因高表达患者远期生存率更长;NOS3与线粒体呼吸链通路高度负相关。吲哚美辛作用后引起线粒体数量减少、形态肿胀、内脊消失、ROS水平显著上升、显著降低UQCRC2蛋白表达。结论吲哚美辛靶向NOS3抑制UQCRC2蛋白表达从而抑制线粒体呼吸链功能,进而在非小细胞肺癌中发挥抑癌作用。

Aim To investigate the mechanisms of indomethacin inhibiting non-small cell lung cancer(NSCLC).Methods CCK-8 was used to detect proliferation rate of NCI-H520 cells.Molecular docking experiment was used to predict the target protein of indomethacin.GEPIA database was used to analyze the relationship between NOS3 mRNA level and prognosis in NSCLC.LinkedOmics database was used to analyze NOS3 enriched genes.KEGG database was used to analyze the signaling pathway,which had high correlation with NOS3.Immunohistochemistry was used to detect the expression level of NOS3.Western blot was used to detect the expression levels of ATP5A,UQCUC2,SDH8 and NDUFB8.Results NSCLC cell vitality was inhibited by 20μmol·L^(-1) indomethacin.Indomethacin could specially bind with NOS3.L-NMMA,a NOS3 inhibitor,could reverse the antitumor effects of indomethacin.Bioinformatic analysis showed that low NOS3 expression level predicted bad prognosis value of patient with NSCLC.NOS3 had high relationship with mitochondrial respiratory chain pathway.Western blot results showed that indomethacin significantly reduced the expression of UQCRC2 protein.Conclusion Indomethacin inhibits NSCLS via NOS3-UQCRC2 axis to destroy mitochondria respiration chain.