摘要
目的 探究Lnc-BM在胃癌发生发展中的作用及分子机制.方法 收集36例胃癌患者的胃癌组织及配对的癌旁正常组织,RT-qPCR检测胃癌组织和癌旁正常组织中Lnc-BM的表达水平.构建Lnc-BM过表达或敲低细胞,平板克隆形成实验和CCK-8实验分析Lnc-BM对胃癌细胞增殖能力的影响,Transwell实验分析Lnc-BM对胃癌细胞迁移和侵袭能力的影响.RNA Pull-down实验分析Lnc-BM与FASTK蛋白的结合,Western blot检测Lnc-BM的表达对FASTK蛋白水平的影响.Western blot实验和Seahorse细胞能量代谢分析检测Lnc-BM过表达或敲降的胃癌细胞中线粒体相关蛋白的表达变化以及线粒体能量代谢的改变.裸鼠荷瘤实验进一步观察Lnc-BM对胃癌细胞体内生长的影响.结果 Lnc-BM在胃癌组织中表达明显高于对应的癌旁正常组织.实验结果显示,过表达Lnc-BM促进胃癌细胞增殖、迁移和侵袭,敲低Lnc-BM抑制胃癌细胞增殖、迁移和侵袭(均P＜0.05).RNA Pull-down结果显示,Lnc-BM可直接结合FASTK蛋白.Western blot实验结果显示,过表达Lnc-BM后FASTK蛋白水平升高,敲低Lnc-BM抑制FASTK蛋白表达(均P＜0.05).与对照组相比,过表达Lnc-BM后,线粒体相关蛋白MT-ND6和TOM20表达升高(均P＜0.05). Seahorse细胞能量代谢分析结果显示,Lnc-BM过表达显著提高细胞的线粒体呼吸能力(均P＜0.05).在Lnc-BM稳定过表达细胞中敲低FASTK,可有效抑制Lnc-BM过表达细胞中增强的线粒体呼吸能力(均P＜0.05).裸鼠皮下荷瘤实验显示,过表达Lnc-BM显著促进肿瘤生长(均P＜ 0.05).结论 Lnc-BM可通过FASTK/MT-ND6轴调节线粒体呼吸功能,促进胃癌进展.
Abstract
Objective To explore the role and molecular mechanism of Lnc-BM in the occurrence and development of gastric cancer(GC).Methods GC tissues and paired adjacent normal tissues of 36 GC patients were collected,and the expression of Lnc-BM was detected by RT-qPCR.Colony formation and CCK-8 assays were used to investigate the proliferation of GC cells.The migration and invasion properties of GC cells were investigated via Transwell assay.RNA pull-down assay was applied to confirm the interaction between FASTK and Lnc-BM.Western blot assay was used to detect FASTK protein level in Lnc-BM overexpressing or knockdown cells.Mitochondrial respiratory capacity and the related proteins expression levels were detected by Seahorse and Western blot assays,respectively.Lnc-BM stably overexpressing GC cells were constructed and then injected subcutaneously into nude mice.The tumor growth was observed.Results Lnc-BM was highly expressed in GC tissues compared with their paired adjacent normal tissues.Lnc-BM overexpression significantly promoted GC cells proliferation migration and invasion,while Lnc-BM knockdown inhibited GC cells proliferation,migration and invasion(P<0.05).RNA pull-down experiment demonstrated that Lnc-BM can directly bind to FASTK.Western blot results indicated that overexpression of Lnc-BM increased the protein levels of FASTK,while knockdown of Lnc-BM inhibited the expression of FASTK(P<0.05).Compared to the control group,overexpression of Lnc-BM increased the levels of mitochondria associated proteins,such as MT-ND6 and TOM20(P<0.05).Seahorse results indicated that overexpression of Lnc-BM enhanced mitochondrial respiratory capacity(P<0.05).Knocking down FASTK in Lnc-BM stably overexpressing cells can reverse the increase in mitochondrial respiratory capacity caused by Lnc-BM overexpression(P<0.05).In vivo,the results of subcutaneously implanted tumor model in nude mouse showed that Lnc-BM overexpression promoted the tumor growth(P<0.05).Conclusion Lnc-BM promotes GC progression by regulating mitochondrial respiratory function through the FASTK/MT-ND6 axis.
国家科技期刊平台
NSTL
维普
万方数据