【作者】 林瀛；

【导师】 陈愉生；

【作者基本信息】 福建医科大学 ， 内科学（呼吸系病）， 2021， 博士

【摘要】 目的:原发性肺癌的发病率及死亡率位列恶性肿瘤首位,非小细胞肺癌脑转移发病率高,患者生活质量差。尽管脑转移治疗手段多样,但患者预后仍不理想。目前关于肺癌脑转移的发生发展机制还不明确。本研究首先通过基因组学及生物信息学方法发现RPTOR与非小细胞肺癌脑转移密切相关;其次利用临床样本、细胞和动物实验,研究RPTOR促非小细胞肺癌脑转移的作用机制;最后探讨RPTOR调控SPHK2/S1P/STAT3轴促非小细胞肺癌脑转移的发病机理,以期通过本研究为非小细胞肺癌脑转移的分子机制提供科学依据。方法:1.采用转录组测序(RNA-seq)方法,研究非小细胞肺癌组织和肺癌脑转移组织中的差异基因,并经新鲜组织RT-PCR筛选,发现肺癌脑转移灶较原发灶组织存在RPTOR表达上调,提示RPTOR基因表达与肺癌脑转移存在相关性,可能为脑转移的关键驱动基因事件。在此基础上利用公开的网络数据库进行相关生信分析佐证,提示RPTOR基因可能与肺癌脑转移相关。2.结合组织样本分析临床病理特征及绘制生存曲线;在细胞水平干扰或过表达RPTOR基因,对肺癌细胞的增殖、迁移侵袭及通过体外血脑屏障能力的影响,Western Blot检测EMT相关功能蛋白改变,细胞转录组测序验证;并利用裸鼠左心室注射肺癌细胞致脑转移,进一步明确RPTOR基因引起肺癌脑转移的作用机制。3.探讨RPTOR通过鞘磷脂代谢通路SPHK2/S1P/STAT3信号轴调控脑转移的发生发展,通过免疫荧光共定位、免疫共沉淀Co-IP、双荧光素酶报告基因检测及染色质免疫共沉淀CHIP实验验证RPTOR通过转录因子YY1与SPHK2相互作用的分子机制。Western Blot检测RPTOR与SPHK2/S1P/STAT3信号轴通路蛋白关系,使用SPHK2通路抑制剂观察RPTOR对肺癌细胞的增殖、迁移侵袭及通过体外血脑屏障能力的影响,相关通路蛋白的改变,验证RPTOR调控SPHK2/S1P/STAT3轴促肺癌脑转移分子机制。结果:1.经石蜡组织转录组测序及RT-PCR筛选,发现肺癌脑转移灶较原发灶组织存在差异基因RPTOR改变,提示RPTOR基因表达与肺癌脑转移相关,在此基础上利用c Bio Portal数据库多个非小细胞肺癌研究数据分析,发现RPTOR基因在非小细胞肺癌中的基因变异以基因突变及扩增为主。对TCGA数据库公开的肺癌数据等进行差异分析,发现RPTOR在肺癌组织配对样本及非配对样本中,肿瘤样本的m RNA表达水平显著高于配对癌旁组织及正常肺组织,差异具有统计学意义。KEGG通路及GSEA富集分析发现RPTOR基因在多条通路显著富集相关,其中SPHK2与鞘磷脂合成通路(Sphingolipid metabolism)显著相关,与我们前期研究结果符合。2.临床样本免疫组化检测发现肺癌脑转移组RPTOR蛋白表达高于无脑转移组,差异具有统计学意义,而高表达RPTOR患者中位生存期显著低于RPTOR低表达者。RT-PCR及WB实验结果表明,RPTOR蛋白在非小细胞肺癌细胞株表达量高于正常肺上皮细胞;稳定过表达RPTOR可引起非小细胞肺癌细胞出现增殖、迁移、侵袭及通过血脑屏障能力增强,相关EMT蛋白出现改变。而敲减RPTOR可引起相反的结果。祼鼠体内实验显示,RPTOR过表达可以促进肺癌细胞脑转移,免疫组化实验结果显示肿瘤细胞核增殖指数、肿瘤平均微血管密度及MMP9蛋白都出现表达增强的趋势,而敲减后肿瘤组织相关指标下调。3.免疫荧光共定位、Co-IP结果提示RPTOR与YY1蛋白存在相互作用,双荧光素酶报告基因检测及CHIP实验结果验证转录因子YY1调控SPHK2启动子转录,RPTOR作为转录辅助因子,通过转录因子YY1与SPHK2相互作用。WB检测信号轴通路关键蛋白改变;验证RPTOR与SPHK2/S1P/STAT3信号轴之间关系,利用SPHK2通路抑制剂可消除RPTOR对肺癌细胞的增殖、迁移侵袭及通过体外血脑屏障能力的影响,验证RPTOR调控SPHK2/S1P/STAT3轴促进肺癌脑转移的机制。结论:1.基于转录组测序及生信分析结果,RPTOR基因与肺癌脑转移显著相关。2.RPTOR基因促进非小细胞肺癌细胞增殖、迁移、侵袭及通过血脑屏障。3.RPTOR作为转录辅助因子,调控转录因子YY1与SPHK2相互作用,并通过鞘磷脂代谢通路SPHK2/S1P/STAT3信号轴调控脑转移的发生发展。更多还原

【Abstract】 ObjectiveThe morbidity and mortality of primary lung cancer ranks first among malignant tumors.The incidence of brain metastasis of non-small cell lung cancer is high and the quality of life of patients is poor.Although there are various treatment methods for brain metastasis,the prognosis of the patients is still not satisfactory.At present,the occurrence and development mechanism of brain metastasis of lung cancer is not clear.In this study,genomics and bioinformatics methods were used to find that RPTOR was related to brain metastasis of non-small cell lung cancer;secondly,clinical sample cells and animal experiments were used to study the mechanism of RPTOR in brain metastasis of non-small cell lung cancer;finally,to explore the pathogenesis of SPHK2/S1P/STAT3 axis promoting brain metastasis of non-small cell lung cancer,in order to provide scientific basis for the molecular mechanism of brain metastasis of non-small cell lung cancer.Methods1.Transcriptome sequencing(RNA-seq)was used to study the differential genes in non-small cell lung cancer tissues and brain metastatic tissues of lung cancer.It was found that the expression of RPTOR in brain metastases of lung cancer was up-regulated than that in primary tissues,suggesting that there is a correlation between RPTOR gene expression and brain metastasis of lung cancer,which may be the key driving gene event of brain metastasis.On this basis,the related bioinformatics analysis was carried out by using the open network database,suggesting that RPTOR gene may be related to brain metastasis of lung cancer.2.Based on the previous research of our group,brain metastasis of lung cancer may be related to ceramide pathway.To explore the regulation of RPTOR on the occurrence and development of brain metastasis through SPHK2/S1P/STAT3 signal axis of sphingomyelin metabolic pathway,we used tissue samples to analyze clinicopathological features and draw survival curves;the interference of RPTOR gene at the cellular level affected the proliferation,migration and invasion of lung cancer cells and the ability to pass the blood brain barrier in vitro.The changes of EMT-related functional proteins were detected by western blot,and the cell transcriptome sequencing was verified.In addition,lung cancer cells were injected into the left ventricle of nude mice to induce brain metastasis,so as to further clarify the mechanism of RPTOR gene causing brain metastasis of lung cancer.3.The molecular mechanism of the interaction between RPTOR and SPHK2 through transcription factor YY1 was further verified by immunofluorescence co-localization,Co-IP,luciferase reporter assay and chromatin immunoprecipitation CHIP.The effects of RPTOR on the proliferation,cell cycle,migration and invasion of lung cancer cells and the ability to pass through the blood-brain barrier in vitro were observed by using SPHK2 pathway inhibitors to verify the relationship between RPTOR regulating SPHK2/S1P/STAT3 axis.Results1.By FFPE RNA-seq,it was found that there were differential gene RPTOR changes in lung cancer brain metastases compared with primary tissues,suggesting that the expression of RPTOR gene was related to lung cancer brain metastasis and may be the key driving gene event of brain metastasis..On this basis,using the data analysis of several non-small cell lung cancer studies in c Bio Portal database,it was found that the main gene variation of RPTOR gene in non-small cell lung cancer was gene mutation and amplification.Based on the difference analysis of lung cancer data published in TCGA database,it was found that the m RNA expression of RPTOR in lung cancer tissue paired samples and unmatched samples was significantly higher than that in paracancerous tissues and normal lung tissues,and the difference was statistically significant.KEGG pathway and GSEA enrichment analysis showed that RPTOR gene was significantly enriched in multiple pathways,among which SPHK2 was significantly correlated with sphingomyelin synthesis pathway(Sphingolipid metabolism),which was consistent with our previous research results.2.Immunohistochemical detection of clinical samples showed that the expression of RPTOR protein in the lung cancer group with brain metastasis was higher than that in the group without brain metastasis,and the median survival time of patients with high expression of RPTOR was significantly lower than that of patients with low expression of RPTOR.RT-PCR and WB assay results showed that the expression of RPTOR protein in non-small cell lung cancer cell line was higher than that in normal lung epithelial cells.The stable overexpression of RPTOR can induce the proliferation,migration,invasion and the ability to cross the blood-brain barrier of NSCLC cells,and the related EMT protein changes.But knocking down RPTOR can cause the opposite result.In vivo experiments showed that overexpression of RPTOR promoted brain metastasis in lung cancer cells.The expression of tumor nuclear proliferation index,tumor mean microvessel density and MMP9 protein all increased,while tumor tissue related indexes were down-regulated after knockdown.3.Immunofluorescence co-localization and immunoprecipitation Co-IP results showed that RPTOR interacted with YY1 protein.The results of Luciferase reporter gene and chromatin immunoprecipitation CHIP confirmed that transcription factor YY1 regulated SPHK2 promoter transcription.RPTOR,as a transcription co-factor,interacted with SPHK2 through transcription factor YY1.Inhibitors of SPHK2 pathway can eliminate the effects of RPTOR on proliferation,cell cycle,migration and invasion of lung cancer cells and the ability to pass through the blood-brain barrier in vitro,and verify the interaction between RPTOR and SPHK2/S1P/STAT3 axis.Conclusions1.Based on the results of RNA-seq,there was a significant correlation between RPTOR gene and brain metastasis of lung cancer.2.The study of RPTOR gene promoting the proliferation,migration,invasion and passage of the blood-brain barrier of non-small cell lung cancer cells.3.RPTOR,as a transcriptional co-factor,regulates the interaction between transcription factor YY1 and SPHK2.RPTOR gene regulates the occurrence and development of brain metastasis through SPHK2/S1P/STAT3 signal axis of sphingomyelin metabolic pathway.更多还原