【作者】 周伟；

【导师】 赵军；

【作者基本信息】 苏州大学 ， 临床医学（专业学位）， 2022， 博士

【摘要】 目的:肺癌(Lung Cancer)是癌症相关死亡最主要的病种,而肺腺癌(Lung Adenocarcinoma,LUAD)是肺癌重要的病理亚型。尽管有外科手术、放疗、化疗和免疫治疗等多种抗癌治疗方式,但中晚期肺腺癌的生存预后仍不理想,肿瘤分泌的外泌体microRNAs(miRs)能够调控血管新生,然而具体作用机制仍未完全阐明,因此有必要进行相关研究,寻找新的治疗靶点。我们前期的生物信息学研究说明NCAPH(non-SMC condensin I complex subunit H)在LUAD中高表达,且是患者预后不良因素,并且预测到miR-1976可能是其上游的调控miR,通过q RT-PCR检测发现miR-1976在LUAD中低表达,并且免疫组化提示miR-1976与LUAD肿瘤微血管密度呈负相关,这些实验结果说明LUAD的血管新生可能受到miR-1976的调控。外泌体是细胞分泌的生物囊泡,平均直径在30到100 nm之间,既往研究提示肿瘤细胞既能分泌促进肿瘤血管新生的外泌体miRs也能分泌抑制肿瘤血管新生的外泌体miRs,肿瘤细胞分泌的外泌体能够被血管内皮细胞摄取从而调控肿瘤血管新生,肿瘤组织血管新生不仅是促进血管新生因素增强的结果,也包括抑制血管新生因素的减弱。因此,本研究目的在于探讨LUAD细胞外泌体miR-1976在肿瘤血管新生的作用并研究具体作用机制。方法:我们采用人类蛋白质图谱数据库(Human Protein Atlas database,HPA数据库)、癌症基因组图谱(The Cancer Genome Atlas,TCGA)和GEO(Gene Expression Omnibus)数据库分析了正常肺组织和LUAD癌组织中NCAPH mRNA和蛋白质的表达量。绘制不同表达水平NCAPH的LUAD患者Kaplan Meier生存曲线。此外,利用c Bio Portal、GEPIA和UALCAN数据库我们获得了LUAD中NCAPH的共表达基因。然后,通过DAVID在线工具对NCAPH的共表达基因进行GO(gene ontology)分析和KEGG(Kyoto Encyclopedia of Genes and Genomes)通路分析,用STRING数据库构建蛋白质-蛋白质相互作用(Protein-Protein Interaction,PPI)网络,并通过Cytoscape软件识别和可视化关键基因(Hub Gene)。为了预测调控NCAPH的miR,使用Target Scan数据库、miRDB和ONCOMIR数据库综合分析来寻找潜在的上游调控miR。本研究纳入了24例LUAD手术切除癌组织标本和正常肺组织标本,进行q RT-PCR检测及CD34免疫组化染色检测。超速离心法获取A549培养基外泌体,并且使用PKH67进行外泌体染色。染色后的外泌体与人脐静脉内皮细胞(Human Umbilical Vein Endothelial Cell,HUVEC)进行共培养,观察人脐静脉内皮细胞对外泌体的摄取情况。使用miR-1976 MIMICS转染A549细胞,获取高表达miR-1976的外泌体。我们进行CCK8实验、Transwell实验、血管形成实验用来明确NCAPH、外泌体miR-1976对HUVECs增殖、迁移、血管形成能力的影响。通过q RT-PCR、Western-Blot实验、双荧光素酶报告实验及RESCUE实验进一步研究了外泌体miR-1976调控NCAPH影响血管新生的机制。最后我们建立了裸鼠皮下成瘤动物模型进一步明确体内环境下外泌体miR-1976对于LUAD肿瘤血管新生的影响。结果:NCAPH在LUAD癌组织中表达水平高于癌旁正常肺组织,高表达NCAPH的患者预后较差。NCAPH共表达HUB基因为CDK1、BUB1、BUB1B、CCNB1、CCNA2、KIF11、TOP2A、CDC45和CDC20,生存分析提示这9个基因均在LUAD中发挥促癌作用。通过生物信息学预测到在LUAD中,miR-1976可能是NCAPH上游的调控miR。基于ENCORI数据库分析结果显示LUAD患者NCAPH mRNA表达水平与miR-1976表达水平的呈负相关。基于24例肺腺癌临床标本分析提示miR-1976在LUAD癌组织中表达水平低于癌旁组织。LUAD癌组织miR-1976表达水平与肿瘤TNM分期相关,晚期LUAD癌组织miR-1976表达水平低于早期LUAD。LUAD患者miR-1976表达水平与癌组织微血管密度(Microvessel Density,MVD)呈负相关。肺腺癌细胞外泌体miR-1976能够被HUVEC摄取,继而升高血管内皮细胞内miR-1976的表达水平。敲减NCAPH能够抑制HUVEC增殖、迁移及血管形成。miR-1976能够通过靶向结合NCAPH 3’UTR抑制其蛋白表达。较正常支气管上皮细胞相比,肺腺癌细胞分泌的外泌体miR-1976明显减少。肺腺癌细胞外泌体miR-1976能够被HUVEC摄取,并且提高HUVEC内miR-1976表达水平。通过双荧光素酶报告实验和RESCUE实验,验证了肺腺癌细胞外泌体miR-1976能够通过NCAPH抑制HUVEC增殖、迁移及血管形成。裸鼠皮下成瘤实验证明肺腺癌细胞来源的外泌体miR-1976在体内条件下能够遏制肿瘤生长,抑制肿瘤血管新生,并且能够下调NCAPH。结论:与正常支气管粘膜上皮相比,肺腺癌细胞外泌体miR-1976表达水平明显降低,外泌体miR-1976能够被肺腺癌肿瘤内血管内皮细胞摄取,通过靶向调控NCAPH抑制肿瘤血管新生。我们的研究为以后临床治疗肺腺癌提供了潜在的新的治疗手段。更多还原

【Abstract】 Objective:Lung cancer is the most common cause of cancer-related death,and lung adenocarcinoma(LUAD)is an important pathological subtype of lung cancer.Despite various anti-cancer treatments such as surgery,radiotherapy,chemotherapy,and immunotherapy,the survival and prognosis of advanced lung adenocarcinoma is still unsatisfactory.Tumor-secreted exosomal microRNAs(miRs)can regulate angiogenesis.However,the relevant mechanism is still not fully elucidated,so it is necessary to conduct related research to find new therapeutic targets.Our previous bioinformatics analysis showed that NCAPH(non-SMC condensin I complex subunit H)was highly expressed in LUAD and was a poor prognostic factor for patients,and it was predicted that miR-1976might be its upstream regulatory microRNA(miR).By q RT-PCR assay,miR-1976 was found to be lowly expressed in LUAD,and we also found that miR-1976 was negatively correlated with tumor microvessel density(MVD)in LUAD by immunohistochemistry.These results suggests that angiogenesis in LUAD may be regulated by miR-1976.Exosomes are biological vesicles secreted by cells,with an average diameter between 30and 100 nm.Previous studies have shown that tumor cells can secrete both exosomal miRs that promote tumor angiogenesis and exosomal miRs that inhibit tumor angiogenesis.The exosomes secreted by tumor cells can be taken up by vascular endothelial cells to regulate tumor angiogenesis.Tumor angiogenesis is not only the result of the enhancement of factors that promote angiogenesis,but also the weakening of factors that inhibit angiogenesis.Therefore,the purpose of this study was to investigate the role of exosomal miR-1976 in LUAD angiogenesis and to study the specific mechanism.Methods:We analyzed NCAPH mRNA and protein expression level in normal and cancer tissues using the Human Protein Atlas database(HPA database),The Cancer Genome Atlas(TCGA),and Gene Expression Omnibus(GEO).The Kaplan Meier survival curves of LUAD patients with different expression levels of NCAPH were drawn.Furthermore,using the c Bio Portal,GEPIA and UALCAN databases we obtained the co-expressed genes of NCAPH in LUAD.Then,GO(gene ontology)analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis were performed on the co-expressed genes of NCAPH by the online tool DAVID,and a protein-protein interaction(PPI)network was constructed with the STRING database,and Cytoscape was used to identify and visualize key genes(Hub Gene).In order to predict miRs(microRNAs)that regulate NCAPH in LUAD,a comprehensive analysis of the Target Scan database,miRDB and ONCOMIR databases was adopted to predict potential upstream regulatory miRs.Twenty-four LUAD patients were enrolled in this study.The surgically resected tumor tissue specimens and adjacent normal lung tissue specimens were used for q RT-PCR detection and CD34 immunohistochemical staining.A549 cell culture medium exosomes were obtained by ultracentrifugation,and exosome staining was performed using PKH67.The stained exosomes were co-cultured with human umbilical vein endothelial cells(HUVECs)to observe the uptake of exosomes by HUVECs.A549 cells were transfected with miR-1976 MIMICS to obtain exosomes highly expressing miR-1976.We performed CCK8 assay,Transwell assay,and Tube Formation assay to clarify the effects of NCAPH and exosomal miR-1976 on the proliferation,migration,and tube formation ability of HUVECs.The mechanism of exosomal miR-1976 regulating NCAPH on angiogenesis was further studied by q RT-PCR,Western-Blot assay,dual luciferase reporter assay and RESCUE assay.Finally,we performed subcutaneous tumorigenesis experiments in nude mice to clarify the effect of exosomal miR-1976 on LUAD angiogenesis in vivo.Results:The expression level of NCAPH in LUAD tumor tissues was higher than that in adjacent normal tissues,and the prognosis of patients with high NCAPH expression was poor.NCAPH co-expressed HUB genes were CDK1,BUB1,BUB1B,CCNB1,CCNA2,KIF11,TOP2A,CDC45 and CDC20,and survival analysis suggested that these 9genes all play a role in promoting cancer in LUAD.It was predicted by combined bioinformatics that miR-1976 might be a regulatory miR of NCAPH in LUAD.The analysis based on ENCORI database showed that the expression level of NCAPH mRNA in LUAD patients was negatively correlated with the expression level of miR-1976.In the specimens of 24 lung adenocarcinoma patients,the expression level of miR-1976 in LUAD cancer tissues was lower than that in adjacent normal tissues.The expression level of miR-1976 in LUAD cancer tissue was correlated with tumor TNM stage,and the expression level of miR-1976 in advanced LUAD cancer tissue was lower than that in early LUAD.The expression level of miR-1976 in LUAD patients was negatively correlated with the microvessel density(MVD)of cancer tissue.LUAD tumor cell exosomal miR-1976 can be taken up by HUVECs,which in turn increases the expression level of miR-1976 in vascular endothelial cells.Knockdown of NCAPH in HUVECs inhibited cell proliferation,migration and tube formation ability.miR-1976 can inhibit its protein expression by targeting the 3’UTR of NCAPH mRNA.Compared with normal bronchial epithelial cells,lung adenocarcinoma cells secreted significantly less exosomal miR-1976.Exosomal miR-1976 secreted by LUAD cell could be taken up by HUVECs and then increased the expression level of miR-1976 in HUVECs.Through dual luciferase reporter assay and RESCUE assay,it was verified that exosomal miR-1976 of lung adenocarcinoma cells can inhibit the proliferation,migration and tube formation of HUVECs through NCAPH.Subcutaneous tumorigenesis experiments in nude mice demonstrated that exosomal miR-1976 derived from lung adenocarcinoma cells could suppress tumor growth,inhibit tumor angiogenesis,and downregulate NCAPH.Conclusion:Compared with normal bronchial epithelium,the expression level of exosomal miR-1976 secreted by lung adenocarcinoma cells was significantly reduced,and exosomal miR-1976 could be taken up by vascular endothelial cells in LUAD and inhibited angiogenesis by targeting NCAPH.Our study provides a potential new therapeutic approach for the treatment of lung adenocarcinoma in the future.更多还原