【作者】 李丽；

【导师】 何平；

【作者基本信息】 中国医科大学 ， 老年医学， 2021， 博士

【摘要】 目的:肺癌是最常见的恶性肿瘤之一,在全球范围内恶性肿瘤导致的死亡中占比最高。肺癌的主要类型为非小细胞肺癌(Non-small-cell lung carcinoma,NSCLC),非小细胞肺癌中的主要组织学类型是肺腺癌。一直以来困扰临床的问题是伴有转移的非小细胞肺癌患者的生存率低,预后恶劣。早诊断、早治疗无疑会使患者的生存率增加并使预后改善;若肺癌被发现时已出现转移,那么找出促进其转移的分子并给予针对治疗,就很必要。环状RNA是一类具有独特闭环结构的非编码RNA,能够避免核酸内切酶的酶切作用,使其拥有很好的稳定性。环状RNA还具有很好的组织特异性和物种保守性,这就意味着环状RNA可能成为一种新型诊断标志物,也预示其可能具有评估疾病预后的价值。本研究拟通过高通量测序筛选出在NSCLC癌组织中高表达的环状RNA,再扩大样本量验证,通过一系列实验明确其生物学特性和作用机制,为NSCLC的诊治提供一个新的视角。研究方法:1.我们陆续采集了2017年7月至2018年1月期间接受肺癌切除术患者(n=41)的癌组织及癌旁组织,这些患者就诊于中国医科大学附属盛京医院胸外科,同时为了后续研究,我们也收集了这些患者相应的临床病理资料。2.选取肺腺癌癌组织3例及其对应的癌旁组织3例进行高通量测序,并将测序结果进行生物信息学分析,筛选出在癌组织中高表达的hsa＿circ＿0067582,hsa＿circ＿0005692和低表达的novel＿circ＿0008866和novel＿circ＿0005280,进而扩大样本量,进行real-time qPCR验证,并依据表达情况对hsa＿circ＿0067582及novel＿circ＿0005280进行临床病理相关性分析。3.选择在NSCLC的癌组织中高表达的hsa＿circ＿0067582做为研究对象。Sanger测序、Rnase R酶耐受实验验证其环状结构、明确其环化位点;核质分离实验确定其细胞内定位;多个肺癌细胞系内完成real-time qPCR实验检测其表达量,筛选出A549细胞株(hsa＿circ＿0067582的表达量最高)和H1299细胞株(hsa＿circ＿0067582的表达量最低)进行后续实验。将hsa＿circ＿0067582的干扰质粒转染入细胞后进行克隆形成实验、MTT实验、细胞划痕实验以及transwell侵袭实验,分别检测其增殖、迁移及侵袭能力。4.在A549细胞内,用RNA-pulldown实验拉下与hsa＿circ＿0067582结合的蛋白质,并将这些蛋白质进行蛋白质谱分析,选择富集较好的RNA结合蛋白hnRNPM作为进一步研究的目的蛋白;拉下来的总RNA进行real-time qPCR实验,验证数据库预测的5个miRNA与hsa＿circ＿0067582是否有结合。RIP实验结合real-time qPCR反向验证hnRNPM与hsa＿circ＿0067582有结合。沉默hsa＿circ＿0067582,验证hnRNPM的m RNA表达量及蛋白表达量。沉默hnRNPM,验证hsa＿circ＿0067582的表达量。提取并清洗TCGA数据库数据,分析肺腺癌及肺鳞癌组织中hnRNPM的表达量及绘制受试者工作特征曲线(ROC)评价其诊断价值,绘制K-M生存曲线判断其预后评估价值。结果:1.NSCLC患者的癌组织及癌旁组织的环状RNA存在差异性表达,具有组织特异性。通过3名肺腺癌患者的癌组织及癌旁组织的高通量测序,分析测序数据得到以下结果:在NSCLC的癌组织及癌旁组织中共有81个差异表达的环状RNA,其中17个在癌组织中高表达,64个在癌组织中低表达。2.通过生物信息学分析,本研究共确定了15种有意义的功能类型。在生物过程类型中,差异基因主要富集在细胞分解代谢过程、含核碱基调节、生物过程、含核碱基代谢物调节、含氮化合物代谢物调节和杂环代谢过程等方面。在细胞成分类型中,差异表达基因主要富集在细胞部分、胞内部分、细胞质和细胞器等。在分子功能类型中,差异表达基因主要富集于催化活性、蛋白结合和转移酶活性。在KEGG途径分析中,吞噬体、肌动蛋白细胞骨架调节和赖氨酸降解存在差异。3.扩大样本量进行real-time qPCR实验,验证了novel＿circ＿0008866和novel＿circ＿0005280在NSCLC癌组织中低表达,hsa＿circ＿0067582在NSCLC癌组织中高表达,均与测序结果一致。Hsa＿circ＿0005692在NSCLC癌组织中低表达,与测序结果不一致。4.NSCLC患者的临床病理分析结果显示:hsa＿circ＿0067582的表达水平与患者是否有淋巴结转移显著相关,有淋巴结转移的患者的表达水平更高;而novel＿circ＿0005280的表达水平与患者的肿瘤直径显著相关,肿瘤直径大的患者表达水平更低。5.Hsa＿circ＿0067582具有明显的Rnase R酶耐受性,其剪切位点为:ACAGTG。6.在各肺癌细胞系内进行real-time qPCR实验,得到的hsa＿circ＿0067582的相对表达量以A549细胞内最高,H1299细胞内最低。7.Hsa＿circ＿0067582主要定位于细胞核中。8.沉默hsa＿circ＿0067582,使NSCLC的增殖、迁移及侵袭能力减低。9.Hsa＿circ＿0067582与RNA结合蛋白hnRNPM相互结合。10.在A549细胞内沉默hsa＿circ＿00675822可显著下调hnRNPM的蛋白表达,但不影响m RNA水平。沉默hnRNPM后hsa＿circ＿0067582的表达量相应减少,hnRNPM与hsa＿circ＿0067582的表达正相关。11.TCGA数据库来源的测序数据分析结果显示,无论是肺腺癌还是肺鳞癌hnRNPM均在癌组织中高表达。12.TCGA数据库显示,hnRNPM对NSCLC具有一定的诊断能力及评估预后能力。结论:1.Novel＿circ＿0005280在人NSCLC癌组织中低表达,表达量与肿瘤直径大小负相关;Hsa＿circ＿0067582在人NSCLC癌组织中高表达,表达量与患者淋巴结是否转移正相关;Hsa＿circ＿0067582和novel＿circ＿0005280具有成为人NSCLC诊断的生物标志物的可能性。2.沉默hsa＿circ＿0067582,使NSCLC的增殖、迁移及侵袭能力减低。3.Hsa＿circ＿0067582与hnRNPM相互结合,并能够调节其蛋白表达,为NSCLC的诊治提供全新的思考方向。更多还原

【Abstract】 Objective: Lung cancer is one of the most common malignant tumors,accounting for the highest proportion of deaths caused by malignant tumors worldwide.The main type of lung cancer is non-small cell lung cancer(NSCLC),among which lung adenocarcinoma is the main histological type.The low survival rate and poor prognosis of patients with metastatic non-small cell lung cancer have always been a difficult clinical problem.Early diagnosis and treatment will undoubtedly increase the survival rate and improve the prognosis of patients.In case of metastasis,it is necessary to identify the molecules that promote metastasis and give targeted therapy.Circular RNA(circRNA)is a non-coding RNA,which has a unique closed-loop structure and can avoid digestion by endonucleases,so it has good stability.Circ RNA also has good tissue specificity and species conservation,which means that circRNA may become a new diagnostic marker,which also indicates that it may have the value of evaluating the prognosis of diseases.In this study,the highly expressed circRNA in non-small cell lung cancer tissues was screened by high-throughput sequencing,and then its biological characteristics and mechanism were confirmed by a series of experiments,which provided a new perspective for the diagnosis and treatment of non-small cell lung cancer.Methods: 1.We collected cancer tissues and adjacent tissues of patients(n=41)who underwent lung cancer resection from July 2017 to January 2018.These patients were admitted to the Department of Thoracic Surgery,Shengjing Hospital of China Medical University.At the same time,for the follow-up study,we also collected the corresponding clinicopathological data of these patients.2.Three cases of lung adenocarcinoma and three corresponding adjacent tissues were selected for high-throughput sequencing,and differentially expressed circRNA was screened,and bioinformatics analysis was performed.Select the highly expressed circRNA in cancer tissues: hsa＿circ＿0067582,hsa＿circ＿0005692;Low expression of circRNAs in cancer tissues were novel＿circ-＿0008866 and novel＿circ＿0005280.After enlarging the sample size,it was verified by real-time qPCR,and the clinicopathological correlation between hsa＿circ＿0067582 and novel＿circ＿0005280was analyzed according to the expression.3.Hsa-circ-0067582,which is highly expressed in tumor tissues,was selected as the research object,and its cyclic structure was verified by Sanger sequencing and Rnase enzyme tolerance test,and its cyclic site was confirmed.Intracellular localization was determined by nucleoplasm separation experiment.The real-time qPCR in multiple lung cancer cell lines verified its expression level,and A549 cell line with the highest expression level and H1299 cell line with the lowest expression level were selected as tool cells.The interference plasmid hsa＿circ＿0067582 was constructed and screened out.After transfection,the cells were subjected to colony formation assay and MTT assay to detect their proliferation ability,wound healing assay to detect their migration ability,and transwell invasion assay to detect their invasion ability.4.Using A549 cells as a tool,the protein binding to hsa＿circ＿0067582 was pulled down by RNA pull-down experiment,and analyzed by protein mass spectrometry.The RNA binding protein hnRNPM with good enrichment was selected as the target protein for further study.The pulled total RNA was verified by real-time qPCR,and the five miRNA predicted by the database were combined with hsa＿circ＿0067582.RIP experiment and real-time qPCR reverse verification hnRNPM and hsa＿circ＿0067582 are combined.The m RNA expression and protein expression of hnRNPM were verified by silencing hsa＿circ＿0067582.The expression of hsa＿circ＿0067582 was confirmed by silencing hnRNPM.Extract and clean TCGA database data,analyze the expression of hnRNPM in lung adenocarcinoma and lung squamous cell carcinoma,draw receiver operating characteristic curve(ROC)to evaluate its diagnostic value,and draw K-M survival curve to evaluate its prognostic value.Results: 1.The expression of circRNAs in cancer tissues and adjacent tissues of patients with NSCLC were different,which is tissue specific.By analyzing the high-throughput sequencing of cancer tissues and adjacent tissues of three patients with lung adenocarcinoma,the following results were obtained: there were 81 differentially expressed circRNAs in NSCLC cancer tissues and adjacent tissues,of which 17 were highly expressed in cancer tissues and 64 were poorly expressed in cancer tissues.2.Through bioinformatics analysis,this study identified 15 significant functional types.In biological process types,the differential genes are mainly concentrated in cell catabolism,nucleobase regulation,biological process,nucleobase-containing metabolite regulation,nitrogenous compound metabolite regulation and heterocyclic metabolism.In the types of cell component,differentially expressed genes are mainly concentrated in cell parts,intracellular parts,cytoplasm and organelles.Among the functional types of molecules,differentially expressed genes mainly focus on catalytic activity,protein binding and transferase activity.In KEGG pathway analysis,there were differences in phagocyte,actin cytoskeleton regulation and lysine degradation.3.The real-time qPCR experiment was carried out by enlarging the sample size,which verified that novel＿circ＿0008866 and novel＿circ＿0005280 were low in NSCLC cancer tissues,and hsa＿circ＿0067582 was high in NSCLC cancer tissues,which were consistent with the sequencing results.The low expression of hsa＿circ＿0005692 in NSCLC was inconsistent with the sequencing results.4.Clinicopathological analysis of NSCLC patients showed that the expression level of hsa＿circ＿0067582 was significantly correlated with lymph node metastasis,and the expression level of patients with lymph node metastasis was higher.However,the expression level of novel＿circ＿0005280 was significantly correlated with the tumor diameter of patients,and the expression level of patients with large tumor diameter was lower.5.Hsa-circ-0067582 has obvious Rnase R resistance,and its Cyclization site is ACAGTG.6.The real-time qPCR experiments were carried out in various lung cancer cell lines,and the relative expression of hsa＿circ＿0067582 was the highest in A549 cells and the lowest in H1299 cells.7.Hsa＿circ＿0067582 is mainly located in the nucleus.8.The silencing of hsa＿circ＿0067582 weakens the proliferation,migration and invasion of NSCLC.9.Hsa-circ-0067582 binds to RNA binding protein hnRNPM.10.The silencing of hsa＿circ＿0067582 in A549 cells can significantly down-regulate the protein expression of hnRNPM,but does not affect the m RNA level.The expression of hsa＿circ＿0067582 decreased after hnRNPM was silenced,and hnRNPM was positively correlated with hsa＿circ＿0067582.11.TCGA database showed that hnRNPM was highly expressed in non-small cell lung cancer tissues,while it was low expressed in adjacent tissues.12.TCGA database shows that hnRNPM has certain diagnostic accuracy and prognostic ability for non-small cell lung cancer.Conclusion: 1.Novel＿circ＿0005280 is poorly expressed in human NSCLC,and the expression level is negatively correlated with tumor diameter.Hsa＿circ＿0067582 is highly expressed in human NSCLC cancer tissues,and the expression level is positively correlated with whether lymph nodes of patients are metastasized.Hsa＿circ＿0067582 and novel＿circ＿0005280 may become biomarkers for the diagnosis of human NSCLC.2.The silencing of hsa＿circ＿0067582 reduced the proliferation,migration and invasion of NSCLC.3.Hsa＿circ＿0067582 can combine with hnRNPM and regulate its protein expression,which provides a new thinking direction for the diagnosis and treatment of NSCLC.更多还原