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类型 基础研究 预答辩日期 2018-05-17
开始(开题)日期 2016-05-31 论文结束日期 2018-03-18
地点 中大医院检验科会议室 论文选题来源 国家自然科学基金项目     论文字数 5 (万字)
题目 环状RNA hsa_circ_0013958的筛选及其在肺腺癌中作用的研究
主题词 肺腺癌,has_circ_0013958,生物学功能,miR-134,ceRNA
摘要 研究背景: 肺癌是严重危害人类健康的第一大恶性肿瘤,其发病率和死亡率均居全球恶性肿瘤的首位。肺腺癌是肺癌中最常见的病理类型,其发病人数呈逐年增多趋势。肺腺癌早期在CT上表现为肺结节,然而病灶早期较小且多位于两肺的外周,给临床医生判断其良、恶性带来极大挑战。因此,积极寻找新型的分子标志物,对肺腺癌的早期诊断具有十分重要的临床意义。环状RNA(CircRNA)具有组织特异性和疾病特异性,研究发现CircRNA在多种肿瘤中具有特定的表达谱,因此CircRNA具有成为肿瘤诊断标志物的潜力。 研究目的: 研究CircRNA作为分子标志物对肺腺癌的诊断价值,并研究其在增殖、凋亡及侵袭转移等方面对肺腺癌的影响,进一步研究其吸附的miRNA分子及可能的ceRNA机制,为肺腺癌的早期诊断提供新的思路。 研究方法: (1)筛查和验证 本研究首先应用CircRNA芯片对3例确诊肺腺癌的组织标本进行分析,在高表达的CircRNA分子中挑选出差异表达最明显的CircRNA(has_circ_0013958),使用qPCR(Quantitative Polymerase Chain Reaction, qPCR)在多种肺腺癌细胞株、49例肺腺癌患者及30例肺腺癌患者血浆等标本中进行验证。 (2)has_circ_0013958诊断效能分析 采用ROC曲线分析has_circ_0013958作为诊断标志物在组织及血浆中的诊断价值。 (3)has_circ_0013958对肺腺癌增殖、凋亡、侵袭的影响 通过CCK-8实验和EDU实验检测has_circ_0013958对肺腺癌细胞增殖的影响,使用流式细胞仪检测has_circ_0013958对细胞凋亡的影响,Transwell实验检测has_circ_0013958对细胞迁移和侵袭的影响。裸鼠荷瘤实验从体内验证has_circ_0013958对肺腺癌的影响,对裸鼠的瘤组织进行免疫组化染色检测Ki67表达,并通过TUNEL法检测细胞凋亡比例。 (4)内源性竞争性RNA(competing endogenous RNA, ceRNA)机制研究 FISH (Fluorescence In Situ Hybridization)实验考察has_circ_0013958在肺腺癌细胞内的分布情况,通过生物信息学数据库分析has_circ_0013958可能的下游miRNA及mRNA分子,进一步通过荧光素酶报告法和Western Blot进行验证。 研究结果: (1)筛查和验证 CircRNA芯片共筛选出差异表达至2倍以上且有统计学差异的CircRNA分子 59种,其中相对正常组织高表达的39种,低表达的20种。其中,has_circ_0013958(hsa_circRNA_100323)高表达最明显,因此我们选择它作为候选的CircRNA进行验证。Northern Blot和PCR产物测序验证qPCR正确后,我们检测了7种肺腺癌细胞株,发现has_circ_0013958相对于正常细胞,在肺腺癌细胞中has_circ_0013958明显升高(P<0.05)。我们检测了49例肺腺癌患者的癌组织及癌旁组织中circ_0013958的表达情况,结果显示,肺腺癌中has_circ_0013958高表达(P<0.05)。为了明确has_circ_0013958在血浆中是否高表达,我们检测30例患者血浆标本,结果显示,30例中 有27例高表达。 (2)has_circ_0013958诊断效能分析 我们对组织标本结果进行ROC分析,结果显示has_circ_0013958的总体ROC曲线的曲线下面积(AUC)是0.815(P<0.001),以0.00101为cutoff值,敏感度和特异度分别为0.755和0.796;I期肺腺癌ROC的AUC是0.750(P<0.001),敏感度和特异度分别为0.583和0.833;II期肺腺癌ROC的AUC是0.766,敏感度和特异度分别为0.813和0.750;III~IV期肺腺癌ROC的AUC为0.874,敏感度和特异度分别是0.762和0.857。血浆标本ROC曲线的AUC是0.794(P<0.001),敏感度和特异度分别为0.667和0.933。 (3)has_circ_0013958对肺腺癌增殖、凋亡、侵袭的影响 has_circ_0013958对增殖的影响:CCK-8实验结果显示,肺腺癌细胞在敲低has_circ_0013958后细胞的OD450值较对照组明显下降(P<0.01)。EDU实验显示在敲低has_circ_0013958后,处于增殖期的细胞比例明显减少(P<0.01)。体内实验显示与对照组相比has_circ_0013958被敲低后能显著抑制肿瘤细胞的增殖(P<0.001)。裸鼠肿瘤组织免疫组化结果显示,has_circ_0013958被敲低后组织的Ki67的表达量明显减少(P<0.01)。 has_circ_0013958对凋亡的影响:流式细胞术结果显示,has_circ_0013958被敲低后细胞的凋亡比例明显增加(P<0.01)。对裸鼠荷瘤实验中的瘤组织进行TUNEL实验,结果显示,has_circ_0013958被敲低后细胞凋亡的比例明显增加(P<0.01)。 has_circ_0013958对侵袭的影响:Transwell实验显示has_circ_0013958被敲低后转移和侵袭的细胞数明显减少(P<0.01)。 (4)ceRNA机制研究 在进行ceRNA机制研究之前,我们首先对has_circ_0013958进行细胞内定位分析。FISH结果显示它主要分布于胞浆中。生物信息学预测分析发现,有5个miRNA分子分别为miR-134、miR-545、miR-629、miR-509、miR-660,可能与has_circ_0013958结合。双荧光素酶报告法实验显示只有miR-134能够与has_circ_0013958分子结合,而在非小细胞肺癌中CCND1是miR-134下游的靶基因之一,据此我们推测可能存在circ_0013958/miR-134/CCND1通路。Western Blot实验证实了我们的假设。 研究结论: (1)我们通过CircRNA芯片筛选获得了在肺腺癌中差异表达的系列CircRNA分子,在肺腺癌细胞、组织及血浆等标本中使用qPCR方法验证了has_circ_0013958高表达;(2)体内及体外实验均表明,has_circ_0013958具有促进肺腺癌细胞增殖、转移和侵袭的作用;同时,has_circ_0013958也具有抑制细胞凋亡的作用;(3)has_circ_0013958通过miR-134影响CCND1蛋白而发挥促进细胞增殖等生物学功能;(4) has_circ_0013958具有成为肺腺癌早期诊断标志物的潜力。
英文题目 Screening of has_circ_0013958 and its role in lung adenocarcinoma
英文主题词 Lung adenocarcinoma, has_circ_0013958, biological function, miR-134, ceRNA
英文摘要 BACKGROUND: Lung cancer is the leading cause of cancer-related deaths, because its morbidity and mortality is the highest in the world. Lung adenocarcinoma (LAC) is the most common pathological type, and its morbidity number is increasing year by year, therefore, it has attracted people’s attention. A large number of patients with LAC is characterized by solitary pulmonary nodules (SPN) on CT. Because the SPNs in early stage are usually small and mostly located in the periphery of the lung, it is a great challenge for clinicians to judge its benign or malignant. Therefore, it is necessary to search some new diagnostic biomarkers. Cyclic RNAs (CircRNAs) are tissue-specific and disease-specific, and they has been identified to be candidate biomarkers in many kind of tumors. Here, we will screen a circRNA with diagnostic value in LAC. OBJECTIVE: The aim of this study is to screen a circRNA with diagnostic value in LAC and then to study its functions and mechanisms. METHODS:In this study, we firstly used CircRNA microarray to analysis the cancer and normal tissues of three cases of patients with LAC, one of the highest CircRNA has_circ_0013958 was selected. The PCR products were verified by Northern Blot and sequencing method. Then, 7 kind of lung adenocarcinoma cell lines, 49 lung adenocarcinoma tissues and 30 lung adenocarcinoma samples were examined by qPCR to verify the expression of has_circ_0013958. The diagnostic value of has_circ_0013958 in tissues and plasma was analyzed by using ROC curve. In order to further study the biological function of has_circ_0013958, three siRNAs of has_circ_0013958 were designed and screened by qPCR. The effect of proliferation of has_circ_0013958 in lung adenocarcinoma cells was detected by CCK-8 and EDU experiments. The effect of has_circ_0013958 on cell apoptosis was detected by flow cytometry, and Transwell assays were used to detect metastasis and invasion functions. The model of human LAC xenografts in nude mouse was made to verify the effect of has_circ_0013958 on tumorigenesis and tumor growth in vivo. After the model was successfully made, cholesterol modified has_circ_0013958 siRNA was intratumorally injected once every 3 days, 10 nmol every time. The length and diameter of tumors were measured by vernier caliper every week for 4 weeks. After the mice were killed, the tumor tissue were removed and detected Ki67 by immunohistochemical staining, apoptosis were detected by TUNEL. In order to further investigate the mechanism of has_circ_0013958, we first detect its content in cytoplasm and nucleus by qPCR, and then it was morphologically verified by FISH method. On this basis, the bioinformatics database was used to analyze the possible downstream miRNA and mRNA molecules of has_circ_0013958, and then a network of interactions between them was constructed. The combination of has_circ_0013958 and miRNA was verified by luciferase assay, and the relationship between has_circ_0013958 and its downstream miRNA and mRNA molecules was verified by Western Blot method. RESULTS:A total of 59 kinds of CircRNA which were differentially expressed to more than 2 times and had statistical significance. Among them, 39 tissues were highly expressed, and 20 were low expressed. Has_circ_0013958 (hsa_circRNA_100323) is one of the most obvious expression of CircRNA, and it was we choose as the candidate biomarker for verification. The qPCR product was proved as correct by Northern Blot and sequencing. On this basis, has_circ_0013958 was highly expressed in 7 lung adenocarcinoma cell lines, 49 lung adenocarcinoma tissues and 30 lung adenocarcinoma patients (P < 0.05). The ROC analysis showed that the area under (AUC) of the overall ROC curve of has_circ_0013958 was 0.815, and the sensitivity and specificity were 0.755 and 0.796 respectively, and the cutoff value was 0.00101; Stage I ROC AUC 0.750 (P < 0.001), sensitivity and specificity were 0.583 and 0.833; stage II ROC AUC is 0.766, the sensitivity and specificity were 0.813 and 0.750; stage III~IV ROC AUC 0.874, sensitivity and specificity were 0.762 and 0.857 respectively. The ROC curve of has_circ_0013958 in plasma samples showed that AUC was 0.794 (P < 0.001), and sensitivity and specificity were 0.667 and 0.933 respectively. In order to perform functional studies, we first screened the siRNA sequence of has_circ_0013958. CCK-8 experiments showed that the lung adenocarcinoma cells were interfered by has_circ_0013958 of siRNA, and the OD450 value of the cells was significantly lower than that of the control group (P < 0.01). EDU experiments showed that after siRNA interference with has_circ_0013958, the percentage of cells in the proliferative phase decreased significantly (P < 0.01). To detect whether has_circ_0013958 can affect apoptosis, we used Annexin V FITC/PI to stain the cells and detected apoptosis by flow cytometry. The results showed that siRNA specific silencing of has_circ_0013958 significantly increased the apoptotic rate of cells (P < 0.01). Transwell experiments showed that the number of cells transferred and attacked by has_circ_0013958 was significantly decreased after silencing (P < 0.01). Tumor formation in nude mice showed that interference with has_circ_0013958 significantly inhibited tumor growth compared with the control group (P < 0.001). Compared with the control group, the expression of Ki67 in the tissues of the nude mice after the interference of has_circ_0013958 was significantly reduced (P < 0.01). TUNEL experiments were carried out on tumor tissues, and the results showed that the proportion of apoptotic cells was significantly increased after has_circ_0013958 interference (P < 0.01). After the clinical research and functional research, we do some mechanism study. The has_circ_0013958 in the nucleus and cytoplasm were detected by qPCR, it was mainly distributed in cytoplasm, FISH experiments further confirmed this conclusion. In order to further explore the mechanism of has_circ_0013958, we use TargetScan and miRanda to predict miRNA molecules and downstream target genes can be combined with the has_circ_0013958 database, five miRNA molecules were predicted: miR-134, miR-545, miR-629, miR-509, miR-660. The dual luciferase showed that only miR-134 can bind with has_circ_0013958, so we confirm that miR-134 was the downstream target of has_circ_0013958. According to the literature, the target gene downstream of miR-134 in non-small cell lung cancer (NSCLC) is CCND1, and we hypothesize that there may be a circ_0013958/miR-134/CCND1 pathway in LAC. Western Blot experiments showed that has_circ_0013958 control of CCND1 needs to be achieved by adsorption of miR-134. Western Blot experiments showed that has_circ_0013958 can affect the expression of CCND1, but it must be achieved by adsorption of miR-134. Taken together, this study demonstrated for the first time that has_circ_0013958 might function as competing endogenous RNA (ceRNA) for miR-134, which could contribute to proliferation through activating CCND1 signaling pathway. CONCLUSIONS: Our data suggest that i) has_circ_0013958 was overexpressed in lung adenocarcinoma cell lines, tissues and plasma samples; ii) Has_circ_0013958 accelerated cell proliferation, migration and inhibit apoptosis in LAC; iii) Has_circ_0013958 might function as competing endogenous RNA (ceRNA) for miR-134, which could contribute to proliferation through activating CCND1 signaling pathway; iv) Has_circ_0013958 would be a promising biomarker for LAC diagnosis
学术讨论
主办单位时间地点报告人报告主题
东南大学附属中大医院 2015年4月17 中大医院学术报告厅 朱晓莉 肺癌治疗的最新进展
武汉大学人民医院 2015年7月17日 武汉大学人民医院检验系会议室 周国华 高通量测序技术(NGS)的进步与临床应用
东南大学医学院博士沙龙 2015年12月31日 东南大学医学院学术报告厅 王西勇 ERCC1-202亚型基因检测
东南大学附属中大医院 2016年1月17日 中大医院学术报告厅 王彩莲 厄洛替尼在EGFR突变或 未突变患者中的作用
东南大学附属中大医院 2016年6月17日 中大医院学术报告厅 张海军 分子靶向药物易瑞沙研究最新进展
东南大学吴国球教授课题组 2016年11月28 医学院学术报告厅 王西勇 A novel circular RNA, hsa_circ_0012673, promotes lung adenocarcinoma proliferation
东南大学吴国球教授课题组 2017年3月18日 医学院学术报告厅 王西勇 CircRNA研究进展
东南大学吴国球教授课题组 2018年1月8日 医学院学术报告厅 王西勇 Circular RNA 0000096 affects cell growth and migration in gastric cancer
     
学术会议
会议名称时间地点本人报告本人报告题目
International Conference on Medical and Health Sciences (ICMHS) 2015年7月26 香港 Endobronchial metastasis from gastric stump cancer: a case report and review of the literature
个体户医疗与分子诊断应用培训班 2015年7月16 武汉 ERCC1 202亚型实时定量PCR检测方法的建立及其与铂类化疗药物耐药的相关性研究
中国临床肿瘤学年会 2016年9月22 厦门 Increased circular RNA hsa_circ_0012673 acts as a sponge of miR-22 to promote lung adenocarcinoma
南京市医学会结核与呼吸系统疾病专科分会学术年会 2016年12月17 南京 Hsa_circ_0013958 in Tissue as Potential Diagnostic and Prognostic Biomarker for Non-Small Cell Lung Cancer
     
代表作
论文名称
Increased circular RNA hsa_circ_0012673 acts as a sponge of miR-22 to promote lung adenocarcinoma pr
ERCC1_202 Is A Prognostic Biomarker in Advanced Stage Non-Small Cell Lung Cancer Patients Treated wi
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
刘煜 正高 教授 博导 中国药科大学
狄斌 正高 教授 博导 中国药科大学
张海军 副高 副教授 博导 东南大学附属中大医院
沈艳飞 正高 教授 博导 东南大学医学院
文红梅 正高 教授 博导 南京中医药大学
      
答辩秘书信息
姓名职称工作单位备注
范小波 其他 讲师 东南大学医学院