胃癌相关环状RNA的鉴定及其诊断价值分析
发布时间：2019-05-23 19:27
【摘要】：目的近年来的研究表明,环状RNA(circular RNA,circRNA)与肿瘤的发生、发展密切相关,参与了肿瘤细胞的增殖、凋亡、浸润和转移等多个过程;同时,circRNA良好的稳定性赋予了其作为肿瘤标志物的明显优势。胃癌一直以来在我国甚至东亚等国的发病率居高不下,虽然早期治疗的预后良好,但早期诊断率却一直较低,因此急需找到一种非创、敏感、特异的分子标志物。因此本论文以胃癌作为研究对象,采用分子生物学实验技术结合生物信息学分析手段,在组织、血浆和细胞3个层面揭示胃癌相关circRNA的表达谱,通过建立基于荧光染料法逆转录-微滴式数字聚合酶链反应(reverse transcription-droplet digital polymerase chain reaction,RT-ddPCR)技术对胃癌相关circRNA进行鉴定,最后探究circRNA作为胃癌分子标志物的临床诊断价值。方法1.先利用circRNA表达芯片,分别获得circRNA在胃癌组织和血浆中的表达谱;再通过TargetScan和miRanda软件预测circRNA与微小RNA(mircroRNA,miRNA)的结合关系,绘制出circRNA-miRNA互作图;最后对组织和血浆表达谱进行交集分析,筛选出作为胃癌标志物的候选circRNA。2.收集121例胃癌患者手术前和术后第10天的空腹血浆、手术中切除的癌组织及其相对应的正常胃黏膜组织、36例通过内镜黏膜下剥离术(endoscopic submucosal dissection,ESD)剥除的异型增生组织及其对应的正常胃黏膜组织;另收集121位健康人空腹血浆作为正常对照。3.建立基于荧光染料法的RT-dd PCR多重RNA检测方法,优化反应条件;同时比较实时荧光定量逆转录-聚合酶链式反应(quantitive reverse transcription-polymerase chain reaction,qRT-PCR)和RT-ddPCR技术的优缺点,分析两者在检测circRNA时的准确性和重复性。4.采用qRT-PCR检测胃癌组织和胃癌细胞株中的2种代表性circRNA(hsa_circ_0001017和hsa_circ_0061276)的表达水平;采用基于染料法的RT-ddPCR检测胃癌患者和正常人血浆中circRNA的拷贝数。5.分析胃癌组织和患者血浆中circRNA表达趋势之间的关系,探究血浆circRNA的来源,并利用细胞上清实验进行验证。分别分析胃癌细胞中circRNA与其宿主基因的表达水平。6.分析circRNA水平与胃癌患者临床病理因素之间的相关性;建立联合受试者工作特征(receiver operating characteristic,ROC)曲线分析hsa_circ_0001017和hsa_circ_0061276的联合诊断效率;利用生存分析和Cox回归模型评估组织和血浆中circRNA对患者术后总体生存期(overall survival,OS)和无病生存期(disease-free survival,DFS)的预测效率。7.构建circRNA过表达载体,检测细胞中circRNA的上调情况,为研究circ RNA导致胃癌发生的机制奠定基础。结果1.通过分析胃癌相关circRNA表达谱,我们发现了343种circRNA在胃癌患者和正常人血浆之间表达差异倍数大于2倍,其中高表达和低表达的分别有171种和172种;而在胃癌组织和正常组织之间表达差异倍数大于2倍的circRNA有308种,其中高表达和低表达者分别为107种和201种。通过交集分析,筛选出17种在胃癌组织和血浆水平同步变化的circRNA(3种高表达,14种低表达)。2.建立了在单孔内使用EvaGreen染料对3种RNA同时定量检测的新方法;发现对组织和血浆circRNA进行定量时,绝对定量RT-ddPCR方法具有较高的稳定性和精确性。3.Hsa_circ_0001017和hsa_circ_0061276在胃癌细胞、胃癌组织中均呈现低表达,其中hsa_circ_0001017在异型增生组织中的表达水平低于胃癌组织;hsa_circ_0001017和hsa_circ_0061276在胃癌患者血浆中低表达,并在术后10天恢复至正常水平。4.Hsa_circ_0001017和hsa_circ_0061276与胃癌患者的主要临床病理因素密切相关。5.细胞培养实验证明,胃癌细胞可分泌circRNA。6.血浆hsa_circ_0001017和hsa_circ_0061276的ROC曲线下面积(area under curve,AUC)分别达到0.851和0.849。联合上述4种circRNA能大大提高胃癌的诊断效率,其AUC达到0.966,特异度和灵敏度分别为0.955和0.957(对应的阴性诊断率和阳性诊断率分别为95.5%和95.7%)。其中,联合血浆hsa_circ_0001017和hsa_circ_006127的AUC为0.912,特异性和灵敏度分别达0.847和0.966。在早期诊断中,组织hsa_circ_0001017的AUC为0.871,特异性和敏感度分别达到0.794和0.811。7.胃癌患者血浆hsa_circ_0001017或hsa_circ_0061276水平可反映OS;术后血浆hsa_circ_0001017或hsa_circ_0061276回复组的DFS较长。经TNM分期发现,血浆hsa_circ_0001017或hsa_circ_0061276的水平也可提示其OS和DFS。血浆hsa_circ_0001017的术前水平和术后回复情况皆可作为患者生存期的独立预测因子。8.成功建立有效的circRNA过表达质粒,为研究circRNA在胃癌发生中的作用奠定了基础。结论本研究是第一个较为系统地探讨胃癌组织和胃癌患者血浆中circRNA表达谱的研究。进一步通过对2种代表性circ RNA(hsa_circ_0001017和hsa_circ_0061276)在血浆、组织和细胞3个层面的表达水平的研究,我们发现这2种cir RNA在胃癌组织和患者血浆中均为低表达,而术后其在血浆水平中回复正常。结果表明,组织hsa_circ_0001017可作为胃癌早期诊断的标志物,联合血浆hsa_circ_0001017和hsa_circ_0061276可作为胃癌非创性诊断标志物并且是独立的预后预测因子,为胃癌诊治提供了新思路。成功建立了基于染料法RT-ddPCR的多重RNA检测方法,为血浆circRNA的检测提供了高效、经济、简便的新技术。
[Abstract]:Objective In recent years, it is shown that circular RNA (circular RNA) is closely related to the occurrence and development of tumor, and is involved in many processes such as proliferation, apoptosis, infiltration and metastasis of tumor cells. The incidence of gastric cancer has been high in China and even in East Asia. Although the prognosis of early treatment is good, the early diagnosis rate has been low, so it is urgent to find a non-invasive, sensitive and specific molecular marker. In this paper, gastric cancer is used as the research object, and the expression profile of the relevant circular RNA of the gastric cancer is revealed at the three levels of tissue, plasma and cell by using the molecular biology experiment technique and the bioinformatics analysis method. The circulating RNA was identified by reverse transcription-drop digital polymerase chain reaction (RT-ddPCR) based on the fluorescent dye method. Method 1. By using the circular RNA expression chip, the expression profiles of the circle RNA in the gastric cancer tissue and the plasma are respectively obtained; the binding relationship between the circle RNA and the microRNA (miRNA) is predicted by the TargetScan and the mirana software, and the circular RNA-miRNA cross-mapping is drawn; and finally, the tissue and the plasma expression spectrum are intersected and analyzed, The candidate circular RNA as a marker for gastric cancer was screened. The normal gastric mucosa and the corresponding normal gastric mucosa were collected in 121 patients with gastric cancer before and after operation and 10 days after operation, and 36 cases of normal gastric mucosa and 36 cases of normal gastric mucosa were removed by endoscopic submucosal dissection (ESD). The fasting plasma of 121 healthy subjects was also collected as a normal control. The RT-dd PCR multiplex RNA detection method based on the fluorescent dye method was established to optimize the reaction conditions, and the advantages and disadvantages of real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RT-ddPCR were compared. The expression level of two representative circular RNA (hsa _ circ _ 0001017 and hsa _ circ _ 0061276) in gastric cancer and gastric cancer cell lines was detected by qRT-PCR, and the copy number of circulating RNA in the plasma of gastric cancer and normal persons was detected by the method of dye-based RT-ddPCR. The relationship between the expression of the circulating RNA in the tissues of the gastric cancer and the plasma of the patient was analyzed, the source of the plasma circular RNA was explored, and the cell supernatant was used for the validation. The expression level of circulating RNA and its host gene in gastric cancer cells was analyzed. The correlation between the level of the circular RNA and the clinicopathological factors of the gastric cancer patients was analyzed, and the combined diagnostic efficiency of hsa _ circ _ 0001017 and hsa _ circ _ 0061276 was analyzed by the curve of the working characteristics (ROC) of the combined subjects. The prediction of the overall survival (OS) and disease-free sural (DFS) in the tissues and plasma was assessed by the survival analysis and the Cox regression model. The expression vector of the circulating RNA was constructed to detect the up-regulation of the circulating RNA in the cell and to lay the foundation for the study of the mechanism of the occurrence of gastric cancer by the circ RNA. Results 1. By analyzing the relevant circular RNA expression profiles of gastric cancer, we found that the expression of 343 circular RNA in the plasma of gastric cancer patients and normal persons was more than 2 times, of which 171 species and 172 species were expressed in high and low expression, respectively; The expression of the circulating RNA was more than 2 times between the gastric cancer tissues and the normal tissues, and the high expression and the low expression were 107 species and 201 species, respectively. By means of the intersection analysis,17 round-RNAs (3 high-expression,14 low-expression) were selected for the simultaneous changes of gastric cancer tissue and plasma level. The novel method for simultaneous quantitative detection of three kinds of RNA by using the EvaGreen dye in a single hole was established, and it was found that the absolute quantitative RT-ddPCR method has high stability and accuracy when the tissue and the plasma circular RNA are quantified, and the Hsa _ circ _ 0001017 and hsa _ circ _ 0061276 have low expression in the gastric cancer cells and the gastric cancer tissues, Hsa _ circ _ 0001017 and hsa _ circ _ 0061276 were found to be lower in the plasma of the patients with gastric cancer. The cell culture experiments show that the gastric cancer cells can secrete a circle of circulating RNA. The area under the ROC curve for plasma hsa _ circ _ 0001017 and hsa _ circ _ 0061276 was 0.851 and 0.849, respectively. The results showed that the four kinds of circular RNA can greatly improve the diagnostic efficiency of the gastric cancer, the AUC of which is 0.966, the specificity and the sensitivity are 0.955 and 0.957 respectively (the corresponding negative diagnosis rate and the positive diagnosis rate are 95.5% and 95.7%, respectively). The AUC of the combined plasma hsa _ circ _ 0001017 and hsa _ circ _ 006127 was 0.912, and the specificity and the sensitivity were 0.847 and 0.966, respectively. In the early diagnosis, the AUC of the tissue hsa _ circ _ 0001017 was 0.871, the specificity and the sensitivity reached 0.794 and 0.811, respectively. The plasma hsa _ circ _ 0001017 or hsa _ circ _ 0061276 in the gastric cancer patients can reflect the OS; the post-operative plasma hsa _ circ _ 0001017 or hsa _ circ _ 0061276 response group is longer DFS. The levels of plasma hsa _ circ _ 0001017 or hsa _ circ _ 0061276 may also be suggestive of OS and DFS by TNM staging. The pre-operative level and post-operative response of plasma hsa _ circ _ 0001017 can be used as an independent predictor of the survival of the patient. The successful establishment of the effective circular RNA overexpression plasmid provides a basis for the study of the role of the circular RNA in the occurrence of gastric cancer. Conclusion This study is the first to study the expression profile of the circulating RNA in the plasma of gastric cancer and gastric cancer. Further, we found that these two cir RNAs were low in plasma, tissue and cell levels by studying the expression levels of two representative ciirc RNA (hsa _ circ _ 0001017 and hsa _ circ _ 0061276) at three levels of plasma, tissue, and cells, and that it was returned to normal in plasma levels after surgery. The results show that the tissue hsa _ circ _ 0001017 can be used as a marker for early diagnosis of gastric cancer, and the combined plasma hsa _ circ _ 0001017 and hsa _ circ _ 0061276 can be used as a non-invasive diagnostic marker for gastric cancer and an independent prognostic factor, which provides a new thought for the diagnosis and treatment of gastric cancer. The method of multiplex RNA detection based on the dye-based RT-ddPCR was successfully established, which provided an efficient, economical and simple new technique for the detection of the plasma circular RNA.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2484162
[Abstract]:Objective In recent years, it is shown that circular RNA (circular RNA) is closely related to the occurrence and development of tumor, and is involved in many processes such as proliferation, apoptosis, infiltration and metastasis of tumor cells. The incidence of gastric cancer has been high in China and even in East Asia. Although the prognosis of early treatment is good, the early diagnosis rate has been low, so it is urgent to find a non-invasive, sensitive and specific molecular marker. In this paper, gastric cancer is used as the research object, and the expression profile of the relevant circular RNA of the gastric cancer is revealed at the three levels of tissue, plasma and cell by using the molecular biology experiment technique and the bioinformatics analysis method. The circulating RNA was identified by reverse transcription-drop digital polymerase chain reaction (RT-ddPCR) based on the fluorescent dye method. Method 1. By using the circular RNA expression chip, the expression profiles of the circle RNA in the gastric cancer tissue and the plasma are respectively obtained; the binding relationship between the circle RNA and the microRNA (miRNA) is predicted by the TargetScan and the mirana software, and the circular RNA-miRNA cross-mapping is drawn; and finally, the tissue and the plasma expression spectrum are intersected and analyzed, The candidate circular RNA as a marker for gastric cancer was screened. The normal gastric mucosa and the corresponding normal gastric mucosa were collected in 121 patients with gastric cancer before and after operation and 10 days after operation, and 36 cases of normal gastric mucosa and 36 cases of normal gastric mucosa were removed by endoscopic submucosal dissection (ESD). The fasting plasma of 121 healthy subjects was also collected as a normal control. The RT-dd PCR multiplex RNA detection method based on the fluorescent dye method was established to optimize the reaction conditions, and the advantages and disadvantages of real-time fluorescence quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RT-ddPCR were compared. The expression level of two representative circular RNA (hsa _ circ _ 0001017 and hsa _ circ _ 0061276) in gastric cancer and gastric cancer cell lines was detected by qRT-PCR, and the copy number of circulating RNA in the plasma of gastric cancer and normal persons was detected by the method of dye-based RT-ddPCR. The relationship between the expression of the circulating RNA in the tissues of the gastric cancer and the plasma of the patient was analyzed, the source of the plasma circular RNA was explored, and the cell supernatant was used for the validation. The expression level of circulating RNA and its host gene in gastric cancer cells was analyzed. The correlation between the level of the circular RNA and the clinicopathological factors of the gastric cancer patients was analyzed, and the combined diagnostic efficiency of hsa _ circ _ 0001017 and hsa _ circ _ 0061276 was analyzed by the curve of the working characteristics (ROC) of the combined subjects. The prediction of the overall survival (OS) and disease-free sural (DFS) in the tissues and plasma was assessed by the survival analysis and the Cox regression model. The expression vector of the circulating RNA was constructed to detect the up-regulation of the circulating RNA in the cell and to lay the foundation for the study of the mechanism of the occurrence of gastric cancer by the circ RNA. Results 1. By analyzing the relevant circular RNA expression profiles of gastric cancer, we found that the expression of 343 circular RNA in the plasma of gastric cancer patients and normal persons was more than 2 times, of which 171 species and 172 species were expressed in high and low expression, respectively; The expression of the circulating RNA was more than 2 times between the gastric cancer tissues and the normal tissues, and the high expression and the low expression were 107 species and 201 species, respectively. By means of the intersection analysis,17 round-RNAs (3 high-expression,14 low-expression) were selected for the simultaneous changes of gastric cancer tissue and plasma level. The novel method for simultaneous quantitative detection of three kinds of RNA by using the EvaGreen dye in a single hole was established, and it was found that the absolute quantitative RT-ddPCR method has high stability and accuracy when the tissue and the plasma circular RNA are quantified, and the Hsa _ circ _ 0001017 and hsa _ circ _ 0061276 have low expression in the gastric cancer cells and the gastric cancer tissues, Hsa _ circ _ 0001017 and hsa _ circ _ 0061276 were found to be lower in the plasma of the patients with gastric cancer. The cell culture experiments show that the gastric cancer cells can secrete a circle of circulating RNA. The area under the ROC curve for plasma hsa _ circ _ 0001017 and hsa _ circ _ 0061276 was 0.851 and 0.849, respectively. The results showed that the four kinds of circular RNA can greatly improve the diagnostic efficiency of the gastric cancer, the AUC of which is 0.966, the specificity and the sensitivity are 0.955 and 0.957 respectively (the corresponding negative diagnosis rate and the positive diagnosis rate are 95.5% and 95.7%, respectively). The AUC of the combined plasma hsa _ circ _ 0001017 and hsa _ circ _ 006127 was 0.912, and the specificity and the sensitivity were 0.847 and 0.966, respectively. In the early diagnosis, the AUC of the tissue hsa _ circ _ 0001017 was 0.871, the specificity and the sensitivity reached 0.794 and 0.811, respectively. The plasma hsa _ circ _ 0001017 or hsa _ circ _ 0061276 in the gastric cancer patients can reflect the OS; the post-operative plasma hsa _ circ _ 0001017 or hsa _ circ _ 0061276 response group is longer DFS. The levels of plasma hsa _ circ _ 0001017 or hsa _ circ _ 0061276 may also be suggestive of OS and DFS by TNM staging. The pre-operative level and post-operative response of plasma hsa _ circ _ 0001017 can be used as an independent predictor of the survival of the patient. The successful establishment of the effective circular RNA overexpression plasmid provides a basis for the study of the role of the circular RNA in the occurrence of gastric cancer. Conclusion This study is the first to study the expression profile of the circulating RNA in the plasma of gastric cancer and gastric cancer. Further, we found that these two cir RNAs were low in plasma, tissue and cell levels by studying the expression levels of two representative ciirc RNA (hsa _ circ _ 0001017 and hsa _ circ _ 0061276) at three levels of plasma, tissue, and cells, and that it was returned to normal in plasma levels after surgery. The results show that the tissue hsa _ circ _ 0001017 can be used as a marker for early diagnosis of gastric cancer, and the combined plasma hsa _ circ _ 0001017 and hsa _ circ _ 0061276 can be used as a non-invasive diagnostic marker for gastric cancer and an independent prognostic factor, which provides a new thought for the diagnosis and treatment of gastric cancer. The method of multiplex RNA detection based on the dye-based RT-ddPCR was successfully established, which provided an efficient, economical and simple new technique for the detection of the plasma circular RNA.
【学位授予单位】：宁波大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2
【参考文献】
相关期刊论文 前3条
1 Jian Zhang;Sha Li;Lu Li;Meng Li;Chongye Guo;Jun Yao;Shuangli Mi;;Exosome and Exosomal MicroRNA: Trafficking, Sorting, and Function[J];Genomics,Proteomics & Bioinformatics;2015年01期
2 张霖;;探讨胃癌的预防[J];中国现代药物应用;2009年01期
3 吴健民;对肿瘤标志物的再认识[J];中华检验医学杂志;2005年01期
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