胃癌相關(guān)環(huán)狀RNA的鑒定及其診斷價值分析
發(fā)布時間：2019-05-23 19:27
【摘要】：目的近年來的研究表明,環(huán)狀RNA(circular RNA,circRNA)與腫瘤的發(fā)生、發(fā)展密切相關(guān),參與了腫瘤細(xì)胞的增殖、凋亡、浸潤和轉(zhuǎn)移等多個過程;同時,circRNA良好的穩(wěn)定性賦予了其作為腫瘤標(biāo)志物的明顯優(yōu)勢。胃癌一直以來在我國甚至東亞等國的發(fā)病率居高不下,雖然早期治療的預(yù)后良好,但早期診斷率卻一直較低,因此急需找到一種非創(chuàng)、敏感、特異的分子標(biāo)志物。因此本論文以胃癌作為研究對象,采用分子生物學(xué)實驗技術(shù)結(jié)合生物信息學(xué)分析手段,在組織、血漿和細(xì)胞3個層面揭示胃癌相關(guān)circRNA的表達譜,通過建立基于熒光染料法逆轉(zhuǎn)錄-微滴式數(shù)字聚合酶鏈反應(yīng)(reverse transcription-droplet digital polymerase chain reaction,RT-ddPCR)技術(shù)對胃癌相關(guān)circRNA進行鑒定,最后探究circRNA作為胃癌分子標(biāo)志物的臨床診斷價值。方法1.先利用circRNA表達芯片,分別獲得circRNA在胃癌組織和血漿中的表達譜;再通過TargetScan和miRanda軟件預(yù)測circRNA與微小RNA(mircroRNA,miRNA)的結(jié)合關(guān)系,繪制出circRNA-miRNA互作圖;最后對組織和血漿表達譜進行交集分析,篩選出作為胃癌標(biāo)志物的候選circRNA。2.收集121例胃癌患者手術(shù)前和術(shù)后第10天的空腹血漿、手術(shù)中切除的癌組織及其相對應(yīng)的正常胃黏膜組織、36例通過內(nèi)鏡黏膜下剝離術(shù)(endoscopic submucosal dissection,ESD)剝除的異型增生組織及其對應(yīng)的正常胃黏膜組織;另收集121位健康人空腹血漿作為正常對照。3.建立基于熒光染料法的RT-dd PCR多重RNA檢測方法,優(yōu)化反應(yīng)條件;同時比較實時熒光定量逆轉(zhuǎn)錄-聚合酶鏈?zhǔn)椒磻?yīng)(quantitive reverse transcription-polymerase chain reaction,qRT-PCR)和RT-ddPCR技術(shù)的優(yōu)缺點,分析兩者在檢測circRNA時的準(zhǔn)確性和重復(fù)性。4.采用qRT-PCR檢測胃癌組織和胃癌細(xì)胞株中的2種代表性circRNA(hsa_circ_0001017和hsa_circ_0061276)的表達水平;采用基于染料法的RT-ddPCR檢測胃癌患者和正常人血漿中circRNA的拷貝數(shù)。5.分析胃癌組織和患者血漿中circRNA表達趨勢之間的關(guān)系,探究血漿circRNA的來源,并利用細(xì)胞上清實驗進行驗證。分別分析胃癌細(xì)胞中circRNA與其宿主基因的表達水平。6.分析circRNA水平與胃癌患者臨床病理因素之間的相關(guān)性;建立聯(lián)合受試者工作特征(receiver operating characteristic,ROC)曲線分析hsa_circ_0001017和hsa_circ_0061276的聯(lián)合診斷效率;利用生存分析和Cox回歸模型評估組織和血漿中circRNA對患者術(shù)后總體生存期(overall survival,OS)和無病生存期(disease-free survival,DFS)的預(yù)測效率。7.構(gòu)建circRNA過表達載體,檢測細(xì)胞中circRNA的上調(diào)情況,為研究circ RNA導(dǎo)致胃癌發(fā)生的機制奠定基礎(chǔ)。結(jié)果1.通過分析胃癌相關(guān)circRNA表達譜,我們發(fā)現(xiàn)了343種circRNA在胃癌患者和正常人血漿之間表達差異倍數(shù)大于2倍,其中高表達和低表達的分別有171種和172種;而在胃癌組織和正常組織之間表達差異倍數(shù)大于2倍的circRNA有308種,其中高表達和低表達者分別為107種和201種。通過交集分析,篩選出17種在胃癌組織和血漿水平同步變化的circRNA(3種高表達,14種低表達)。2.建立了在單孔內(nèi)使用EvaGreen染料對3種RNA同時定量檢測的新方法;發(fā)現(xiàn)對組織和血漿circRNA進行定量時,絕對定量RT-ddPCR方法具有較高的穩(wěn)定性和精確性。3.Hsa_circ_0001017和hsa_circ_0061276在胃癌細(xì)胞、胃癌組織中均呈現(xiàn)低表達,其中hsa_circ_0001017在異型增生組織中的表達水平低于胃癌組織;hsa_circ_0001017和hsa_circ_0061276在胃癌患者血漿中低表達,并在術(shù)后10天恢復(fù)至正常水平。4.Hsa_circ_0001017和hsa_circ_0061276與胃癌患者的主要臨床病理因素密切相關(guān)。5.細(xì)胞培養(yǎng)實驗證明,胃癌細(xì)胞可分泌circRNA。6.血漿hsa_circ_0001017和hsa_circ_0061276的ROC曲線下面積(area under curve,AUC)分別達到0.851和0.849。聯(lián)合上述4種circRNA能大大提高胃癌的診斷效率,其AUC達到0.966,特異度和靈敏度分別為0.955和0.957(對應(yīng)的陰性診斷率和陽性診斷率分別為95.5%和95.7%)。其中,聯(lián)合血漿hsa_circ_0001017和hsa_circ_006127的AUC為0.912,特異性和靈敏度分別達0.847和0.966。在早期診斷中,組織hsa_circ_0001017的AUC為0.871,特異性和敏感度分別達到0.794和0.811。7.胃癌患者血漿hsa_circ_0001017或hsa_circ_0061276水平可反映OS;術(shù)后血漿hsa_circ_0001017或hsa_circ_0061276回復(fù)組的DFS較長。經(jīng)TNM分期發(fā)現(xiàn),血漿hsa_circ_0001017或hsa_circ_0061276的水平也可提示其OS和DFS。血漿hsa_circ_0001017的術(shù)前水平和術(shù)后回復(fù)情況皆可作為患者生存期的獨立預(yù)測因子。8.成功建立有效的circRNA過表達質(zhì)粒,為研究circRNA在胃癌發(fā)生中的作用奠定了基礎(chǔ)。結(jié)論本研究是第一個較為系統(tǒng)地探討胃癌組織和胃癌患者血漿中circRNA表達譜的研究。進一步通過對2種代表性circ RNA(hsa_circ_0001017和hsa_circ_0061276)在血漿、組織和細(xì)胞3個層面的表達水平的研究,我們發(fā)現(xiàn)這2種cir RNA在胃癌組織和患者血漿中均為低表達,而術(shù)后其在血漿水平中回復(fù)正常。結(jié)果表明,組織hsa_circ_0001017可作為胃癌早期診斷的標(biāo)志物,聯(lián)合血漿hsa_circ_0001017和hsa_circ_0061276可作為胃癌非創(chuàng)性診斷標(biāo)志物并且是獨立的預(yù)后預(yù)測因子,為胃癌診治提供了新思路。成功建立了基于染料法RT-ddPCR的多重RNA檢測方法,為血漿circRNA的檢測提供了高效、經(jīng)濟、簡便的新技術(shù)。
[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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
本文編號：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.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.2
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相關(guān)期刊論文 前3條
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3 吳健民;對腫瘤標(biāo)志物的再認(rèn)識[J];中華檢驗醫(yī)學(xué)雜志;2005年01期
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