长链非编码RNA在肝细胞肝癌的发病机制中作用及其诊断价值研究

发布时间：2018-06-13 23:23

本文选题：肝细胞肝癌 + 长链非编码RNA　；参考：《南京医科大学》2016年博士论文

【摘要】：肝细胞性肝癌(Hepatocellular carcinoma, HCC)是我国最常见的恶性肿瘤之一,其死亡率位列癌症相关肿瘤死亡率第三位。HCC具有起病隐匿、恶性程度高、进展迅速、易转移、病死率高、总体预后不佳等特点,严重危害人类健康。因此,探索肝癌发生以及其早期转移的具体机制对于肝癌早期诊断及提高患者预后具有重要价值。既往针对于肝癌发生发展的机制研究多集中于传统的蛋白编码基因,主要涉及遗传学,表观遗传学以及相关信号通路的调控。随着高通量测序技术发展,研究者已经揭示在人类基因组中非编码RNA (Non-coding RNA, ncRNA)比例占据了95%以上,蛋白编码RNA以及功能性非编码RNA间复杂相互作用在肝癌的发生发展中具有重要的作用。长链非编码RNA (long non-coding RNA, lncRNA)作为ncRNA的重要组成成分之一,也越来越受到重视。目前已经揭示lncRNA可以通过多种途径参与表观遗传,基因转录及转录后水平的调控并且具有作为早期疾病诊断标记物的潜力。生物信息学研究发现具有特殊位置关系的蛋白编码基因以及长链非编码RNA间具有潜在的顺式调控关系,该现象被称为FlanklOkb模式,即在同一染色体基因座中,在某一蛋白编码基因的侧翼10kb之内存在长链非编码RNA,则该lncRNA具有潜在调控该蛋白编码基因的能力。既往研究表明,角蛋白19 (Keratin 19, KRT19)是胆管／肝脏祖细胞(Hepatic progenitor cell, HPC)的重要标记物,但在肝细胞性肝癌中表达时提示其预后较差,目前推测可能与KRT19介导的肿瘤细胞转移相关,但具体机制尚不清楚。本研究主要基于生物信息学理论聚焦于KRT19上游25kb处lncRNA (Linc00974),探索其是否能够介导KRT19参与对肝细胞性肝癌的发生发展的调控及其具体机制。此外,KRT19外周循环可溶性片段目前已作为肺癌的肿瘤标记物在临床应用,但因在肝癌中灵敏性及特异性不高而未用于肝癌相关诊断。现有传统的生物标志物对于肝癌的早期发现及进展具有有明显的局限性。因此,本研究拟通过高通量芯片技术,探索在HCC患者外周血浆中是否存在具有肝癌发生发展相关差异lncRNA,并探索其作为生物标记物的潜能。本研究中我们利用生物信息学分析、表达谱芯片结合经典分子生物学技术等,在分子水平、细胞水平、实验动物水平以及临床样本中探索由表观遗传所介导的长链非编码RNALinc00974对KRT19异常表达的肝癌生物学行为的影响。研究发现：较癌旁组织而言,Linc00974在肝癌中呈现明显高表达状态。鉴于Linc00974尚未在人类疾病中报道,在证实Linc00974缺乏蛋白编码能力并可稳定存在于HCC相关细胞株基础上,通过胞浆胞核RNA分离提取的方式,进一步确认Linc00974主要定位于细胞浆中,为后续研究Linc00974具体调控机制提供了理论基础。结合生物信息学分析与已发表文献,我们发现异常表达的长链非编码RNA通常由其启动子区异常甲基化进而影响其转录水平表达。通过生物信息学预测我们发现,在Linc00974上游启动子区存在明显富集的CpG岛(CpG island),提示Linc00974的异常表达可能由于其上游CpG岛的异常甲基化引起,经亚硫酸氢盐测序法检测发现,在Linc00974异常高表达的肝癌组织中呈现明显甲基化抑制水平。随后我们在150例经临床病理确诊为HCC患者的癌及癌旁组织中通过实时荧光定量PCR (Quantitative Real-time PCR, qRT-PCR)免疫印迹、免疫组织化学检测KRT19表达水平,研究发现,在肝癌患者中存在27例为KRT19阳性表达,123例为阴性表达,Pearson相关性分析提示,在肝癌人群中,Linc00974与KRT19表达呈现明显一致的正相关。结合肝癌患者相关临床信息分析同样提示该长链非编码RNA与肝癌患者肿瘤生长,TNM分期以及肿瘤转移密切相关。进一步通过体外细胞功能研究发现,异常表达的Linc00974在KRT19特异性表达的肝癌相关细胞株中可以明显通过上调KRT19表达促进细胞增殖以及侵袭能力并伴随细胞凋亡抑制以及细胞周期阻滞,而对KRT19阴性的肝癌细胞株则无明显影响。利用上述KRT19特异性表达肝癌细胞株进行裸鼠皮下原位荷瘤以及尾静脉注射肺定植模型,发现Linc00974既可促进肝癌细胞的体内生长,又能促进肝癌细胞侵袭入血,引起肿瘤远期转移,并且该效应依赖于KRT19表达。为进一步探索Linc00974对KRT19具体调控机制,我们首先利用RNA免疫共沉淀技术(RNA Immunoprecipitation, RIP)探索Linc00974是否可以通过直接绑定KRT19蛋白进而影响其功能及相关效应产生。RIP研究显示Linc00974无法直接与KRT19蛋白结合。结合Linc00974亚细胞定位及incRNA相关调控机制报道,通过运用生物信息学预测,研究组发现,在Linc00974与KRT19间存在潜在共同绑定miRNA(miR-624),进一步通过荧光素酶报告基因实验结合表达谱芯片及后期大数据信号通路富集发现,miR-624可以作为“桥梁样”作用,与Linc00974、KRT19形成内源性竞争性RNA环路(competing endogenous RNA loop,ceRNA loop)。通过体外试验证实,在miR-624存在的肝癌细胞中,Linc00974通过吸附miR-624而发挥内源性“海绵样”作用,诱导KRT19表达,并进一步活化NOTCH及TGF-p信号通路。最终研究表明Linc00974可通过KRT19依耐性信号通路促进肝癌细胞生长以及侵袭-转移级联反应,证明Linc00974在肝癌的发生发展中发挥着重要作用。既往研究发现,AFP作为传统的肝癌诊断标记物,在诊断早期肝癌以及肝癌早期转移过程中其特异性相对较低,因此,探索具有更高特异性及灵敏性的微创肝癌术前标记物对肝癌早期诊断以及早期转移预或AFP阴性肝癌患者的补充诊断具有重要的临床意义。在本研究中,我们首先基于前期KRT19及Linc00974相关临床检测及分析,进一步探索Linc00974是否可以作为肝癌诊断相关血浆标记物；其次,通过运用高通量长链非编码RNA转录组芯片技术在肝癌患者术前／术后血浆样本进行筛选,在严密质控基础上,对差异表达的血浆lncRNA在随机临床样本中进行定量验证,通过风险评分方法、受试者工作特征曲线(Receiver operating characteristic curve, ROC)分析结合曲线下积分及聚类分析来评估血浆lncRNA对肝癌的诊断能力。研究发现：1.在肝癌患者血浆中,Linc00974仅以片段方式存在。Linc00974血浆特异性片段(Linc00974 Fraction 1,Linc00974F-1)在肝癌患者术前呈现明显高表达,肿瘤切除后,血浆Linc00974F-1表达显著下降。结合KRT19血浆可溶性片段CYFRA21-1分别进行ROC曲线分析及聚类分析显示,联合Linc00974F-1与CYFRA21-1诊断肝癌其曲线下面积仅为0.764。而联合两者指标在预测肿瘤生长尤其在区分是否为小肝癌水平以及预测肿瘤是否发生转移时其曲线下面积分别为0.834及0.866,提示联合两者指标可明显联合Linc00974F-1及CYFRA21-1可作为潜在预测早期肝癌以及肝癌转移相关的生物标记物。2.与健康对照相比,肝癌患者术前血浆中存在明显异常的lncRNA表达谱系。联合肝癌患者术后血浆lncRNA表达谱分析,筛选出13条满足潜在肝癌术前诊断标记物特征的lncRNA进行后续验证。基于随机选择标准,设定20对肝癌患者及健康对照作为测试组及147/180例(肝癌患者/健康对照)样本作为验证组。采用风险分层结合ROC曲线分析发现：最终筛选出的RP11-160H22.5, XLOC_014172及LOC149086可稳定表达于肝癌患者及健康对照血浆中并且在肝癌患者术前呈现明显高表达状态,而在肿瘤切除后可显著下降并且在肝硬化、慢性肝炎患者中无异常表达。联合三者进行肝癌术前诊断其曲线下面积可达0.896,其诊断灵敏度和特异度分别为91%/92%。进一步分析三者指标与肝癌患者临床信息关联发现,XLOC_014172及LOC149086联合可作为潜在肝癌早期转移标记物,其ROC曲线下面积可达0.934,灵敏度和特异度分别为90%/90%。少数肝癌患者的术后血浆RP11-160H22.5, XLOC_014172及LOC149086反常性升高,结合临床信息统计及相关文献报道分析,其升高可能与患者术前肿瘤早期转移,通过相关载体诸如外泌小体等引起的继发性升高相关。综上所述,我们的研究对原发性肝癌发生发展,特别是肿瘤生长及转移中功能性非编码RNA参与的表观遗传调控作用进行了深入而广泛的探讨,为进一步理解该过程中纷繁复杂的信号通路提供了新的思路和视角,同时,通过高通量检测结合现有肿瘤标记物为肝癌的早期诊断以及早期转移预警提供了新的靶点。
[Abstract]:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China. The mortality rate of cancer related cancer mortality is third.HCC, which has the characteristics of insidious onset, high malignancy, rapid progression, easy metastasis, high mortality and poor overall prognosis. The specific mechanism of its early metastasis is of great value for the early diagnosis of liver cancer and the improvement of the prognosis of the patients. Previous studies on the mechanism of the development of liver cancer mainly focus on the traditional protein coding genes, mainly involving genetics, epigenetics and related signaling pathways. With the development of high throughput sequencing technology, researchers It has been revealed that the proportion of non coded RNA (Non-coding RNA, ncRNA) occupies more than 95% in the human genome. The complex interaction between protein encoded RNA and functional non coded RNA plays an important role in the development and development of liver cancer. Long chain non coded RNA (long non-coding RNA, lncRNA) is one of the important components of ncRNA. More attention has been made. It has been revealed that lncRNA can participate in epigenetic, gene transcription and post transcriptional regulation and have potential as a marker for early diagnosis of disease through a variety of pathways. Bioinformatics studies have found that a protein encoding gene with a special location and a potential for long chain uncoded RNA The phenomenon is known as the FlanklOkb model, that is, in the same chromosome loci, the memory of the flanking 10KB of a protein encoding gene is in the long chain non coding RNA, then the lncRNA has the potential to regulate the gene encoding the protein. Previous studies have shown that keratin 19 (Keratin 19, KRT19) is a bile duct / liver progenitor cell (Hepat). IC progenitor cell, HPC) is an important marker, but the expression in HCC indicates a poor prognosis. It is presumed that it may be associated with KRT19 mediated tumor cell metastasis, but the specific mechanism is not clear. This study focuses on the bioinformatics theory focusing on lncRNA (Linc00974) at 25KB in the upstream of KRT19, to explore whether it can be found. Mediating the involvement of KRT19 in the regulation of the development of hepatocellular carcinoma and its specific mechanism. In addition, the KRT19 peripheral circulating soluble fragment is currently used as a tumor marker for lung cancer, but it is not used for the diagnosis of liver cancer because of the low sensitivity and specificity in the liver cancer. The existing traditional biomarkers for liver cancer In this study, we use high throughput chip technology to explore the existence and development of lncRNA in the peripheral plasma of HCC patients and explore its potential as a biomarker. In this study, we use bioinformatics analysis, expression spectrum chip to combine classic Molecular biology techniques, in the molecular level, cell level, experimental animal level and clinical samples, explore the effects of epigenetic long chain non coding RNALinc00974 on the biological behavior of liver cancer with abnormal expression of KRT19. The study shows that Linc00974 is highly expressed in liver cancer compared with the paracancerous tissue. Linc00974 has not been reported in human disease. On the basis of confirming the coding ability of Linc00974 deficiency protein and stable on the basis of HCC related cell lines, it is further confirmed that Linc00974 is mainly located in the cytoplasm through the separation and extraction of cytoplasmic nucleus RNA, which provides a theoretical basis for subsequent research on the specific regulation mechanism of Linc00974. It is found that the abnormal expression of long chain non coding RNA is usually based on the abnormal methylation of its promoter region and then affects its transcriptional level. By bioinformatics, we found that there is an obvious enrichment of CpG Island (CpG Island) in the promoter region of the upstream of Linc00974, suggesting that the abnormal expression of Linc00974 can be expressed. Due to the abnormal methylation of the CpG island in the upper reaches of its upstream, it was detected by the hydrogen sulfite sequencing method and showed significant methylation inhibition in the abnormal high expression of Linc00974 in the liver cancer tissues. Then we passed real-time fluorescent quantitative PCR (Quantitative Real-time P) in 150 cases of HCC patients with clinicopathological diagnosis of cancer and para cancer. CR, qRT-PCR) immunoblotting and immunohistochemical detection of KRT19 expression. It was found that there were 27 cases of KRT19 positive expression and 123 negative expression in patients with liver cancer. Pearson correlation analysis suggested that the expression of Linc00974 and KRT19 showed a positive correlation in the liver cancer population. It is suggested that the long chain noncoding RNA is closely related to tumor growth, TNM staging and tumor metastasis in patients with liver cancer. Further through in vitro cell function study, it is found that abnormal expression of Linc00974 in the hepatocellular carcinoma related cell lines with KRT19 specific expression can be obviously promoted by up regulation of KRT19 to promote cell proliferation and invasiveness. The inhibition of apoptosis and cell cycle arrest, but no obvious effect on KRT19 negative liver cancer cell lines. Using the KRT19 specific expression of hepatoma cell lines in nude mice in situ tumor bearing tumor and tail vein injection lung colonization model, it is found that Linc00974 can promote the growth of liver cancer cells in vivo, and promote the invasion of hepatoma cells. In order to further explore the specific regulation mechanism of Linc00974 to KRT19, we first use RNA immunoprecipitation (RNA Immunoprecipitation, RIP) to explore whether Linc00974 can directly bind KRT19 protein and then affect its function and related effects to produce.RIP research, in order to further explore the specific regulation mechanism of KRT19. It was shown that Linc00974 could not be directly associated with KRT19 protein. Combined with Linc00974 subcellular localization and incRNA related regulatory mechanism, the research group found that there was a potential CO binding miRNA (miR-624) between Linc00974 and KRT19 by using bioinformatics prediction, and further through the fluorescein reporter gene experiment combined with the expression spectrum chip and the later stage. The enrichment of large data signal pathway shows that miR-624 can act as a "bridge like" function and form endogenous competitive RNA loop (competing endogenous RNA loop, ceRNA loop) with Linc00974 and KRT19. Induction of KRT19 expression and further activation of NOTCH and TGF-p signaling pathways. The final study shows that Linc00974 can promote the growth of hepatoma cells and the invasion and metastasis cascade reaction through the KRT19 impatience signaling pathway. It is proved that Linc00974 plays an important role in the development of liver cancer. Previous studies have found that AFP is a diagnostic marker for the traditional liver cancer. The specificity is relatively low in the diagnosis of early liver cancer and the early metastasis of liver cancer. Therefore, it is important to explore the early diagnosis of liver cancer with higher specificity and sensitivity to the early diagnosis of liver cancer and the supplementary diagnosis of early metastasis or AFP negative liver cancer. In the previous KRT19 and Linc00974 related clinical tests and analysis, we further explore whether Linc00974 can be used as a plasma marker for the diagnosis of liver cancer; secondly, screening the plasma samples before and after operation by high throughput long chain non coded RNA transcriptional chip technology on the basis of strict quality control. The plasma lncRNA was quantified in a randomized clinical sample. By the risk scoring method, the Receiver operating characteristic curve (ROC) analysis combined with the curve integral and cluster analysis to evaluate the diagnostic ability of plasma lncRNA to liver cancer. The study found that 1. in the plasma of the liver cancer patients, Linc00974 only The.Linc00974 plasma specific fragment (Linc00974 Fraction 1, Linc00974F-1) was highly expressed before operation in the patients with liver cancer. After tumor resection, the expression of Linc00974F-1 in plasma was significantly decreased. The ROC curve analysis and cluster analysis of KRT19 plasma soluble fragment CYFRA21-1, respectively, showed that Linc00974F-1 and CYFRA21- were combined with CYFRA21-. 1 the area under the curve of the diagnosis of liver cancer is only 0.764. and the combination of the two indexes is 0.834 and 0.866 respectively in predicting the growth of tumor, especially whether it is small liver cancer and whether the tumor has metastasize. It is suggested that the combination of the two indexes can be combined with Linc00974F-1 and CYFRA21-1 as a potential predictor of early liver disease. .2. related biomarkers associated with metastasis of cancer and liver cancer compared with healthy controls, there was a significant abnormal lncRNA expression pedigree in the plasma of the patients with liver cancer. The plasma lncRNA expression profile of the patients with liver cancer after operation was analyzed, and 13 lncRNA were screened for subsequent validation of the characteristics of the diagnostic markers for the potential hepatocellular carcinoma. 20 cases of liver cancer patients and healthy controls were set as test groups and 147/180 cases (liver cancer patients / health control) samples were used as validation groups. The risk stratification combined with ROC curve analysis showed that the final screening of RP11-160H22.5, XLOC_014172 and LOC149086 could be stably expressed in liver cancer patients and healthy control plasma and in patients with liver cancer. There was a significant state of high expression before the resection and no abnormal expression in the patients with liver cirrhosis and chronic hepatitis after the tumor resection. The area under the curve for the diagnosis of liver cancer was up to 0.896 in the three cases, and the sensitivity and specificity of the diagnosis were 91%/92%. further analysis of the correlation between the three indexes and the clinical information of the patients with liver cancer. It is found that the combination of XLOC_014172 and LOC149086 can be used as an early metastasis marker for potential liver cancer. The area under the ROC curve is up to 0.934. The sensitivity and specificity are respectively the increase of RP11-160H22.5, XLOC_014172 and LOC149086 in the postoperative plasma of a few 90%/90%. patients with 90%/90%.. The early metastasis of the tumor may be associated with the secondary elevation caused by the related vector, such as the external secretory body. To sum up, our study has carried out a thorough and extensive discussion on the epigenetic regulation of the development of primary liver cancer, especially the functional non coded RNA involved in tumor growth and metastasis. It provides new ideas and perspectives in understanding the complex signal pathways in the process, and provides new targets for early diagnosis of liver cancer and early warning of early metastasis by high throughput detection combined with existing tumor markers.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

【相似文献】