长链非编码RNA在脊柱转移瘤及肝细胞癌中的作用与临床意义研究
发布时间：2018-09-06 20:32
【摘要】：随着环境污染及食品安全问题日趋严重,吸烟、饮酒等不良生活习惯逐渐向年轻化发展,致使肿瘤发病率逐年增高。目前肿瘤已经成为威胁人类健康的重大疾病。我国不管发病率还是死亡率均居世界前列,给国民健康造成了极大的危害。肿瘤及其相关并发症的研究越来越被研究者们所重视。虽然近些年在肿瘤诊断方法及治疗手段上已取得长足的进步。但对于肺癌、肝癌、胰腺癌等恶性肿瘤的治疗效果、预后和远期生存率仍然较差。其主要原因归结为对肿瘤发生发展机制的认识不够深入。脊柱为肿瘤骨转移最为常见的部位,高达70%的癌症患者会发生脊柱转移。脊柱转移瘤可导致脊髓压迫及病理性骨折,使患者瘫痪,完全失去自理能力。严重影响患者的生活质量,从而加速病情的进展。至今国内外学者对肿瘤脊柱转移的机制尚不清楚,致使缺乏有效的治疗方法。因此脊柱转移瘤的治疗仍然是困扰医学界的一大难题。手术虽然能有效促进恢复,但并发症多,创伤较大且短时间内容易复发,使部分患者在术后一月内死亡。因此,探寻介导肿瘤脊柱转移的关键靶分子,是提高脊柱转移瘤病人临床疗效,改善患者预后及生存质量的新方向。部分肿瘤具有较强的嗜骨性,晚期容易发生骨转移,例如肝癌、肺癌、乳腺癌等。脊柱为肝癌骨转移最为常见的部位,发生脊柱转移患者存活率不到3%。肝细胞癌(hepatocellular carcinoma,HCC)是一种全球范围内常见的恶性肿瘤,占原发性肝癌的90%以上。患者常常伴有肝内及肝外转移。HCC早期临床症状及体征多不典型,很难及早发现。明确诊断时大多病程已进入中、晚期,失去手术机会。因其恶性程度高、早期发现难、进展速度快、治疗复杂、术后复发及转移率较高等特点,使HCC患者疗效不佳,预后较差。以往对HCC研究主要以microRNA及编码蛋白的RNA为主,长链非编码RNA研究相对较少。目前国内外对HCC的分子机制研究还不够深入,因此我们需进一步对HCC在发生、发展及转移过程中的分子生物学机制进行探索。寻找可预测HCC发生的分子标志物,为HCC诊疗开辟新途径。人类基因组是由庞大且复杂的核苷酸序列排列而成。目前已知可编码蛋白的基因仅占人类基因组序列的2%,其余98%不具备编码蛋白能力的序列被认为是基因组中无用的“噪声”。这类从基因组转录而来,且不编码蛋白的RNA分子称为非编码RNA(non-coding RNA,ncRNA)。早期,研究者仅仅对编码RNA进行关注,但近年来人们的目光逐渐被非编码RNA所吸引。随着研究的不断深入,发现长链非编码RNA(long non-coding RNA,lncRNA)与人类多种疾病如阿尔茨海默症、肿瘤、亨廷顿氏病的发生、发展存在着重要的关系。LncRNA转录本长度大于200nt,且不具备蛋白编码的能力。最新研究表明,lncRNA在生命活动过程中扮演着重要角色,可通过调控mRNA和蛋白表达水平影响生物学行为。目前越来越多的肿瘤相关lncRNA得以发现,其通过多种方式调控着肿瘤的生长、转移、侵润与凋亡,成为全新肿瘤标志物及药物靶标,显著推进了肿瘤研究的进展。Ji等通过测序技术发现了肺腺癌转移相关转录本(metastasis-associated lung adenocarcinoma transcript 1,MALAT 1),对225例Ⅲ期非小细胞肺癌(non-small cell lung cancer,NSCLC)患者进行筛查。发现70例转移患者的样本中MALAT1过表达,且具有病程及组织特异性。提示MALAT 1可作为Ⅰ期NSCLC患者潜在标志物。该基因在其他肿瘤中也陆续被发现,但在NSCLC患者中的表达最为显著。MALAT 1可通过激活肿瘤转移相关基因来促进肿瘤转移,还可参与mRNA前体的可变剪切和丝氨酸/精氨酸剪接因子的磷酸化修饰,来影响基因的功能及表达。随着对肿瘤基因组学及转录组学研究的不断深入,研究者发现不但同种肿瘤中可存在不同的lncRNA,而且同一lncRNA也可在不同肿瘤中差异表达。HULC最早发现在肝癌细胞中过表达,可作为原发性肝癌较为敏感的分子标志物,其在前列腺癌、胃癌中也出现异常表达情况。H19作为一个原癌基因,在多种消化系统肿瘤中表达量升高。综上所述,lncRNA在肿瘤学研究中发挥着至关重要的作用。目前,脊柱转移瘤患者多采取微创治疗方案,脊柱后路开放式手术相对较少。因此组织样本较为珍贵,收集相对困难,且骨组织RNA较难提取。致使国内外对于lncRNA在脊柱转移瘤中的研究尚处空白。LncRNA在HCC中的研究仍然较少,并且绝大多数lncRNA调控机制尚不明确。由此提示我们,是否可以通过高通量测序技术进行筛选及验证,找到全新的关键lncRNA作为肿瘤诊断及治疗靶标,为脊柱转移瘤和HCC临床诊疗提供新策略。基于此我们开展了以下三部分研究,现总结如下。第一章:脊柱转移瘤转录组lncRNA表达谱分析目的:建立脊柱转移瘤差异表达lncRNA及mRNA表达谱,并对其长度及外显子数量进行预测。明确差异表达mRNA可富集的分子功能、生物学过程和疾病通路相关信息。方法:对脊柱转移瘤患者配对组织(肿瘤组织及癌旁组织)中RNA进行提取,利用高通量测序技术构建脊柱转移瘤中差异表达lncRNA及mRNA表达谱。随后通过pathway分析及GO分析对差异表达mRNA的分子功能、参与生物学过程及疾病通路进行富集。最后对差异表达lncRNA长度及外显子数目进行预测,并通过对比与编码基因间位置关系,将差异表达lncRNA分类。结果:通过高通量测序技术,在脊柱转移瘤组织中找到171条差异表达lncRNA和2929条差异表达mRNA,其中有66条全新未被报道的lncRNA。对差异表达mRNA进行pathway分析和GO分析。结果显示,脊柱转移瘤中差异表达的mRNA可富集到31种疾病,79种通路及1535种分子功能,其中包括肿瘤相关疾病及肿瘤相关通路。进一步明确了脊柱转移瘤中异常表达mRNA参与的分子功能、生物学过程和对疾病通路的调节。171条差异表达lncRNA中,大部分外显子数目小于5个,长度小于10000nt,均少于mRNA。其中Antisense共有50条,已知的19条,未知的31条;Sence共有53条,已知的51条,未知的2条;Bidirectionsl共有6条,已知的4条,未知的2条;Intergenic共有40条,已知的20条,未知的20条;Intronic共有22条,已知的8条,未知的14条。结论:成功构建了脊柱转移瘤lncRNA、mRNA表达谱。在171条差异表达lncRNA中发现66条全新未被报道的lncRNA。明确了2929条差异表达mRNA参与的分子功能、生物学过程和对疾病通路的调节。为肿瘤学研究及后续实验提供了宝贵的数据资源。第二章:lnc000489在脊柱转移瘤患者血浆中的表达及临床特征相关性分析目的:通过在血浆中验证,找出脊柱转移瘤患者血浆中高表达的lncRNA。了解其分子功能、生物学过程和疾病通路相关信息。明确其在脊柱转移瘤中的诊断价值。方法:从前期构建的脊柱转移瘤lncRNA表达谱中挑选20条全新的lncRNA。将脊柱转移瘤患者血浆设为实验组,健康志愿者血浆设为对照组。通过qRT-PCR实验检测候选lncRNA在血浆中表达量,统计分析后找出在脊柱转移瘤中具有显著差异的lncRNA。利用ROC曲线计算灵敏度和特异性。随后对差异表达lncRNA进行共表达分析,并对共表达mRNA进行pathway和GO分析。最后统计分析差异表达lncRNA与临床特征间的相关性。结果:脊柱转移瘤患者血浆中lnc00489表达量相对于健康志愿者显著升高(P0.05),ROC曲线分析显示lnc00489对脊柱转移瘤具有一定的诊断价值。与lnc00489存在共表达关系的mRNA共有51条,可富集到实体肿瘤、卵巢癌、口腔癌、前列腺癌等多种肿瘤相关疾病和Wnt、Hh、Hedgehog signaling pathway、Basal cell carcinoma等肿瘤相关通路,进一步说明了lnc00489可能与肿瘤的发生、发展及转移存在相关性。Lnc00489表达量与临床特征进行相关性分析,显示lnc00489与转移椎体个数及是否伴有内脏转移显著相关。转移椎体数量越多病情相对严重。肿瘤主要的转移途径为血运转移,内脏及椎体血供相对丰富。综上所述lnc00489可能在肿瘤脊柱转移的过程中发挥着重要作用,其有望成为潜在的血浆标志物,为脊柱转移瘤的诊断及治疗提供了新思路。结论:Lnc00489表达量在脊柱转移瘤患者血浆中显著升高,对脊柱转移瘤具有一定的诊断价值。与lnc00489共表达的mRNA可富集到多种肿瘤相关疾病和通路。血浆中lnc00489表达量与转移椎体数量和是否伴有内脏转移具有相关性,其有望成为诊断脊柱转移瘤全新的血浆分子标志物。第三章:lnc34822在HCC患者血浆中表达及临床特征相关性研究目的:从构建的lncRNA数据库中找到一条在HCC中差异表达的lncRNA,明确其在HCC中的诊断价值。方法:利用前期建立的lncRNA数据库,结合相关文献调研筛选出10条高表达lncRNA。通过在HCC患者组织及血浆中验证,明确是否存在高表达的lncRNA。ROC曲线分析计算诊断灵敏度和特异性。利用qRT-PCR实验检测候选lncRNA在组织、血浆及细胞系(Hep3B、HepG-2、SMMC-7721、Huh7.5、LO2)中表达量。统计分析差异表达lncRNA在血浆中的稳定性,与外周血细胞间关系,不同细胞系中表达情况,在HCC患者术前、术后及复发时表达量的变化情况和与临床特征间的相关性。结果:有报道称,同种lncRNA可在不同肿瘤中进行调控,同种肿瘤也可受到多种不同lncRNA的影响。结合文献调研从构建的脊柱转移瘤lncRNA表达谱中筛选出10条高表达lncRNA。经过在HCC患者组织及血浆中验证后,发现lnc34822在HCC中表达量显著上调(P0.01),并且可以在血浆中稳定表达。Lnc34822在肝癌细胞系中表达量明显高于正常肝细胞系,且与外周血细胞没有相关性。ROC曲线分析显示,lnc34822对HCC具有一定的诊断价值。Lnc34822在HCC患者术后血浆中表达量明显降低,而复发时又迅速升高,由此推断lnc34822有望成为一个动态监测HCC患者病情进展的血浆标志物。通过与临床信息相关性分析发现lnc34822血浆中表达量与AFP及肝上肿瘤个数呈正相关。肝癌患者血液中AFP含量显著升高,目前已成为临床上广泛应用的肿瘤标志物。肝上肿瘤数量越多病情越严重。进一步说明了lnc34822有望成为HCC潜在的血浆诊断标志物。结论:Lnc34822在HCC患者血浆中稳定高表达,且与外周血细胞没有相关性,对HCC具有一定的诊断价值。血浆中lnc34822表达量可动态监测HCC患者病情进展,其有望成为诊断HCC全新的血浆分子标志物。通过以上研究,我们成功构建了全新lncRNA数据库,为肿瘤学研究提供了宝贵的数据资源。并从中找到与脊柱转移瘤和HCC发生、发展相关的lncRNA,为脊柱转移瘤和HCC临床诊断及治疗提供了新思路。
[Abstract]:With the environmental pollution and food safety becoming more and more serious, bad habits such as smoking and drinking are gradually becoming younger and younger. The incidence of cancer is increasing year by year. Although great progress has been made in the diagnosis and treatment of cancer in recent years, the prognosis and long-term survival rate of lung cancer, liver cancer, pancreatic cancer and other malignant tumors are still poor. Spine is the most common site of bone metastasis. Up to 70% of cancer patients have spinal metastasis. Spinal metastasis can cause spinal cord compression and pathological fracture, paralysis, complete loss of self-care ability. It seriously affects the quality of life of patients, thus accelerating the progress of the disease. The mechanism of spinal metastasis is still unclear, resulting in the lack of effective treatment. Therefore, the treatment of spinal metastasis is still a major problem in the medical community. Although surgery can effectively promote recovery, it has many complications, large trauma and short-term recurrence, so that some patients died within one month after surgery. The key target molecule of spinal metastasis is to improve the clinical efficacy, prognosis and quality of life of patients with spinal metastases. Less than 3%. Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, accounting for more than 90% of primary liver cancer. Patients often have intrahepatic and extrahepatic metastases. The early clinical symptoms and signs of HCC are often atypical and difficult to detect. Most of the definite diagnosis of HCC has entered the middle, late stage, lost the opportunity for surgery. Because of its high degree of malignancy, difficulty in early detection, rapid progress, complex treatment, high rate of recurrence and metastasis, HCC patients have poor prognosis and poor curative effect. Therefore, we need to further explore the molecular biology mechanism of HCC in the process of occurrence, development and metastasis. To find molecular markers that can predict the occurrence of HCC will open up a new way for the diagnosis and treatment of HCC. Two percent of the sequences, and 98 percent of the other sequences that do not encode proteins, are considered useless "noise" in the genome. RNA molecules transcribed from the genome and that do not encode proteins are called non-coding RNA (ncRNA). In the early days, researchers focused only on coding RNA, but in recent years, people's attention has gradually been focused on non-coding RN. As the research progresses, it has been found that long non-coding RNA (lncRNA) plays an important role in the development of human diseases such as Alzheimer's disease, tumor and Huntington's disease. Nowadays, more and more tumor-associated lncRNAs have been found to regulate the growth, metastasis, invasion and apoptosis of tumors in various ways. They have become new tumor markers and drug targets, and have greatly promoted the progress of tumor research. Ji et al. identified metastasis-associated lung adenocarcinoma transcript 1 (MALAT 1) by sequencing and screened 225 patients with stage III non-small cell lung cancer (NSCLC). These results suggest that MALAT-1 may be a potential marker for stage I NSCLC patients. The gene has been found in other tumors, but it is most significantly expressed in NSCLC patients. MALAT-1 can promote tumor metastasis by activating Tumor Metastasis-related genes, and can also participate in the alterable splicing of mRNA precursors and phosphorylation repair of serine/arginine splicing factors. With the development of tumor genomics and transcriptome, researchers have found that not only different lncRNA can be found in the same tumor, but also the same lncRNA can be differentially expressed in different tumors. HULC was first found to be overexpressed in hepatocellular carcinoma cells, which can be regarded as a more sensitive primary hepatocellular carcinoma. H19, as a proto-oncogene, is highly expressed in a variety of digestive system tumors. In summary, lncRNA plays a vital role in oncology research. At present, spinal metastases are treated with minimally invasive treatment and posterior spinal opening. Therefore, tissue samples are relatively rare, collection is relatively difficult, and it is difficult to extract RNA from bone tissues. As a result, the study of lncRNA in spinal metastases is still blank at home and abroad. Sequencing techniques were screened and validated to identify novel key lncRNA targets for the diagnosis and treatment of spinal metastases and HCC, providing new strategies for clinical diagnosis and treatment. Methods: RNA was extracted from paired tissues (tumor tissues and adjacent tissues) of patients with spinal metastases, and high-throughput sequencing was used to construct spinal rotation. Differentially expressed lncRNA and mRNA expression profiles in tumor metastasis were then analyzed by pathway analysis and GO analysis to enrich the molecular functions of differentially expressed mRNA and participate in biological processes and disease pathways. Results: 171 differentially expressed lncRNA and 2929 differentially expressed mRNAs were found in spinal metastases by high-throughput sequencing, of which 66 were completely new and unreported. Pathway analysis and GO analysis of differentially expressed mRNAs were performed. The results showed that differentially expressed mRNAs in spinal metastases were enriched in 31 diseases and 79 common diseases. Pathway and 1 535 molecular functions, including tumor-related diseases and tumor-related pathways, further clarify the molecular functions, biological processes and regulation of the abnormal expression of mRNA in spinal metastases. There are 50 ntisense, 19 known, 31 unknown; 53 Sence, 51 known, 2 unknown; 6 Bidirectionsl, 4 known, 2 unknown; 40 Intergenic, 20 known, 20 unknown; 22 Intronics, 8 known, 14 unknown. Conclusion: We have successfully constructed lncRNA, mRNA of spinal metastases. Expression profiles. Among 171 differentially expressed lncRNAs, 66 novel unreported lncRNAs were found. The molecular functions, biological processes and regulation of disease pathways of 2929 differentially expressed mRNAs were identified. This provides valuable data for oncology research and follow-up experiments. Chapter 2: The expression of lnc000489 in the plasma of patients with spinal metastases. Objective: To identify the high expression of lncRNA in the plasma of patients with spinal metastases by validating its clinical features. The expression of candidate lncRNA in plasma was detected by qRT-PCR assay. After statistical analysis, significant differences in lncRNA expression in spinal metastases were found. The sensitivity and specificity were calculated by ROC curve. Then, the differential expression of lncR was detected. Results: The expression of lnc00489 in the plasma of patients with spinal metastases was significantly higher than that of healthy volunteers (P 0.05). ROC curve analysis showed that lnc00489 had certain diagnostic value for spinal metastases. There are 51 mRNAs co-expressed with lnc00489, which can be enriched in many tumor-related diseases such as solid tumors, ovarian cancer, oral cancer, prostate cancer and other tumor-related pathways such as Wnt, Hh, Hedgehog signaling pathway, Basal cell carcinoma. This further indicates that lnc00489 may be associated with the occurrence, development and metastasis of tumors. Correlation analysis between Lnc00489 expression and clinical features showed that lnc00489 was significantly associated with the number of metastatic vertebrae and visceral metastasis. Lnc00489 may be a potential plasma marker for the diagnosis and treatment of spinal metastases. Conclusion: The expression of Lnc00489 is significantly elevated in the plasma of patients with spinal metastases and has diagnostic value for spinal metastases. Tumor-related diseases and pathways. The expression of lnc00489 in plasma is correlated with the number of metastatic vertebrae and visceral metastasis. It is expected to be a new plasma molecular marker for the diagnosis of spinal metastases. Chapter 3: Expression of lnc34822 in plasma of HCC patients and its correlation with clinical features. Objective: To find the correlation between the expression of lncRNA in the constructed lncRNA database. Methods: Ten highly expressed lncRNA were screened from the previously established lncRNA database and the related literature. The sensitivity and specificity of diagnosis were determined by validating the presence of highly expressed lncRNA in tissues and plasma of HCC patients. The expression of candidate lncRNA in tissues, plasma and cell lines (Hep3B, HepG-2, SMMC-7721, Huh7.5, LO2) was detected by qRT-PCR. The stability of the differentially expressed lncRNA in plasma, the relationship between the differentially expressed lncRNA and peripheral blood cells, the expression in different cell lines, the changes of the expression in HCC patients before, after and after HCC, and the recurrence of the expression of lncRNA were statistically analyzed. Results: It has been reported that homologous lncRNA can be regulated in different tumors, and homologous tumors can also be affected by a variety of different lncRNA. Ten highly expressed lncRNA were screened from the constructed lncRNA expression profiles of spinal metastases based on literature investigation. After being verified in HCC patients'tissues and plasma, lnc34822 was found. The expression of Lnc34822 in HCC cell line was significantly higher than that in normal liver cell line, and there was no correlation between Lnc34822 and peripheral blood cells. It is concluded that lnc34822 may be a plasma marker for monitoring the progression of HCC.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2227456
[Abstract]:With the environmental pollution and food safety becoming more and more serious, bad habits such as smoking and drinking are gradually becoming younger and younger. The incidence of cancer is increasing year by year. Although great progress has been made in the diagnosis and treatment of cancer in recent years, the prognosis and long-term survival rate of lung cancer, liver cancer, pancreatic cancer and other malignant tumors are still poor. Spine is the most common site of bone metastasis. Up to 70% of cancer patients have spinal metastasis. Spinal metastasis can cause spinal cord compression and pathological fracture, paralysis, complete loss of self-care ability. It seriously affects the quality of life of patients, thus accelerating the progress of the disease. The mechanism of spinal metastasis is still unclear, resulting in the lack of effective treatment. Therefore, the treatment of spinal metastasis is still a major problem in the medical community. Although surgery can effectively promote recovery, it has many complications, large trauma and short-term recurrence, so that some patients died within one month after surgery. The key target molecule of spinal metastasis is to improve the clinical efficacy, prognosis and quality of life of patients with spinal metastases. Less than 3%. Hepatocellular carcinoma (HCC) is a common malignant tumor worldwide, accounting for more than 90% of primary liver cancer. Patients often have intrahepatic and extrahepatic metastases. The early clinical symptoms and signs of HCC are often atypical and difficult to detect. Most of the definite diagnosis of HCC has entered the middle, late stage, lost the opportunity for surgery. Because of its high degree of malignancy, difficulty in early detection, rapid progress, complex treatment, high rate of recurrence and metastasis, HCC patients have poor prognosis and poor curative effect. Therefore, we need to further explore the molecular biology mechanism of HCC in the process of occurrence, development and metastasis. To find molecular markers that can predict the occurrence of HCC will open up a new way for the diagnosis and treatment of HCC. Two percent of the sequences, and 98 percent of the other sequences that do not encode proteins, are considered useless "noise" in the genome. RNA molecules transcribed from the genome and that do not encode proteins are called non-coding RNA (ncRNA). In the early days, researchers focused only on coding RNA, but in recent years, people's attention has gradually been focused on non-coding RN. As the research progresses, it has been found that long non-coding RNA (lncRNA) plays an important role in the development of human diseases such as Alzheimer's disease, tumor and Huntington's disease. Nowadays, more and more tumor-associated lncRNAs have been found to regulate the growth, metastasis, invasion and apoptosis of tumors in various ways. They have become new tumor markers and drug targets, and have greatly promoted the progress of tumor research. Ji et al. identified metastasis-associated lung adenocarcinoma transcript 1 (MALAT 1) by sequencing and screened 225 patients with stage III non-small cell lung cancer (NSCLC). These results suggest that MALAT-1 may be a potential marker for stage I NSCLC patients. The gene has been found in other tumors, but it is most significantly expressed in NSCLC patients. MALAT-1 can promote tumor metastasis by activating Tumor Metastasis-related genes, and can also participate in the alterable splicing of mRNA precursors and phosphorylation repair of serine/arginine splicing factors. With the development of tumor genomics and transcriptome, researchers have found that not only different lncRNA can be found in the same tumor, but also the same lncRNA can be differentially expressed in different tumors. HULC was first found to be overexpressed in hepatocellular carcinoma cells, which can be regarded as a more sensitive primary hepatocellular carcinoma. H19, as a proto-oncogene, is highly expressed in a variety of digestive system tumors. In summary, lncRNA plays a vital role in oncology research. At present, spinal metastases are treated with minimally invasive treatment and posterior spinal opening. Therefore, tissue samples are relatively rare, collection is relatively difficult, and it is difficult to extract RNA from bone tissues. As a result, the study of lncRNA in spinal metastases is still blank at home and abroad. Sequencing techniques were screened and validated to identify novel key lncRNA targets for the diagnosis and treatment of spinal metastases and HCC, providing new strategies for clinical diagnosis and treatment. Methods: RNA was extracted from paired tissues (tumor tissues and adjacent tissues) of patients with spinal metastases, and high-throughput sequencing was used to construct spinal rotation. Differentially expressed lncRNA and mRNA expression profiles in tumor metastasis were then analyzed by pathway analysis and GO analysis to enrich the molecular functions of differentially expressed mRNA and participate in biological processes and disease pathways. Results: 171 differentially expressed lncRNA and 2929 differentially expressed mRNAs were found in spinal metastases by high-throughput sequencing, of which 66 were completely new and unreported. Pathway analysis and GO analysis of differentially expressed mRNAs were performed. The results showed that differentially expressed mRNAs in spinal metastases were enriched in 31 diseases and 79 common diseases. Pathway and 1 535 molecular functions, including tumor-related diseases and tumor-related pathways, further clarify the molecular functions, biological processes and regulation of the abnormal expression of mRNA in spinal metastases. There are 50 ntisense, 19 known, 31 unknown; 53 Sence, 51 known, 2 unknown; 6 Bidirectionsl, 4 known, 2 unknown; 40 Intergenic, 20 known, 20 unknown; 22 Intronics, 8 known, 14 unknown. Conclusion: We have successfully constructed lncRNA, mRNA of spinal metastases. Expression profiles. Among 171 differentially expressed lncRNAs, 66 novel unreported lncRNAs were found. The molecular functions, biological processes and regulation of disease pathways of 2929 differentially expressed mRNAs were identified. This provides valuable data for oncology research and follow-up experiments. Chapter 2: The expression of lnc000489 in the plasma of patients with spinal metastases. Objective: To identify the high expression of lncRNA in the plasma of patients with spinal metastases by validating its clinical features. The expression of candidate lncRNA in plasma was detected by qRT-PCR assay. After statistical analysis, significant differences in lncRNA expression in spinal metastases were found. The sensitivity and specificity were calculated by ROC curve. Then, the differential expression of lncR was detected. Results: The expression of lnc00489 in the plasma of patients with spinal metastases was significantly higher than that of healthy volunteers (P 0.05). ROC curve analysis showed that lnc00489 had certain diagnostic value for spinal metastases. There are 51 mRNAs co-expressed with lnc00489, which can be enriched in many tumor-related diseases such as solid tumors, ovarian cancer, oral cancer, prostate cancer and other tumor-related pathways such as Wnt, Hh, Hedgehog signaling pathway, Basal cell carcinoma. This further indicates that lnc00489 may be associated with the occurrence, development and metastasis of tumors. Correlation analysis between Lnc00489 expression and clinical features showed that lnc00489 was significantly associated with the number of metastatic vertebrae and visceral metastasis. Lnc00489 may be a potential plasma marker for the diagnosis and treatment of spinal metastases. Conclusion: The expression of Lnc00489 is significantly elevated in the plasma of patients with spinal metastases and has diagnostic value for spinal metastases. Tumor-related diseases and pathways. The expression of lnc00489 in plasma is correlated with the number of metastatic vertebrae and visceral metastasis. It is expected to be a new plasma molecular marker for the diagnosis of spinal metastases. Chapter 3: Expression of lnc34822 in plasma of HCC patients and its correlation with clinical features. Objective: To find the correlation between the expression of lncRNA in the constructed lncRNA database. Methods: Ten highly expressed lncRNA were screened from the previously established lncRNA database and the related literature. The sensitivity and specificity of diagnosis were determined by validating the presence of highly expressed lncRNA in tissues and plasma of HCC patients. The expression of candidate lncRNA in tissues, plasma and cell lines (Hep3B, HepG-2, SMMC-7721, Huh7.5, LO2) was detected by qRT-PCR. The stability of the differentially expressed lncRNA in plasma, the relationship between the differentially expressed lncRNA and peripheral blood cells, the expression in different cell lines, the changes of the expression in HCC patients before, after and after HCC, and the recurrence of the expression of lncRNA were statistically analyzed. Results: It has been reported that homologous lncRNA can be regulated in different tumors, and homologous tumors can also be affected by a variety of different lncRNA. Ten highly expressed lncRNA were screened from the constructed lncRNA expression profiles of spinal metastases based on literature investigation. After being verified in HCC patients'tissues and plasma, lnc34822 was found. The expression of Lnc34822 in HCC cell line was significantly higher than that in normal liver cell line, and there was no correlation between Lnc34822 and peripheral blood cells. It is concluded that lnc34822 may be a plasma marker for monitoring the progression of HCC.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7
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