基于全转录组深度测序的垂体ACTH腺瘤mRNA及microRNA表达谱分析

发布时间：2018-06-13 12:09

本文选题：垂体ACTH腺瘤 + 信号通路　；参考：《北京协和医学院》2016年博士论文

【摘要】：背景：垂体腺瘤是常见的颅内良性肿瘤之一,发病率居颅内肿瘤第二位,约占颅内肿瘤的10%-15%,并呈现逐年增加的趋势。随着肿瘤不断生长,可压迫蝶鞍区周围结构,如视神经、海绵窦、脑底动脉、下丘脑等,甚至累及额叶、脑干,而导致严重的功能障碍。同时,肿瘤生长还可导致垂体激素分泌紊乱。按照外周血激素水平,垂体腺瘤分为无功能型垂体腺瘤(NFPA)和功能型垂体腺瘤,包括：泌乳素腺瘤(PRL)、促肾上腺皮质激素腺瘤(Cushing)、生长激素腺瘤(GH)、促甲状腺激素腺瘤(TSH)、促性腺激素腺瘤(PGA)以及混合激素分泌腺瘤等。其中垂体促肾上腺皮质激素腺瘤(ACTH-PAs)临床又称为库欣病(Cushing's Disease)是一种伴有促肾上腺皮质激素(ACTH)分泌的功能型垂体腺瘤约占全部垂体腺瘤的14%,约占库欣综合征(Cushing's syndrome)的70%。该病在欧美国家的发病率为39／百万,我国尚缺乏大规模流行病学数据。由于该病的诊断主要基于实验室激素水平检查、患者临床症状体征、影像学检查及病理学免疫组织化学检测,因此对于库欣病的早期诊断十分困难,容易误诊。患者常常由于高皮质醇血症而引起多种严重并发症,如高血压、糖尿病、高脂血症、骨质疏松及精神抑郁等就诊。在治疗上,对于绝大多数库欣病人而言,经鼻蝶窦入路垂体腺瘤切除术仍然是临床首选治疗方法,放射治疗以及帕瑞肽、酮康唑等药物治疗常作为难治性库欣病的术后辅助治疗方式。虽然文献报道外科手术成功率在65%-90%,但是由于肿瘤自身生物学行为的不同及术者操作水平的差异,肿瘤复发率为3%-47%,平均复发时间为16～49个月,复发患者预后较差,病死率高。近年来针对库欣病的分子水平研究主要集中在肿瘤的发生、发展、肿瘤的侵袭性及激素分泌等方面,但目前对于库欣病的发病机制尚不完全清楚,因此临床上亟待寻找有助于库欣病诊断、治疗及判断预后的潜在靶点。本研究通过新一代测序技术(Next generation sequencing, NGS),对垂体ACTH腺瘤全转录组进行高通量测序,在转录组水平筛选相关差异表达基因和microRNA;并与患者临床表型进行关联分析,探究垂体ACTH腺瘤所致临床表型的分子机理,此外,考虑到血清游离miRNA具有作为肿瘤标志物的潜力,筛选差异表达的血清miRNA,并对差异表达的血清miRNA进行了验证,为深入研究库欣病的发病机制,筛选诊断标志物提供依据。以期最终实现垂体腺瘤患者个性化治疗。第一部分：基于全转录组深度测序的垂体ACTH腺瘤mRNA差异表达分析目的：通过新一代高通量测序技术对垂体ACTH腺瘤行转录组测序,探究垂体ACTH腺瘤与瘤周正常垂体组织间的差异表达基因,以期揭示肿瘤发生发展过程中可能的存在分子机制,并为后续的microRNA组学研究提供完整的分子生物学基础。方法：收集单一中心68例垂体ACTH患者的组织标本(肿瘤及瘤周正常垂体),20例无功能垂体腺瘤,18例生长激素型垂体腺瘤和8例泌乳素型垂体腺瘤,同时采集患者相关临床信息。运用新一代测序技术对9对配对样本(肿瘤和瘤周正常垂体组织)进行转录组深度测序,通过生物信息学分析,获取垂体ACTH腺瘤基因差异表达谱。进一步对筛选出的差异表达基因行GO term和PATHWAY分析,并挑选差异表达显著基因进行验证和功能机制研究,探究其在垂体ACTH腺瘤中可能存在的分子调节机制。结果：通过对垂体ACTH腺瘤mRNA-seq数据做生物信息学分析,我们筛选出166个上调的差异表达基因和288个下调的差异表达基因,发现了库欣病肿瘤发生发展、激素分泌和肿瘤侵袭性等相关的基因和相关信号通路。并从中挑选未经报道的TIMP3基因做进一步验证,发现与瘤周正常垂体组织相比TIMP3在mRNA和蛋白水平的表达在各类型垂体腺瘤中显著下调,并与肿瘤的体积、侵袭性、Ki-67等显著相关。结论：通过建立垂体ACTH腺瘤基因的差异表达谱,揭示库欣病可能存在的发病机制及临床表型相关机制,并为后续的库欣病microRNA组学分子靶向研究提供理论基础。第二部分：基于全转录组深度测序的垂体ACTH腺瘤microRNAs差异表达分析目的：运用新一代测序技术,建立垂体ACTH腺瘤microRNA差异表达谱。鉴定相关microRNA在垂体ACTH腺瘤组织中及患者血清中的表达情况,以期作为诊断库欣病和预后监测的分子标志物。方法：经蝶窦入路手术治疗库欣病患者,术中收集垂体ACTH腺瘤患者肿瘤标本及瘤周垂体组织共9对。运用Illumina Genome Analyzer Ⅱx行全转录组microRNA测序,结合生物信息学分析建立垂体ACTH腺瘤差异表达的microRNA谱。并在55例肿瘤组织和15例瘤周正常垂体组织中运用荧光定量PCR进行验证,将筛选得到的microRNA在197例病人和120例健康人的血清中运用荧光定量PCR行验证检测,并进行靶基因的预测及与第一部分mRNA-seq结果做关联分析,在组织水平作相关基因功能验证,得到一组microRNA panel作为针对库欣病诊断和预后观察的分子标志物。结果：所有9对样品建库成功,Illumina Genome Analyzer Ⅱx深度测序平均读取10M/样品。我们发现,垂体ACTH腺瘤与瘤周垂体相比,有67个microRNA显著差异表达。通过对测序数据分析发现,垂体ACTH腺瘤相对瘤周正常垂体组织比较,根据Fold change2, Recurrence≥5,发现67个差异表达的microRNA,其中包括39个上调的microRNA和28个下调的microRNA,在肿瘤和瘤周正常垂体组织中验证得到6个上调(miR-9-5p, miR-9-3p, miR-190b, miR-137, miR-885-5p, miR-592)和1个下调的mir-551b-3p,并且在患者和正常人的血清中进行检测,也得到了1个下调的mir-551b-3p,其表达水平与组织中一致。受试者工作特征曲线分析显示,mir-551b-3p对于库欣病的诊断具有较高的敏感性和特异性。血清中mir-551b-3p的表达水平与患者的肿瘤体积,血皮质醇和ACTH水平显著相关。结论：研究结果表明microRNA的差异表达可能参与垂体ACTH腺瘤的发生发展。microRNA有可能成为库欣病人特异性的,外周血无创诊断的分子标志物。
[Abstract]:Background: pituitary adenoma is one of the most common benign intracranial tumors, with the incidence of second of intracranial tumors, accounting for 10%-15% of intracranial tumors and increasing trend year by year. As the tumor grows, it can oppress the surrounding structure of the sella region, such as the optic nerve, cavernous sinus, the cerebral artery, the hypothalamus, and even the frontal and brain stem. At the same time, tumor growth can also lead to a disorder of pituitary hormone secretion. According to peripheral blood hormone levels, pituitary adenomas are divided into nonfunctional pituitary adenomas (NFPA) and functional pituitary adenomas, including prolactin adenoma (PRL), adrenocorticotropin adenoma (Cushing), growth hormone adenoma (GH), thyroid stimulating hormone adenoma (TSH), and promoting pituitary adenomas (TSH). Gonadotropin adenoma (PGA) and mixed hormone secretory adenoma. The pituitary adrenocorticotropin adenoma (ACTH-PAs) is also known as Cushing's disease (Cushing's Disease), a functional pituitary adenoma secreted by adrenocorticotropin (ACTH), about 14% of all pituitary adenomas, accounting for Cushing's syndrome (Cushing's syndrome). The incidence of 70%. in European and American countries is 39 per million. There is still a lack of large-scale epidemiological data in our country. Because the diagnosis of this disease is mainly based on laboratory hormone level examination, clinical symptoms and signs, imaging examination and pathological examination of Pathology, it is very difficult for early diagnosis of Cushing's disease and easy to be misdiagnosed. Patients often cause a variety of severe complications due to hypercortisol, such as hypertension, diabetes, hyperlipidemia, osteoporosis, and depression. In the treatment of most Cushing patients, transsphenoidal pituitary adenoma resection is still the preferred treatment, radiotherapy, Parein, and ketkang. Azoles are often used as adjuvant therapy for refractory Cushing's disease. Although the literature reports that the success rate of surgery is 65%-90%, the recurrence rate of the tumor is 3%-47%, the recurrence time is 16~49 months due to the difference of the biological behavior of the tumor and the difference of the operation level. The prognosis of the recurrent patients is poor and the mortality is high. Recent studies on the molecular level of Cushing's disease are mainly focused on the occurrence, development, invasiveness and hormone secretion of the tumor, but the pathogenesis of Cushing's disease is not yet completely clear, so it is urgent to find the potential targets for the diagnosis, treatment and prognosis of Cushing's disease. Next generation sequencing (NGS), high flux sequencing of pituitary ACTH adenoma complete transcriptional group, screening related differentially expressed genes and microRNA at the level of the transcriptional group, and associated with the clinical phenotype of the patients, to explore the molecular mechanism of the clinical phenotype of pituitary ACTH adenoma, and to consider the free miRNA in serum. The differential expression of serum miRNA was screened as the potential of tumor markers, and the differential expression of serum miRNA was verified to provide a basis for the in-depth study of the pathogenesis of Cushing's disease and screening diagnostic markers. The first part: the pituitary ACTH gland based on the full transcriptional sequence based on the full transcriptional sequence. Differential expression analysis of tumor mRNA Objective: To explore the differentially expressed genes between pituitary ACTH adenomas and normal pituitary tissues by a new generation of high-throughput sequencing technology to explore the differentially expressed genes between pituitary ACTH adenomas and normal pituitary tissues, in order to reveal the possible molecular mechanisms in the process of tumor development and to provide a complete study for subsequent microRNA studies. Molecular biology basis. Methods: a single center 68 case of pituitary ACTH patients (tumor and tumor Zhou Zhengchang pituitary), 20 non functional pituitary adenomas, 18 growth hormone pituitary adenomas and 8 prolactin pituitary adenomas were collected, and the patient's clinical information was collected. 9 pairs of paired samples (tumors and) were used in the new generation sequencing technique. The differential expression profiles of pituitary ACTH adenomas were obtained by bioinformatics analysis. The differentially expressed genes were further analyzed by GO term and PATHWAY analysis, and the differentially expressed genes were selected to verify and function the mechanism of the pituitary adenoma, and to explore the possibility of it in pituitary ACTH adenomas. Results: through bioinformatics analysis of the mRNA-seq data of pituitary ACTH adenoma, we screened 166 up-regulated differentially expressed genes and 288 down regulated differentially expressed genes, and found genes and related signaling pathways related to the development of Cushing's disease tumor, hormone secretion and tumor invasion. The unreported TIMP3 gene was selected to further verify that the expression of TIMP3 at mRNA and protein levels was significantly downregulated in all types of pituitary adenomas compared with normal pituitary tissue, and was significantly related to the volume, invasiveness and Ki-67 of the tumor. Conclusion: the differential expression profile of the gene of the ACTH adenoma of the body was established to reveal Cushing's disease. Possible pathogenesis and clinical phenotype related mechanisms, and provide a theoretical basis for subsequent study of Cushing's disease microRNA molecular targeting. The second part: analysis of differential expression of microRNAs in pituitary ACTH adenomas based on full transcriptional sequence analysis: using a new generation sequencing technology to establish a microRNA differential table for pituitary ACTH adenomas To identify the expression of microRNA in the pituitary adenoma and the sera of the pituitary ACTH, as a molecular marker for the diagnosis of Cushing's disease and prognosis. Methods: transsphenoidal approach to the patients with Cushing's disease, 9 pairs of pituitary ACTH adenomas and pituitary tissue were collected during the operation. Illumina Genome was used. Analyzer II X line full transcriptional group microRNA sequencing, combined with bioinformatics analysis to establish the microRNA spectrum of differential expression of pituitary ACTH adenoma, and using fluorescence quantitative PCR in 55 cases of tumor tissue and 15 cases of Zhou Zhengchang pituitary tissue, the selected microRNA was used in the serum of 197 patients and 120 healthy people. PCR was tested and the target gene was predicted and associated with the first part of the mRNA-seq results. A group of microRNA panel as a molecular marker for the diagnosis and prognosis of Cushing's disease was obtained at the tissue level. Results: all 9 pairs of samples were built successfully and Illumina Genome Analyzer II X was deep. 10M/ samples were read by degree sequencing. We found that there were 67 significant differences in the expression of the pituitary ACTH adenoma compared with the peritumoral pituitary. Through analysis of the sequencing data, the pituitary ACTH adenoma was compared to the Zhou Zhengchang pituitary tissue, and 67 differentially expressed microRNA were present according to Fold change2, Recurrence more than 5, including 39 up-regulated adenomas. MicroRNA and 28 down regulated microRNA were tested for 6 up-regulated (miR-9-5p, miR-9-3p, miR-190b, miR-137, miR-885-5p, miR-592) and 1 down-regulated mir-551b-3p in the tumor and normal pituitary tissue, and were detected in the sera of the patients and normal people, and 1 down-regulated mir-551b-3p were also obtained, the expression level and the tissue of the tissues. The results showed that mir-551b-3p had high sensitivity and specificity for the diagnosis of Cushing's disease. The expression level of mir-551b-3p in serum was significantly related to the tumor volume, plasma cortisol and ACTH levels in the patients. Conclusion: the results showed that the differential expression of microRNA may be involved in pituitary ACTH adenomas. The occurrence and development of.MicroRNA is likely to be a specific marker of Cushing's disease and a molecular marker for noninvasive diagnosis of peripheral blood.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R736.4

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