miRNA-22调控髓母细胞瘤增殖与凋亡的分子机理研究

发布时间：2018-09-19 14:08

【摘要】：髓母细胞瘤（medulloblastoma， MB）是最常见的儿童中枢神经系统肿瘤（占儿童颅内肿瘤的12%-25%），，常起源于小脑蚓部或后髓帆，为WHOIV级肿瘤。儿童MB发病的高峰是3岁和7岁，MB在成人发病率较低（仅0.4%-1%），发病多在20-40岁。男女发病比约为1.3：1。临床表现以颅内压增高的症状为主，头痛伴有持续的呕吐，当肿瘤压迫小脑时尚会出现共济失调的体征，肿瘤还可随脑脊液向脊髓蛛网膜下腔播散，使患者病情急剧恶化。目前治疗方法以手术辅以放化疗的综合治疗为主，这在很大程度上改善了患者预后，但随之而来的是长期放化疗的毒副作用，如神经内分泌障碍、生长发育迟缓、神经心理及认知功能障碍等。因此研究MB发病的分子机制，探索可用于治疗的分子生物学新靶点有着重要的意义。已有研究表明microRNAs是一类具有转录后调控作用的20-24个核苷酸的内源性非编码小RNA，广泛参与到细胞的增殖、凋亡、分化、发育以及肿瘤的发生和发展。Ferretti E等对34例MB及14例正常小脑组织利用实时定量PCR技术，建立了MBmiRNA表达文库，其中位于染色体17p13.3的miR-22呈现低表达，而17p的缺失是MB染色体异常的一种重要的表现形式，这初步提示了miR-22在髓母细胞瘤的发生、发展中起着重要的作用。为进一步深入研究miR-22参与MB发病的分子机制，我们进一步扩大MB样本量加以验证，并进行miR-22生物学功能分析，下游靶点筛查、验证及生物学功能分析，初步阐明miR-22在髓母细胞瘤发病过程中的生物学作用。实验方法：第一部分选取MB临床标本、原代培养细胞及MB细胞系利用qRT-PCR的方法检测miR-22表达情况。并采用荧光原位杂交（Fluoresence in situ hybridization，FISH）技术，初步探讨染色体17p位点是否存在杂合性缺失。第二部分体外培养髓母细胞瘤细胞系并利用慢病毒表达载体进行上调和下调miR-22的表达，行CCK-8检测细胞增殖活性，并行流式细胞仪检测细胞凋亡。同时采用荷瘤小鼠模型在体研究miR-22过表达后对MB的影响。对过表达miR-22的MB细胞系行基因芯片筛查分析，并采用双荧光素酶报告系统验证miR-22新靶点。第三部分在MB临床标本、原代细胞及MB细胞系中检测PAPST1的表达，并在MB细胞系中下调PAPST1表达后CCK-8检测细胞增殖活性的变化。结果: 1. miR-22在MB中低表达（1）70%（19/27）的MB临床标本以及3株MB细胞系qRT-PCR结果提示存在miR-22的低表达（正常小脑组织表达水平的20%以下）。（2）19%（5/27）的MB标本存在17p13.3的丢失。 2. miR-22表达对MB细胞系增殖与凋亡的影响（1）上调miR-22可以抑制MB细胞系的增殖活性，相反下调其表达可以增强MB细胞系的增殖活性。（2）miR-22过表达可以促进MB细胞系的凋亡。（3）miR-22过表达可以抑制裸鼠肿瘤的生长促进瘤细胞凋亡。（4）PAPST1是miR-22的生物学新靶点基因芯片提示miR-22上调后显著抑制了PAPST1的表达，双荧光素酶报告实验证实PAPST1是miR-22的生物学新靶点。 3. PAPST1在MB中的表达及其对MB细胞增殖特性的影响（1）67%（18/27）的MB临床标本以及2株MB细胞系qRT-PCR结果提示存在PAPST1的高表达（正常小脑组织表达水平的2倍以上）。（2）PAPST1免疫组织化学显示棕褐色的颗粒分布于细胞浆，且着色明显强于正常小脑组织。（3）下调PAPST1表达后MB细胞系增殖活性降低。结论： miR-22在髓母细胞瘤中常表达下调，其表达水平通常与髓母细胞瘤体外增殖活性呈负相关，过表达miR-22在体内、体外均可以诱导髓母细胞瘤凋亡，抑制肿瘤生长。 PAPST1是miR-22的新靶点，在髓母细胞瘤中普遍高表达，其表达下调可以抑制髓母细胞瘤的增殖活性。
[Abstract]:Medulloblastoma (MB) is the most common central nervous system tumor in children (12% - 25% of intracranial tumors in children). It usually originates from the cerebellar vermis or posterior medullary velum and is a WHO grade IV tumor. The peak incidence of MB in children is 3 and 7 years old, and the incidence of MB in adults is low (only 0.4% - 1%). The incidence of MB in men and women is about 1.3:1. The clinical manifestations are mainly symptoms of increased intracranial pressure, headache accompanied by persistent vomiting. When the tumor oppresses the cerebellum, there will be ataxia. The tumor can also spread to the spinal subarachnoid space with cerebrospinal fluid, making the patient's condition deteriorate sharply. It improves the prognosis of patients, but the side effects of long-term radiotherapy and chemotherapy, such as neuroendocrine disorders, growth retardation, neuropsychological and cognitive dysfunction, etc. Therefore, it is important to study the molecular mechanism of MB pathogenesis and explore new molecular biological targets for treatment.
Studies have shown that microRNAs are endogenous, non-coding, 20-24 nucleotides with post-transcriptional regulation. They are widely involved in cell proliferation, apoptosis, differentiation, development, and tumorigenesis and development. Among them, the expression of microRNA-22 on chromosome 17p13.3 is low, and the deletion of 17p is an important manifestation of abnormal MB chromosome, which preliminarily suggests that microRNA-22 plays an important role in the occurrence and development of medulloblastoma. To validate and analyze the biological function of microRNAs-22, screening downstream targets, validation and biological function analysis, and preliminarily elucidate the biological role of microRNAs-22 in the pathogenesis of medulloblastoma.
Experimental methods:
In the first part, MB clinical specimens were selected, primary cultured cells and MB cell lines were used to detect the expression of microRNA-22 by qRT-PCR, and fluorescence in situ hybridization (FISH) technique was used to investigate the presence of loss of heterozygosity at chromosome 17p locus.
In the second part, Medulloblastoma cell lines were cultured in vitro and up-regulated and down-regulated by lentiviral expression vector. Cell proliferation activity was detected by CCK-8 and apoptosis was detected by flow cytometry. The effect of overexpression of miR-22 on MB was studied in vivo using tumor-bearing mice model. Gene chip screening and analysis, and dual luciferase reporter system was used to verify the new target of miR-22.
In the third part, the expression of PAPST1 was detected in MB clinical specimens, primary cells and MB cell lines, and the changes of cell proliferation activity were detected by CCK-8 after down-regulation of PAPST1 expression in MB cell lines.
Result:
1. low expression of miR-22 in MB
(1) 70% (19/27) of MB clinical specimens and 3 strains of MB cell lines showed low expression of microRNAs-22 (less than 20% of normal cerebellar tissue).
(2) there was a loss of 17p13.3 in MB specimens of 19% (5/27).
Effect of 2. miR-22 expression on proliferation and apoptosis of MB cell line
(1) Up-regulation of miR-22 can inhibit the proliferation of MB cell lines, but down-regulation of its expression can enhance the proliferation of MB cell lines.
(2) over expression of miR-22 can promote the apoptosis of MB cell line.
(3) over expression of miR-22 can inhibit tumor growth and promote apoptosis in nude mice.
(4) PAPST1 is a new biological target for miR-22.
Microarray analysis indicated that up-regulation of microarray RNA-22 significantly inhibited the expression of PAPST1. Double luciferase assay confirmed that PAPST1 was a new biological target of microarray RNA-22.
Expression of 3. PAPST1 in MB and its effect on proliferation of MB cells
(1) 67% (18/27) of MB clinical specimens and 2 strains of MB cell lines showed high expression of PAPST1 (more than twice the expression level in normal cerebellum).
(2) PAPST1 immunohistochemistry showed that brown granules were distributed in the cytoplasm, and the staining was stronger than that of normal cerebellum.
(3) after downregulation of PAPST1 expression, the proliferation activity of MB cell line decreased.
Conclusion: Mi-22 is often down-regulated in medulloblastoma, and its expression level is usually negatively correlated with the proliferative activity of medulloblastoma in vitro. Overexpression of Mi-22 can induce apoptosis and inhibit the growth of medulloblastoma in vitro.
PAPST1 is a novel target of microRNAs-22, which is highly expressed in medulloblastoma and its down-regulation can inhibit the proliferation of medulloblastoma.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.41

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