脑胶质瘤中调控LRIG1表达的微小RNA筛选及机制研究

发布时间：2018-08-12 15:45

【摘要】：脑胶质瘤是中枢神经系统最常见的肿瘤,恶性胶质瘤(WHO Ⅲ IV级),特别是胶质母细胞瘤预后极差。手术切除联合术后放化疗是目前治疗恶性胶质瘤的标准方案。尽管经过积极的治疗,患者的预后仍然较差。全面认识胶质瘤发生和进展的分子机制是寻找治疗靶点的关键所在。多亮氨酸重复区免疫球蛋白样蛋白1 (Leucine-rich repeats and immunoglobulin-like domains 1, LRIG1)是一种广泛的酪氨酸激酶受体(Receptor tyrosine kinase, RTK)抑制蛋白。正常情况下,生长因子与其受体结合后除了激活与细胞增殖相关的基因外,还诱导内源性负反馈调节因子LRIG1的表达,使信号通路激活的程度和时间受到严格的控制。在胶质瘤组织和细胞系中,LRIG1的表达普遍降低,而且WHO分级越高其降低程度越明显。体内外研究表明,过表达LRIG1对胶质瘤生长表现出明显的抑制效应。不仅如此,它还能够增强胶质瘤细胞对放化疗的敏感性。因此,在胶质瘤中LRIG1被认为是一个抑癌基因。但是,目前LRIG1表达下调的机制仍不清楚。微小RNA (microRNA)是一类由约22个核苷酸组成的非编码小分子RNA,它通过识别并结合靶mRNA 3'端非翻译区(3'untranslated region,3'UTR)的种子序列进而发挥下调靶基因表达的作用。大量的研究证实异常表达的miRNA在胶质瘤的发生和进展中发挥着至关重要的作用,而且生物信息学分析发现在LRIG1的3'UTR存在着多个miRNA的潜在作用位点。因此,本研究拟筛选出与LRIG1下调相关的miRNA,并尝试以miRNA为靶点重建内源性LRIG1的表达。在第一部分中,通过生物信息学分析并结合目前已有的文献报道初步筛选出4个在胶质瘤中发挥促癌效应且与LRIG1具有潜在作用的miRNA,即miR-19a、 miR-93、 miR-106b及miR-23a、进一步采用荧光定量PCR和蛋白免疫印迹法检测了人脑胶质瘤标本中上述4个：niRNA与LRIG1蛋白的表达,相关性分析表明miR-19a、 miR-23a与LRIG1蛋白的表达呈显著负相关,相关系数分别为-0.664(P=0.036)、-0.678(P=0.031)。以上结果提示胶质瘤中miR-19a、 miR-23a可能是LRIG1的转录后调控因子。第二部分中,采用反义寡核苷酸敲低人脑胶质瘤细胞系中miR-19a或miR-23a的表达。蛋白免疫印迹检测表明下调miR-19a后,U87和U251细胞中LRIG1表达明显增高；而下调miR-23a后,LRIG1的表达无明显改变。经荧光素酶报告实验证实LRIG1是miR-19a的直接作用靶点。为了进一步探讨LRIG1是否为miR-19a的功能靶点,本部分中进行了逆转实验。其结果提示干扰内源性LRIG1可以拮抗反义miR-19a的抗增殖作用和促凋亡效应。以上结果提示LRIG1在一定程度上介导了反义miR-19a对胶质瘤细胞的抑制作用。第三部分中,探讨反义miR-19a对裸鼠皮下移植瘤生长及内源性LRIG1表达的影响。皮下成瘤实验表明转染反义miR-19a后U87细胞在体内的生长受到明显抑制。接种后30d,反义miR-19a组皮下肿瘤的重量为1.44±0.23g,对照组为3.59±0.62g(P0.05)。免疫组化分析表明反义miR-19a组皮下肿瘤中LRIG1阳性率较对照组明显增高,差异有统计学意义。以上结果提示反义miR-19a可以抑制U87细胞在体内的生长并可以提高内源性LRIG1的表达。综上所述,脑胶质瘤中过度表达的miR-19a是导致LRIG1蛋白下调的直接原因之一。体内外实验表明,下调miR-19a可以抑制U87细胞的生长,这可能与反义miR-19a促进内源性LRIG1表达有关。图注：正常情况下,LRIG1可以促进多种酪氨酸激酶受体发生泛素化并经蛋白溶酶体途径降解。脑胶质瘤中过度表达的miR-19a通过下调LRIG1蛋白从而破坏了酪氨酸激酶受体信号通路的负反馈环路,可能是促进胶质瘤的发生和发展的因素之一。(RTKs:酪氨酸激酶受体；LRIG1:多亮氨酸重复区免疫球蛋白样蛋白1；RISC:RNA诱导的沉默复合体；CBL蛋白介导受体泛素化)
[Abstract]:Glioma is the most common tumor of the central nervous system. Malignant gliomas (WHO grade III IV), especially glioblastoma, have a very poor prognosis. Surgical resection combined with postoperative radiotherapy and chemotherapy is the standard treatment for malignant gliomas. Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) are a wide range of tyrosine kinase receptor (RTK) inhibitors. Normally, growth factors bind to their receptors in addition to In addition to activating the genes related to cell proliferation, LRIG1 also induces the expression of LRIG1, an endogenous negative feedback regulator. The degree and time of signal pathway activation are strictly controlled. LRIG1 is considered to be a tumor suppressor gene in gliomas. However, the mechanism of down-regulation of LRIG1 expression remains unclear. MicroRNA is a non-coding group of about 22 nucleotides. Small molecule RNA, which plays a role in down-regulating target gene expression by recognizing and binding the seed sequence of the 3'untranslated region (3'UTR) of the target mRNA, has been demonstrated by numerous studies to play a crucial role in the genesis and progression of gliomas, and bioinformatics analysis has found that the 3'untranslated region (3'UTR) of the target mRNA plays a key role in the development of gliomas. Therefore, we intend to screen the microRNAs associated with the downregulation of LRIG1, and try to reconstruct the expression of endogenous LRIG1 by targeting microRNAs. In the first part, we preliminarily screened four microRNAs that play a carcinogenic role in glioma through bioinformatics analysis and combined with existing literature reports. The potential roles of LRIG1 in microRNAs, namely, microRNAs-19a, microRNAs-93, microRNAs-106b and microRNAs-23a, were further examined by fluorescence quantitative PCR and Western blotting. The expression of niRNA and LRIG1 protein in human glioma specimens was detected. Correlation analysis showed that the expressions of microRNAs-19a, microRNAs-23a and LRIG1 protein were negatively correlated with each other. These results suggest that microRNAs-19a and microRNAs-23a may be posttranscriptional regulators of LRIG1 in gliomas. In the second part, antisense oligonucleotides were used to knock down the expression of microRNAs-19a or microRNAs-23a in human gliomas. Western blot analysis showed that the expression of LRIG1 in U87 and U251 cells was down-regulated after microRNAs-19a. LRIG1 was confirmed to be the direct target of microRNA-19a by luciferase reporter assay. In order to further explore whether LRIG1 is the functional target of microRNA-19a, reversal experiments were carried out in this part. The results suggest that interfering with endogenous LRIG1 can antagonize the anti-proliferation of antisense microRNA-19a. These results suggest that LRIG1 mediates the inhibitory effect of antisense microRNA-19a on glioma cells to some extent. In the third part, the effects of antisense microRNA-19a on the growth and endogenous LRIG1 expression of subcutaneous xenografts in nude mice were investigated. 30 days after inoculation, the weight of subcutaneous tumors in antisense microwave-19a group and control group was 1.44 + 0.23 g and 3.59 + 0.62 g respectively (P 0.05). Immunohistochemical analysis showed that the positive rate of LRIG1 in antisense microwave-19a group was significantly higher than that in control group. The above results suggested that antisense microwave-19a could inhibit the growth of U87 cells in vivo. In conclusion, overexpression of microRNAs in gliomas is one of the direct reasons for the down-regulation of LRIG1 protein. In vivo and in vitro experiments show that down-regulation of microRNAs can inhibit the growth of U87 cells, which may be related to antisense microRNAs-19a promoting the expression of endogenous LRIG1. Overexpression of microRNA-19a in gliomas destroys the negative feedback loop of tyrosine kinase receptor signaling pathway by down-regulating LRIG1 protein, which may be one of the factors promoting the occurrence and development of gliomas. IG1: polyleucine repeat immunoglobulin-like protein 1; RISC: RNA-induced silencing complex; CBL protein-mediated receptor ubiquitination
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R739.41

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