【摘要】：腦膠質(zhì)瘤是中樞神經(jīng)系統(tǒng)最常見(jiàn)的腫瘤,惡性膠質(zhì)瘤(WHO Ⅲ IV級(jí)),特別是膠質(zhì)母細(xì)胞瘤預(yù)后極差。手術(shù)切除聯(lián)合術(shù)后放化療是目前治療惡性膠質(zhì)瘤的標(biāo)準(zhǔn)方案。盡管經(jīng)過(guò)積極的治療,患者的預(yù)后仍然較差。全面認(rèn)識(shí)膠質(zhì)瘤發(fā)生和進(jìn)展的分子機(jī)制是尋找治療靶點(diǎn)的關(guān)鍵所在。多亮氨酸重復(fù)區(qū)免疫球蛋白樣蛋白1 (Leucine-rich repeats and immunoglobulin-like domains 1, LRIG1)是一種廣泛的酪氨酸激酶受體(Receptor tyrosine kinase, RTK)抑制蛋白。正常情況下,生長(zhǎng)因子與其受體結(jié)合后除了激活與細(xì)胞增殖相關(guān)的基因外,還誘導(dǎo)內(nèi)源性負(fù)反饋調(diào)節(jié)因子LRIG1的表達(dá),使信號(hào)通路激活的程度和時(shí)間受到嚴(yán)格的控制。在膠質(zhì)瘤組織和細(xì)胞系中,LRIG1的表達(dá)普遍降低,而且WHO分級(jí)越高其降低程度越明顯。體內(nèi)外研究表明,過(guò)表達(dá)LRIG1對(duì)膠質(zhì)瘤生長(zhǎng)表現(xiàn)出明顯的抑制效應(yīng)。不僅如此,它還能夠增強(qiáng)膠質(zhì)瘤細(xì)胞對(duì)放化療的敏感性。因此,在膠質(zhì)瘤中LRIG1被認(rèn)為是一個(gè)抑癌基因。但是,目前LRIG1表達(dá)下調(diào)的機(jī)制仍不清楚。微小RNA (microRNA)是一類由約22個(gè)核苷酸組成的非編碼小分子RNA,它通過(guò)識(shí)別并結(jié)合靶mRNA 3'端非翻譯區(qū)(3'untranslated region,3'UTR)的種子序列進(jìn)而發(fā)揮下調(diào)靶基因表達(dá)的作用。大量的研究證實(shí)異常表達(dá)的miRNA在膠質(zhì)瘤的發(fā)生和進(jìn)展中發(fā)揮著至關(guān)重要的作用,而且生物信息學(xué)分析發(fā)現(xiàn)在LRIG1的3'UTR存在著多個(gè)miRNA的潛在作用位點(diǎn)。因此,本研究擬篩選出與LRIG1下調(diào)相關(guān)的miRNA,并嘗試以miRNA為靶點(diǎn)重建內(nèi)源性LRIG1的表達(dá)。在第一部分中,通過(guò)生物信息學(xué)分析并結(jié)合目前已有的文獻(xiàn)報(bào)道初步篩選出4個(gè)在膠質(zhì)瘤中發(fā)揮促癌效應(yīng)且與LRIG1具有潛在作用的miRNA,即miR-19a、 miR-93、 miR-106b及miR-23a、進(jìn)一步采用熒光定量PCR和蛋白免疫印跡法檢測(cè)了人腦膠質(zhì)瘤標(biāo)本中上述4個(gè)：niRNA與LRIG1蛋白的表達(dá),相關(guān)性分析表明miR-19a、 miR-23a與LRIG1蛋白的表達(dá)呈顯著負(fù)相關(guān),相關(guān)系數(shù)分別為-0.664(P=0.036)、-0.678(P=0.031)。以上結(jié)果提示膠質(zhì)瘤中miR-19a、 miR-23a可能是LRIG1的轉(zhuǎn)錄后調(diào)控因子。第二部分中,采用反義寡核苷酸敲低人腦膠質(zhì)瘤細(xì)胞系中miR-19a或miR-23a的表達(dá)。蛋白免疫印跡檢測(cè)表明下調(diào)miR-19a后,U87和U251細(xì)胞中LRIG1表達(dá)明顯增高；而下調(diào)miR-23a后,LRIG1的表達(dá)無(wú)明顯改變。經(jīng)熒光素酶報(bào)告實(shí)驗(yàn)證實(shí)LRIG1是miR-19a的直接作用靶點(diǎn)。為了進(jìn)一步探討LRIG1是否為miR-19a的功能靶點(diǎn),本部分中進(jìn)行了逆轉(zhuǎn)實(shí)驗(yàn)。其結(jié)果提示干擾內(nèi)源性LRIG1可以拮抗反義miR-19a的抗增殖作用和促凋亡效應(yīng)。以上結(jié)果提示LRIG1在一定程度上介導(dǎo)了反義miR-19a對(duì)膠質(zhì)瘤細(xì)胞的抑制作用。第三部分中,探討反義miR-19a對(duì)裸鼠皮下移植瘤生長(zhǎng)及內(nèi)源性LRIG1表達(dá)的影響。皮下成瘤實(shí)驗(yàn)表明轉(zhuǎn)染反義miR-19a后U87細(xì)胞在體內(nèi)的生長(zhǎng)受到明顯抑制。接種后30d,反義miR-19a組皮下腫瘤的重量為1.44±0.23g,對(duì)照組為3.59±0.62g(P0.05)。免疫組化分析表明反義miR-19a組皮下腫瘤中LRIG1陽(yáng)性率較對(duì)照組明顯增高,差異有統(tǒng)計(jì)學(xué)意義。以上結(jié)果提示反義miR-19a可以抑制U87細(xì)胞在體內(nèi)的生長(zhǎng)并可以提高內(nèi)源性LRIG1的表達(dá)。綜上所述,腦膠質(zhì)瘤中過(guò)度表達(dá)的miR-19a是導(dǎo)致LRIG1蛋白下調(diào)的直接原因之一。體內(nèi)外實(shí)驗(yàn)表明,下調(diào)miR-19a可以抑制U87細(xì)胞的生長(zhǎng),這可能與反義miR-19a促進(jìn)內(nèi)源性LRIG1表達(dá)有關(guān)。圖注：正常情況下,LRIG1可以促進(jìn)多種酪氨酸激酶受體發(fā)生泛素化并經(jīng)蛋白溶酶體途徑降解。腦膠質(zhì)瘤中過(guò)度表達(dá)的miR-19a通過(guò)下調(diào)LRIG1蛋白從而破壞了酪氨酸激酶受體信號(hào)通路的負(fù)反饋環(huán)路,可能是促進(jìn)膠質(zhì)瘤的發(fā)生和發(fā)展的因素之一。(RTKs:酪氨酸激酶受體；LRIG1:多亮氨酸重復(fù)區(qū)免疫球蛋白樣蛋白1；RISC:RNA誘導(dǎo)的沉默復(fù)合體；CBL蛋白介導(dǎo)受體泛素化)
[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
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R739.41

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