【作者】 张祖萍；

【导师】 李桂源；

【作者基本信息】 中南大学 ， 病理与病理生理学， 2010， 博士

【副题名】1.LRRC4在脑胶质瘤中失活的表观修饰的分子机制 2.脑胶质瘤DNA甲基化谱的初步构建

【摘要】 [LRRC4基因的前期研究结果]LRRC4(Genbank登录号为AF196976)是我室采用EST介导的定位候选克隆策略结合5’RACE的方法从染色体7q31-32克隆的一个富亮氨酸重复(Leucine-rich repeat, LRR)超家族新成员。多组织膜Northern Blot和组织芯片分析显示LRRC4在人、鼠组织均表现出脑特异表达的特点。Northern-blot和RT-PCR结果显示LRRC4基因不仅在多种恶性胶质瘤细胞系(如U251、U87、SF126、SF767、BT325和M17)中表达缺失；而且在87.5%(21/24)原发性胶质瘤中存在显著表达缺失和下调。外源性的LRRC4基因的转染可使U251生长速度减慢；细胞周期阻滞在G0/G1期；软琼脂集落形成率下降；裸鼠成瘤体积明显缩小和成瘤时间明显延迟。进一步研究显示,LRRC4通过LRR结构域调控ERK/AKT/NF-κB和JNK2/c-Jun/p53信号通路共同将U251细胞阻滞在G0/G1期,抑制细胞的增殖和侵袭。以上结果表明：LRRC4不仅是脑组织特异性基因,而且是与胶质瘤密切相关的抑瘤基因。前期研究表明：LRRC4基因虽然在原发性胶质瘤组织和多种恶性胶质瘤细胞系中表达下调甚至缺失,但其编码区并未发生突变、缺失、重排。【LRRC4基因启动子成功定位和克隆]为了揭示LRRC4基因在胶质瘤细胞和组织中表达下调的分子机制,我们开展了LRRC4基因的转录调控机制研究。采用高特异性的启动子预测软件PromoterInspector和PromoterScan对其启动子进行了预测分析,同时采用CpGplot软件对LRRC4基因5’端调控区的CpG岛进行了分析,三者预测结果大部分重叠,综合分析我们初步锁定LRRC4基因启动子区可能位于其翻译起始位点上游2151bp至101bp以内,因此我们设计引物扩增LRRC4基因-2475/-101的调控区片段,并构建成荧光素酶报告载体pGL3--2475/-101。荧光素酶活性分析系统结果表明,pGL3--2475/-101在Cos7和Hela细胞具有与pGL3-control几乎同等活性,这一结果表明LRRC4基因-2475/-101区域内包含了LRRC4基因的启动子片段。为了进一步定位LRRC4基因的启动子片段,以pGL3--2475/-101为模板,分别在它的5’或3’端缺失部分序列,构建了一系列缺失突变体荧光素酶的报告载体,并将它们分别转染Cos7和Hela细胞中,发现-835/-293区域是LRRC4基因发挥启动子活性的必需序列,缺失这一区域LRRC4基因启动子则丧失了全部的启动活性。为了验证该结果我们又构建了pGL3-835/-293/eGFP报告载体,转染Cos7和Hela细胞,发现LRRC4基因的-835/-293区域能驱动eGFP在Cos7和Hela细胞中的表达,从另一方面证明的-835/-293区域的启动活性。总之,通过以上研究我们成功地定位和克隆了LRRC4基因的启动子。[LRRC4基因启动子在胶质瘤中呈高甲基化状态]生物信息学分析LRRC4基因的启动子序列,发现该序列不含有TATA盒和CAAT盒,GC含量高达70%左右,为一典型的CpG岛,该结果提示LRRC4基因启动子甲基化可能参与其转录调控。采用甲基化特异性PCR (MS-PCR)对2株胶质瘤细胞、30例胶质瘤组织和3例正常脑组织进行检测,结果显示在胶质瘤细胞SF767和SF126中LRRC4基因的启动子呈完全的甲基化状态,在30例胶质瘤病人组织中均呈不同程度的部分甲基化状态,而在3例正常人脑组织中则呈完全非甲基化状态。为了明确LRRC4基因的启动子序列中究竟哪些位点发生了甲基化,选取胶质瘤细胞SF767和SF126,两例胶质瘤组织及一例正常脑组织进行了亚硫酸氢钠测序,结果表明与正常的脑组织相比,在胶质瘤细胞和组织中LRRC4基因的启动子区存在高密度的甲基化位点。该结果提示LRRC4基因启动子甲基化可能为肿瘤特异性的,它有可能成为区别胶质瘤组织与正常脑组织的一个重要的分子标志物。[5-Aza-CdR能逆转LRRC4基因在胶质瘤细胞中的表达]为了弄清楚LRRC4基因启动子甲基化和其在胶质瘤中失活之间的功能上联系,采用不同浓度甲基化酶抑制剂5-Aza-CdR处理胶质瘤细胞SF126和SF767,观察了LRRC4基因启动子甲基化改变与其表达的关系,结果表明5-Aza-CdR能逆转LRRC4基因启动子甲基化状态,并且能够上调LRRC4基因的表达水平。该结果从反面证明了启动子甲基化对LRRC4基因表达的抑制作用,另一方面也证明了LRRC4基因启动子甲基化是其在胶质瘤中表达缺失的重要分子机制。在5-Aza-CdR处理胶质瘤细胞逆转LRRC4基因表达的同时,我们还观察了5-Aza-CdR对SF126和SF767细胞增殖及细胞周期的影响,发现5-Aza-CdR对SF126和SF767细胞增殖具有明显的抑制作用,它能使胶质瘤细胞G0/G1期细胞数量明显增加,S期和G2/M期的细胞数量明显减少,从而出现G0/G1期的细胞阻滞。总之,5-Aza-CdR抑制了胶质瘤细胞SF126和SF767生长,并且逆转了LRRC4基因在胶质瘤中甲基化状态,恢复其转录活性,从而能够发挥LRRC4基因的肿瘤抑制作用,这为LRRC4基因作为胶质瘤去甲基化治疗的潜在靶标提供了科学依据。【LRRC4基因启动子甲基化抑制转录因子SP1和E2F1与其结合]为了进一步分析LRRC4基因启动子甲基化抑制其表达的分子机制,采用在线软件MatInspector分析了LRRC4基因启动子区(-835bp至-293bp)潜在的转录因子结合位点,发现该区域可能存在多种转录因子结合位点,我们选取了五个位点设计探针,进行了凝胶迁移实验(EMSA),我们只在E2F1(-655/-631)和Spl(-568/-547)两个位点检测出了特异性结合条带,说明E2F1和Sp1参与了LRRC4基因的转录调控。为了确定甲基化是否影响了E2F1和Sp1两个转录因子的结合,利用SssI甲基转移酶处理了E2F1(-655/-631)和Sp1(-568/-547)结合位点的双链寡核苷酸探针,然后进行EMSA实验,发现其特异性结合条带消失,说明DNA甲基化抑制了E2F1和Sp1两个位点与转录因子的结合。为了反映体内真实情况,我们又进行了染色质免疫共沉淀实验(CHIP),结果发现未经5-Aza-CdR处理SF126和SF767细胞,经SP1和E2F1抗体沉淀的基因组DNA中未能扩增出LRRC4基因启动子序列；5-Aza-CdR处理SF126和SF767细胞后,在经SP1和E2F1抗体沉淀的基因组DNA中却特异地扩增出LRRC4基因启动子序列。因此,LRRC4基因启动子区受转录因子SP1和E2F1的调控,胶质瘤中由于LRRC4基因启动子的甲基化,干扰转录因子SP1和E2F1与其结合,抑制了LRRC4基因的转录。[与LRRC4基因启动子甲基化相关联的组蛋白修饰]在表观遗传学机制中,DNA甲基化修饰和组蛋白修饰都不是孤立事件,它们往往相互影响、相互作用共同调控着基因表达。为了分析与LRRC4基因启动子甲基化相关联的组蛋白修饰,采用H3乙酰化抗体,H3K4三甲基化抗体及H3K9三甲基化抗体对5-Aza-CdR处理前后的胶质瘤细胞SF126和SF767进行了染色质免疫共沉淀实验(CHIP)。结果表明未经5-Aza-CdR处理的胶质瘤细胞,只在经H3K9三甲基化抗体沉淀的gDNA中特异地扩增出LRRC4基因启动子区片段,而经H3乙酰化抗体和H3K4三甲基化抗体沉淀的gDNA中却未能特异地扩增出LRRC4基因启动子区片段。在经5μM5-Aza-CdR处理胶质瘤细胞4天后,H3乙酰化抗体和H3K4三甲基化抗体沉淀的gDNA中才特异地扩增出LRRC4基因启动子区片段,而经H3K9三甲基化抗体沉淀的gDNA中扩增出LRRC4基因启动子区片段的强度有所减弱。说明5-Aza-CdR处理胶质瘤细胞后在LRRC4基因启动子减少甲基化H3K9的水平,同时提高乙酰化H3和甲基化H3K4的水平。因此我们得出结论：H3K9三甲基化与LRRC4基因启动子的甲基化状态密切关联,与LRRC4基因转录抑制相关；而H3乙酰化和H3K4三甲基化与LRRC4基因启动子的去甲基化状态密切关联,与LRRC4基因转录激活相关。第二部分脑胶质瘤DNA甲基化谱的初步构建每种类型的肿瘤都有特定的DNA甲基化模式,不同肿瘤或同一肿瘤不同发生阶段,基因组DNA上CpG岛甲基化状态的差异,构成了肿瘤特定DNA甲基化谱。胶质瘤DNA甲基化谱建立不仅有助于全面揭示胶质瘤发生发展的分子机制,更重要的是可为胶质瘤的早期诊断、治疗及预后评价等提供非常有价值的线索。目前虽然在胶质瘤的异常甲基化相关基因研究方面取得了一些进展,但大部分研究只是针对几个或一群基因采取候选基因研究方法。迄今,尚无学者对胶质瘤的DNA异常甲基化在全基因组层面上进行直接、全面系统分析。缺少高通量的筛选技术一直是阻碍基因组水平研究DNA甲基化谱系的关键因素。本研究采用最新发展的甲基化DNA免疫沉淀(MeDIP)结合启动子区及CpG岛芯片(NimbleGen, HG18 CpG Promoter)高通量技术对年龄、性别相匹配的6例胶质瘤病人的瘤组织(T1,T2,T3,T4,T5,T6)和4例正常人的脑组织的脑白质(N1,N2,N3,N5),进行初步筛选。在我们界定的高甲基化基因(位点)和低甲基化基因(位点)标准下,共筛选出高甲基化位点562个,其中涉及基因数325个；低甲基化位点108个,其中涉及基因数74个,它们在染色体上分布比较均匀；这些甲基化异常基因参与了细胞通讯、信号转导、细胞粘附、细胞迁移、凋亡、代谢、转运、细胞蛋白翻译后修饰及神经系统发育等广泛的生物学过程,同时还涉及多条与肿瘤发生有关的信号通路,如MAPK signaling pathway, Wnt signaling pathway, Jak-STAT signaling pathway及Cell cycle等,因此,这些甲基化异常的基因可能与胶质瘤的发生发展有着密切的关系。为了验证甲基化芯片结果,我们挑选了甲基化区域为CpG岛且位于启动子区的8个基因(ANKDD1A, SST, SIX3, GAD1, PHOX2B, HIST1H3E, PCDHA13和PCDHA8)采用MassArray检测系统在40例胶质瘤组织(包括T1,T2,T3,T4,T5,T6)、11例正常脑组织(包括N1,N2,N3,N5)及4株胶质瘤细胞系(U251、U87、SF126和SF767)进行了验证和检测。ANKDD1A,SST,HIST1H3E,PHOX2B,PCDHA13, GAD1, SIX3和PCDHA8在正常脑组织平均甲基化率分别为13.29%±2.12%,9.72%±2.42%,16.34%±4.13%,11.81%±3.76%,23.06%±4.49%,21.39%±3.13%,31.46%±4.28%和31.66%±12.77%；在胶质瘤组织中平均甲基化率分别为36.24%±23.71%,35.08%±19.44%,51.36%±21.25%,29.22%±15.13%,50.38%±19.15%,41.67%±25.56%,55.77%±21.77%和69.42%±19.49%；在胶质瘤细胞系中平均甲基化率分别为76.14%±27.99%,47.64%±31.61%,72.55%±25.49%,62.21%±16.80%,22.44%±18.00%,63.05%±20.35%,58.09%±30.00%和55.00%±21.29%。除了PCDHA13基因甲基化水平在胶质瘤细胞系与正常脑组织中无差别外(p>0.05),其他基因在胶质瘤组织和胶质瘤细胞中甲基化水平明显高于正常脑组织,且具有统计学意义(p<0.01)。而且,除了PCDHA8和PCDHA13基因外,其他基因在胶质瘤细胞细胞系中的甲基化水平明显高于胶质瘤组织,且具有统计学差异(p<0.01)。在NimbleGen启动子区及CpG岛芯片上,LRRC4基因探针覆盖区位于其翻译起始位点上游-1472bp至-2218bp一个CpG岛处,芯片筛选结果显示该区域在胶质瘤中不存在甲基化。为了进一步证实我们克隆的LRRC4基因启动子活性区(其翻译起始位点上游-835bp至-293bp)在胶质瘤中的甲基化状态,我们采用MassArray检测系统在上述40例胶质瘤组织,11例正常脑组织及4株胶质瘤细胞系进行了检测。结果显示该区域在正常脑组织、胶质瘤组织及胶质瘤细胞系中平均甲基化率分别为15.47%±3.96%,38.29%±21.69%及68.57%±25.46%。LRRC4基因的启动子活性区域(-835bp至-293bp)在胶质瘤组织和胶质瘤细胞系中甲基化水平明显高于正常脑组织,且具有显著统计学差异(p<0.01),该结果与前一章研究结果一致。因此,我们认为LRRC4基因确实是胶质瘤中的高甲基化基因,可以归结为胶质瘤DNA高甲基化谱基因之一。总之,通过本研究筛选出了胶质瘤中异常甲基化的基因,为胶质瘤DNA甲基化谱建立奠定了基础,同时也为寻找胶质瘤的诊断、治疗或预后等分子标志物提供线索。更多还原

【Abstract】 [Background of LRRC4]LRRC4 (GenBank accession number is AF196976), a novel member of LRR superfamily, was identified by us at 7q31-32 employing a strategy of combination EST-mediated screening and 5’RACE. We found that LRRC4 specifically expresses in human and mouse normal brain tissues by Northern-blot and RT-PCR assay. But its expression was not only deleted in several malignant glioma cell lines, but also deleted or down-regulated in 87.5% primary gliomas biopsies. It had the potential to suppress tumorigenesis of U251 malignant glioma cells in vivo and cell proliferation in vitro by a tetracycline-inducible expression system. It was also found that LRRC4 requires a functional LRR cassette domain to suppress U251 cell proliferation and invasion by regulating the ERK/AKT/NF-κB and JNK2/c-Jun/p53 pathways. In conclusion, LRRC4 is not only a brain-specific gene but also a tumor suppressor gene for glioma. In order to get further proof for a suppressor, molecular mechanism of LRRC4 down-regulation in glioma had been analyzed in primary gliomas and glioma cell lines. However, we did not find any genetic alteration such as mutation, deletion and rearragment in LRRC4 coding region.[Cloning and location of LRRC4 promoter]In order to explore the mechanism of LRRC4 down-regulation in glioma, we carried out research on LRRC4 transcription regulation. The LRRC4 promoter region in the 5’end of humans was predicted using the PromoterInspector and PromoterScan programs. The CpG island was found using CpGplot from the European Molecular Biology Open Software Suite. The three program prediction results have some overlapping region. Then a genomic DNA fragment that spanned positions-2475 to-101 relative to the initiation codon ATG of LRRC4 was amplified by PCR. The PCR product was cloned into pGL3-enhancer luciferase report vector and was named as pGL3--2475/-101. Luciferase activity analysis showed that pGL3 -2475/-101 had similar luciferase activity as pGL3-control.The result suggested that the region-2475/-101 include the LRRC4 promoter. In order to further locate the LRRC4 promoter, four deletion constructs were created (pGL3-1483/-101, pGL3-835/-101, pGL3-293/-101 and pGL3-835/-293), originating from the construct pGL3-2475/-101. Then they were transfected into Cos7 and Hela cells and we found the region-835/-293 was necessary for LRRC4 promoter activity. In order to verify the result, we constructed pGL3-835/-293/eGFP plasmid and it was transfected into Cos7 and Hela cells. The result showed that eGFP expression could be drived by the sequence-835/-293 in Cos7 and Hela cells. In a word, we successfully cloned and located the LRRC4 promoter through the study above.[Methylation of LRRC4 promoter in glioma】Bioinformatics analysis shows that the LRRC4 promoter region has no TATA box or CAAT box. But it has high G/C content (approximately 70%) and characteristics of a CpG island. The result suggested the possibility that LRRC4 might be regulated through changes in the methylation status. Two glioma cell lines,30 primary glioma biopsies and three tissue specimens of normal brain were examined for LRRC4 methylation by MS-PCR. Both SF767 and SF126 cell lines showed complete methylation of the LRRC4 promoter. And the LRRC4 promoter was free from methylation in the three normal brain tissue samples, but was methylated to different extents in the 30 primary gliomas. To determine a more detailed map of the methylation in the LRRC4 promoter, we performed bisulfite sequencing around the promoter region of the LRRC4 gene in two glioma biopsies, two glioma cell lines and a normal brain tissue studied above. The results revealed densely methylated CpG sites within the promoter regions compared to normal brain tissue. All the results suggested that LRRC4 methylation is a tumor-specific event. It may distinguish tumors from normal tissue and serve as a promising biomarker for diagnosis. 【LRRC4 expression induced by 5-Aza-CdR]To demonstrate a functional association between LRRC4 promoter methylation and its gene inactivation, a DNA demethylating agent, 5-Aza-2’-deoxycytidine (5-Aza-CdR), was used to treat SF126 and SF767 cell lines. Methylation-specific PCR was used to examine the methylation status changes of LRRC4 promoter in SF126 and SF767 cell lines. LRRC4 mRNA expression in SF126 and SF767 cell lines treated by 5-Aza-CdR was detected by RT-PCR. The results indicated that LRRC4 promoter aberrant hypermethylation can be reversed and LRRC4 expression can be induced by 5-Aza-CdR. The results demonstrated that LRRC4 promoter methylation suppressed its expression and promoter methylation is the important molecular mechanism of LRRC4 inactivation in glioma. At the time, we also observed the effect on glioma cell lines cell growth and cell cycle of 5-Aza-CdR.5-Aza-CdR displayed a growth inhibitory effect on SF126 and SF767 cells and cell cycles were blocked at G0/G1 phase after 5-Aza-CdR treatment. Taken together, glioma cell lines SF126 and SF767 cell growth could be inhibited and cell cycles could be blocked by 5-Aza-CdR; methylation can be reversed and LRRC4 expression can be induced by 5-Aza-CdR. All these data suggests that LRRC4 may serve as a demethylation therapeutic target in glioma.[Methylation interruptting SP1 and E2F1 binding with LRRC4 promoter]To explore the mechanism of promoter methylation-mediated LRRC4 inactivation in glioma, transcription factors binding sites of LRRC4 promoter was analyzed by MatInspector program. There were several transcription factors in LRRC4 promoter. Five transcription factors binding sites were chosen for EMSA. However, specific binding bands could be detected only at E2F1(-655/-631) and SP1(-568/-547) binding sites. The result implied that E2F1 and SP1 involved in LRRC4 transcription regulation. In order to observe the effect on transcription factors binding of LRRC4 promoter methylation, oligonucleotide probes for E2F1(-655/-631) and SP1(-568/-547) binding sites were treated by methylase M.SssI and EMSA were carried out. Previous specific binding band disappeared. The results suggested that DNA methylation interrupted transcription fators E2F1 and SP1 binding with DNA. We also performed CFHP to verify the result in vivo. In glioma cells untreated by 5-Aza-CdR, E2F1 and SP1 binding with LRRC4 promoter could not be observed. However, when glioma cells were treated by 5-Aza-CdR for four days, we could observe the E2F1 and SP1 binding with LRRC4 promoter. Therefore we draw the conclusion that LRRC4 transcription are regulated by transcription factors E2F1 and SP1 and methylation interferes E2F1 and SP1 binding with LRRC4 promoter, which suppresses LRRC4 trancription. [Histone modification correlated with LRRC4 promoter methylation]Both DNA methylation and histone modification are not isolated epigenetic events. There are cross-talks between them. In order to analyze histone modifation correlated with LRRC4 promoter methylation, we carried out CHIP in glioma cells treated or untreated by 5-Aza-CdR using H3 acetylation, trimethyl-H3(lys4) and trimethyl-H3(Lys9) antibodies. In glioma cells untreated by 5-Aza-CdR, LRRC4 promoter could be amplified only from DNA immunoprecipitated by trimethyl-H3 (Lys9) antibodies. In glioma cells treated by 5-Aza-CdR for four days, the level of trimethyl-H3(Lys9) around LRRC4 promoer was decreased while the level of H3 acetylation and trimethyl-H3(lys4) were increased. On the basis of these data, we draw the following conclusions that trimethyl-H3(Lys9) is correlated with LRRC4 promoter methylation, which suppresses LRRC4 expression and H3 acetylation and trimethyl-H3(lys4) are correlated with LRRC4 promoter demethylation, which activates LRRC4 expression.DNA methylation patterns appear to have tumor-type specificity. Genome-wide different CpG island methylation status in different type tumor constructs tumor-specific DNA methylation profile. Geome-wide profile of DNA methylation in gliomas not only contributes to uncover all around the molecular mechanism of glioma initiation and development, but also provide very important and valuable clue such as biomarker for early diagnosis and prognosis, or useful target for therapy. Though some progress has been made in research on methylation genes in gliomas, candidate genes approach was adopted in most study to aim at several or a flock of genes. So far, no study is aimed directly at genome wide to analyze completely and systematically aberrant methylation genes in gliomas. In some degree, lacking high-throughput methods is the bottleneck in this investigation.In this study, a novel and high-throughput technique called MeDIP combination with CpG island microarrays were applied to screen aberrant methylation genes in 6 glioma biopsies(T1,T2,T3,T4,T5,T6) and 4 normal brain tissue(N1,N2,N3,N5). Under our standardization, we identified 562 hypermethylation loci and 108 hypomethylation loci, including 325 hypermethylation genes and 74 hypomethylation genes. They distribute evenly on every chromosome and susceptible chromosome is not found. The differential methylation genes involve in extensive biological process such as cell communication, signal transduction, cell adhesion, cell migration, apoptotic program, metabolic process, transport, post-translational protein modification, nervous system development, and etc. At the same time they also refer to several cell pathways such as MAPK signaling pathway, Wnt signaling pathway, Jak-STAT signaling pathway and Cell cycle. Therefore, the differential methylation genes may contribute to initiation and development of glioma.In order to validate the results of microarrays,8 hypermethylation genes (ANKDD1A, SST, SIX3, GAD1, PHOX2B, HIST1H3E, PCDHA13 and PCDHA8), whose methylation region is a CpG island and locate in promoter, were chosen to verify and investigate in 40 glioma biopsies,11 normal brain tissue and 4 glioma cell lines using MassArray system. ANKDD1A, SST, SIX3, GAD1, PHOX2B, HIST1H3E, PCDHA13 and PCDHA8 methylation level in normal tissue is 13.29%±2.12%,9.72%±2.42%,16.34%±4.13%, 11.81%±3.76%,23.06%±4.49%,21.39%±3.13%,31.46%±4.28% and 31.66%±12.77% respectively. However, ANKDD1 A, SST, SIX3, GAD1, PHOX2B, HIST1H3E, PCDHA13 and PCDHA8 methylation level in glioma biopsies is 36.24%±23.71%,35.08%±19.44%,51.36%±21.25%, 29.22%±15.13%,50.38%±19.15%,41.67%±25.56%,55.77%±21.77% and 69.42%±19.49% respectively; and in glioma cell lines is 76.14%±27.99%,47.64%±31.61%,72.55%±25.49%,62.21%±16.80%, 22.44%±18.00%,63.05%±20.35%,58.09%±30.00% and 55.00%± 21.29% respectively. Except PCDHA13, methylation level of genes in glioma biopsies and glioma cell lines is significantly increased compared to normal brain tissue (p<0.01). Except PCDHA8 and PCDHA13, methylation level of genes in glioma cell lines is higher than those in glioma biopsies(p<0.01).There is a group of probes against LRRC4 on CpG island microarray of NimbleGen. The probes covering region is a CpG island and located LRRC4 translation initiation site upstream from 1472bp to 2218bp. The CpG island microarray screening results showed that the region aginst LRRC4 is unmethylated in glioma. To furtherly verify methylation of LRRC4 active promoter region spanned positions-835 to-293 relative to the initiation codon ATG of LRRC4 in glioma, MassArray is also carried out to detect this region in 40 glioma biopsies,11 normal brain tissue and 4 glioma cell lines studied above. The LRRC4 promoter average methylation level is 15.47%±3.96%,38.29%±21.69% and 68.57%±25.46% in normal brain tissue, glioma biopsies and glioma cell lines, respectively. Methylation level of LRRC4 active promoter in glioma biopsies and glioma cell lines is significantly increased compared to normal brain tissue (p<0.01). The result is in accordance with our previous study. Based on the research, we can draw the conclusion that LRRC4 is one of the methylated genes in glioma and we can classify LRRC4 into DNA methylation profile of glioma. Taken together, we have identified aberrant methylation genes in gliomas. This not only lays a foundation for mapping geome-wide aberrant DNA methylation in gliomas, but also provides important clues to find biomarker for diagnosis and prognosis, or useful target for therapy.更多还原