【作者】 徐小延；

【导师】 袁正伟；

【作者基本信息】 中国医科大学 ， 遗传学， 2018， 博士

【摘要】 目的:唇腭裂是一种常见的先天性畸形,其发生率约为1‰~4‰,我国为1.84‰。疾病分型包括单纯性唇裂、单纯性腭裂和唇腭裂;而根据其是否伴有其他组织结构的异常,可划分为综合征型唇腭裂(syndromic cleft lip with or without cleft palate,SCL/P)和非综合征型唇腭裂(nonsyndromic cleft lip with orwithout cleft palate,NSCL/P),二者分别占唇腭裂总数的30%和70%。唇腭裂病因尚不清楚,是环境因素和遗传因素相互作用的复杂多基因疾病。若能找到该病的致病位点或易感基因,对于研究其发生机制和胚胎早期诊断、干预都具有十分重要的意义。DNA甲基化是最早发现的基因表观修饰方式之一,DNA甲基化在大多数情况下可以抑制基因的表达,反之去甲基化则可以使基因重新活化和表达。目前,DNA甲基化检测技术有中低通量的BSP(Bisulfite Sequencing PCR)和MSP(Methylation-Specific PCR)、中高通量的MethyLight和高通量的全基因组甲基化分析(ChIP on chip)。本课题我们利用甲基化芯片技术筛查单纯性唇裂(NSCL)胎儿局部病变组织的异常甲基化位点,并利用焦磷酸测序技术进行大样本验证初步确定候选基因,最后在大鼠成肌细胞系中进行候选基因的功能鉴定,期望能找出单纯性唇裂的相关基因,为今后的产前诊断提供依据。研究方法:1、标本收集。(1)NSCL组:2012年12月之前于中国医科大学附属盛京医院产前诊断中心诊断为NSCL的引产胎儿病变组织,共16例。孕周为22-30周。(2)正常对照组:选取周龄与NSCL组相匹配的非唇腭裂引产胎儿唇部组织,共15例。2、甲基化芯片技术筛查NSCL组织异常甲基化位点。选择周龄相匹配的NSCL组和对照组胎儿各3例(女性胎儿2例,男性胎儿1例),取唇部组织提取DNA,进行甲基化芯片检测。本实验中采用的是Illumina公司的450K Infinium Methylation BeadChip芯片。3、甲基化芯片结果生物信息学分析。应用MINI-R软件、GO注释、Pathway代谢通路注释、多样品间表达模式聚类分析等生物信息学方法对差异位点和基因进行功能分析。4、Real-time PCR对候选基因在组织标本中的表达差异分析。由于甲基化芯片结果筛选出大量甲基化异常位点,我们从二个标准确定候选基因:(1)以往文献报道和唇腭裂发生相关的基因,同时在本实验芯片结果中患者组存在异常高甲基化水平的位点,并且统计分析显示有较强显著差异(p<0.05|beta.difference|>0.14))的;(2)芯片结果甲基化水平差异的基因中,参与重要细胞通路的转录因子。在本实验中,我们先采用Real-time PCR的方法扩大样本量对这上述标准筛选出的候选基因在16例患者组和15例对照组中的表达情况进行检测。5、焦磷酸测序技术对候选基因异常甲基化位点的验证。对于芯片结果甲基化水平异常且Real-time PCR验证表达下调的的基因相关位点,设计引物,提取DNA,用甲基化转化试剂盒进行转化、纯化。以转化后的DNA样本做模板,进行PCR。取10μl PCR产物上PyroMark Q96实时定量焦磷酸系列分析仪,导出实验结果并分析。6、Western Blot和免疫组化技术检测候选基因在患者与正常对照组中的表达。取患者组织16例和对照组15例,采用Western Blot方法验证候选基因在患者组和对照组之间的蛋白表达差异。同时将其余组织固定用于免疫组化染色,检测候选基因在病变组织中的表达。7、大鼠成肌细胞中候选基因的功能研究。利用siRNA-脂质体3000转染大鼠成肌细胞系,干扰Dab1基因的表达,然后利用Real-time PCR、Western Blot检测转染后12h、24h不同时间RNA、蛋白表达情况,确定对Dab1基因沉默的效率。利用细胞划痕实验、Transwell方法检测Dab1基因沉默后细胞迁移能力的改变,利用流式细胞仪检测细胞凋亡的改变,利用MTT法检测细胞增殖率的改变。结果:1、甲基化芯片对病变组织标本中甲基化差异位点的筛选。(1)NSCL胎儿的病变组织与正常对照组织相比存在大量的甲基化异常位点,其中高甲基化位点3661个,低甲基化位点1218个。这些甲基化异常位点存在于2849个基因之中,在除了Y染色体以外的各条染色体上均有分布。众多的异常高甲基化位点所在基因参与了重要生物学功能,可能导致了一部分相关基因的表达异常,从而直接或者间接影响了胚胎发育过程中颅面部的形成。从位点分布的位置来看,在筛选出来的甲基化水平差异位点中,分布于基因本体区、CpG岛的位点占很大比例,另外也有一些在TSS1500区域和岛前区域。(2)在已有研究报道和唇腭裂发病相关的基因中,结合我们的芯片结果,显示有异常高甲基化水平(p<0.05)的基因有MSX1、BMP7、EGFR、PAX9、GLI2、IRF6。(3)通过检索Pathway的主要公共数据库分析差异基因参与的主要信号转导通路中,REELIN细胞通路中的二个关键转录因子(DAB1和FYN)在患者组均存在高甲基化的位点;鉴于REELIN为该通路的主要成员,因此在后续研究中我们同时将REELIN与DAB1、FYN同时列为研究对象。2、Real-time PCR检测候选基因mRNA在病变组织中表达情况。利用Real-time PCR的方法对9个候选基因在病变组织中的表达情况进行检测,标本来源为16例患者组和15例对照组。结果表明DAB1、FYN、REELIN基因在NSCL胎儿病变组织中的表达情况与对照组相比,患者组表达下调且有显著差异(p<0.05);MSX1、GLI2、IRF6基因表达上调,有显著差异(p<0.05);BMP7、EGFR、PAX9则没有显著差异。3、扩大样本量焦磷酸测序验证芯片结果。我们对Real-time PCR结果中显示表达显著下调的两个基因(DAB1和FYN)的甲基化异常相关位点,进行了焦磷酸测序验证,标本来源为16例患者组和15例对照组。结果显示位于DAB1基因体区CpG岛的二个cg位点甲基化水平存在显著差异(P<0.05);相关性分析结果表明:这二个位点中的一个cg位点,其甲基化程度与DAB1基因的表达水平呈负相关(P<0.01,r=-0.6076)。即患者组存在高甲基化现象,并且基因表达量在组织中下调,而FYN的相关位点的甲基化水平差异并没有统计学意义。4、Western Blot和免疫组化检测候选基因的蛋白表达情况。应用Western Blot和免疫组化的方法对病变组织中的DAB1和FYN蛋白表达进行检测,结果发现在患者组织中两个蛋白表达量均显著降低(P<0.001)。5、大鼠成肌细胞中Dab1基因沉默效率检测。利用Real-time PCR和Western Blot对大鼠成肌细胞系进行Dab1基因siRNA沉默12h、24h、48h后进行RNA、蛋白表达水平检测,结果显示转染后24h mRNA表达水平最低,48h蛋白表达量最低。6、大鼠成肌细胞Dab1基因沉默后细胞迁移能力、凋亡、增殖情况的检测。细胞划痕实验、Transwell迁移实验结果均提示Dab1基因被沉默后,细胞的迁移水平被抑制;流式细胞仪检测凋亡,结果表明凋亡水平没有明显改变;MTT法检测细胞增殖率结果表明,增殖率也没有差别。所以提示Dab1基因表达水平的抑制,影响了局部细胞的迁移能力,从而导致了面部形成过程中组织的融合异常。结论:1、NSCL胎儿的病变组织存在大量的甲基化异常位点,在除Y染色体以外的各条染色体上均有分布。甲基化异常位点主要分布于基因本体区和CpG岛,少部分分布在TSS1500区域和岛前区域。2、REELIN信号通路基因表达异常与NSCL发生有关。3、NSCL胎儿的病变组织中DAB1基因异常的高甲基化导致DAB1低表达是NSCL病理改变发生的原因之一。4、siRNA干扰大鼠成肌细胞中Dab1基因的表达,可引起细胞的迁移能力明显降低,但对凋亡、增殖方面没有影响。更多还原

【Abstract】 Objectives:Cleft lip（CL）is a common congenital deformity with 1‰-4‰morbidityis.In China,its morbidity at birth is about 1.84‰.The category of CL includes simple cleft lip,simple palate cleft and cleft palate and lip.According to whether it is accompanied with abnormality of other tissues,it can be categorized as syndromic cleft lip with or without cleft palate（SCLP）and nonsyndromic cleft lip with or without cleft palate（NSCLP）,which takes up to 30%and 70%respectively of the total amounts of CL/P.The pathogenicity of cleft palate and lip is very complicated since it includes the interaction of environment and genetics and also is regulated by many factors.If the pathogenic locus or susceptibility genes of CL could be found,it will be very helpful to the early diagnosis,risk forecasting,prevention and intervention of nonsyndromic cleft lip（NSCL）.DNA methylation is one of the epihenetic modifications found in genome.DNA methylation can suppress gene expressions normally,whereas demethylation can induce the re-activations and expressions of genes.Till now,technologyies used in DNA methylation inspection included Bisulfite Sequencing PCR（BSP）method,Methylation-Specific PCR（MSP）method,MethyLight method and ChIP onchip,etc.among them,CHIP onchip has become an important platform for candidate genes screening of multe-genes complicated genetic diseases.In the study,we applied the methylation chip technology to screen the abnormal methylation locus in partial lesion tissues from NSCL fetus and the candidate locus were preliminarily validated through pyrosequencing among enlarged samples.Functions of candidate genes were studied in primary rat myoblast.It is expected to confirm the candidate genes of NSCL for future prenatal diagnosis.Methods:1.Samples Collection.（1）NSCL Group:16 lesion issues from odinopoeia NSCL fetus diagnosed by Prenatal Diagnosis Center of 2^nd Affiliated Hospital of CMU before 2012 Dec,whose gestational weeks were from 22 to 30.（2）Normal control Group:15 lip tissues from non-CL odinopoeia fetus with matched gestational weeks.2.Screening of the abnormal methylation lotus using chip technology.3 cases were selected（2 female fetuses cases,1 male fetus case）from NSCLgroup and control group respectively.DNA was extracted from cleft tissues and performed methylation chip.The Illumina 450K Infinium Methylation BeadChip was utilized in the experiment.3.Bioinformatics analysis of methylation chip results.MINI-R software,GO annotation,Pathway metabolic pathway annotation,cluster analysis of multi-samples expression modes and other bioinformatics were applied for functional analysis on candidate genes.4.Detection of candidate genes expressions in tissues samples by Real-Time PCR.We identified the candidate genes based on two criteria.First,the genes that have ever been reported to be related with CL morbidity and the ones which showed significantly different methylation level in our chip results with strong significant difference per statistical analysis（p<0.05,|beta.difference|>0.14）.Second,those that play important role in cell pathway.In our experiment,we used RealTime-PCR method to detect the expression of candidate genes in 16 cases of NSCL group and 15 cases of control group.5.Validation of abnormal methylation genes using pyrosequencing.DNA was extracted and purified by methylation conversion Kit.PCR was run with conversed DNA samples as templates:10μL of PCR products were analyzed on the PyroMark Q96 real-time quantitate pyrophosphoric acid series analysis meter.6.Candidate gene proteins were detected with Western Blot and immunohistochemistry in the NSCL and control groups.Protein levels from NSCL group and control group were validated by Western Blot technology.At the same time,lesion tissues were utilized using immunohistochemistry.7.Functional studies of candidate genes in primary rat myoblast.Dab1 gene expression in primary rat myoblast was silenced by SiRNA l.Transfection efficiency at different time was measured by Real-Time PCR and Western Blot method.Cell migration capability was detected by cell wound scratch assay and transwell method.Cell apoptosis level was detected by flow cytometry.Cell proliferation capability was detected by MTT method.Results:1.The screening of methylation discrepancy genes in the lesion tissue samples by methylation chips.（1）There existed lots of abnormal methylation genes in the NSCL odinopoeia fetus comparing with the normal tissues,among which there were 3661 high methylation gene locus and 1218 low methylation gene locus.These loci distributed in2849 genes and spread widely in chromosomes.Most abnormal methylation genes were distributed in the gene noumenon region and CpG islands.There were also some abnormal methylation level genes located in TSS1500 and island shore.（2）Several genes that showed abnormal methylation level（p<0.05）included MSX1,BMP7,EGFR,PAX9,GLI2 and IRF6 have been reported to be related to CLP morbidity.（3）Two key transcription factors in REELIN cell pathway,both DAB1 and FYN,showed high methylation levels.In view of REELIN being the key member of the pathway,we will regard REELIN,DAB1 and FYN all as research objects in the following research.2.The mRNA expression of candidate gene was detected in tissue lesions by Real-Time PCR.We used real-time PCR to detect the expression of 9 candidate genes in tissue lesions.The result showed that DAB1,FYN and REELIN genes expression were decreased in NSCL group（p<0.05）.MSX1,GLI2 and IRF6 gene expression levels were increased in NSCL group（p<0.05）.BMP7,while no difference was found fir EGFR and PAX9 genes.3.The chip results were validated by pyrosequencing.We enlarged the samples numbers and pyrosequenced DAB1 and FYN which showed strongly significant decrease in NSCL samples.The result showed that there really existed significant methylation level discrepancy（p<0.05）for DAB1 gene.The correlation analysis result showed that the methylation degree of one of the two loci presented negative relationship with the DAB1gene’s expression level（P<0.01,r=-0.6076）.Namely,there existed high methylation phenomenon in NSCL group compared with control group,while there was no statistical significance for FYN.4.DAB1 and FYN proteins in the lesion tissues were detected using Western Blot and immunohistochemistry.In our study,we detected the protein level of DAB1 and FYN in tissues using Western Blot and immunohistochemical,and the protein showed significantly decreased in NSCL tissues（P<0.001）.5.The silence efficiency of siRNA-Dab1 was measured in primary rat myoblast.The Dab1 levels were detected 12 hrs,24 hrs and 48 hrs after SiRNA-Dab1 transfection.The RNA expression level reached the lowest level at 24 hrs after transfection.Dab1 protein expression is mostly inhibited at 48 hrs after transfection.6.The measurement of cell transfer capability and apoptosis status of primary rat myoblast after DAB1 gene was inhibited.The cell wound scratch assay and Transwell assay results indicated that the cell transfer level was suppressed after the DAB1 gene was silenced.The apoptosis detection result by streaming cell meter showed no change in apoptosis level.The result of cell proliferation inspected by MTT method indicated that there was neither difference in proliferation.So it can be assumed that the high methylation of DAB1 gene may affect its expression level,which could influence cellular transfer capability and result in the tissue fusion abnormality during facial formation.Conclusion:1.There were plenty of abnormal methylation loci widely spread in genome in NSCL lesion tissues.The abnormal methylation loci mainly distributed in gene noumenon region and CpG Island,few was found in TSS1500 and island shore.2.The abnormal gene expression of REELIN signals pathway is related with NSCL morbidity.3.The low DAB1 expression resulted from its abnormal high methylation in NSCL lesion tissue is one of the factors related to the occurrence of NSCL.4.In primary rat myoblast,decrease of Dab1 gene expression can result in the significant reduction of cell migration capability,but it has no impact on apoptosis and proliferation.更多还原