Advances in microfluidic-based DNA methylation analysis

DNA methylation has been extensively investigated in recent years,not least because of its known relationship with various diseases.Progress in analytical methods can greatly increase the relevance of DNA methylation studies to both clinical medicine and scientific research.Microflu-idic chips are excellent carriers for molecular analysis,and their use can provide improvements from multiple aspects.On-chip molecular analysis has received extensive attention owing to its advantages of portability,high throughput,low cost,and high efficiency.In recent years,the use of novel microfluidic chips for DNA methylation analysis has been widely reported and has shown obvious superiority to conventional methods.In this review,wefirst focus on DNA methylation and its applications.Then,we discuss advanced microfluidic-based methods for DNA methylation analysis and describe the great progress that has been made in recent years.Finally,we summarize the advantages that microfluidic technology brings to DNA methylation analysis and describe several challenges and perspectives for on-chip DNA methylation analysis.This review should help researchers improve their understanding and make progress in developing microfluidic-based methods for DNA methylation analysis.

Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips

To understand how differentially methylated genes（DMGs）might affect the pathogenesis of Kashin-Beck disease（KBD）.Genome-wide methylation profiling of whole blood from 12matched KBD and controls pairs was performed using a high-resolution Infinium 450 K methylation array.In total,97 CpG sites were differentially

Experimental Study on Malignant Transformation of Human Bronchial Epithelial Cells Induced by Glycidyl Methacrylate and Analysis on its Methylation

Objective To establish the model of human bronchial epithelial cells （16HBE） malignant transformation induced by glycidyl methacrylate （GMA） and define the different methylation genes at different stages. Methods DNA was extracted at different 16HBE malignant phases and methylation at different stages were detected using Methylation chip of Promoter Microarray Methylation＇. Methylation-specific PCR （MSP） was methylation status of some genes, and then compared with the control groups. changes of genes DNA ＇NimbleGen HG18 CpG used to observe the Results The result showed that GMA induced 16HBE morphorlogical transformation at the dose of 8 I^g/mL, and cell exposed to GMA had 1 374 genes in protophase, 825 genes in metaphase, 1 149 genes in anaphase, respectively; 30 genes are all methylation in the 3 stages; 318 genes in protophase but not in metaphase and anaphase; 272 genes in metaphase but not in protophase and anaphase; 683 genes in anaphase but not in metaphase and protophase; 73 genes in protophase and metaphase but not in anaphase; 67 genes in protophase and anaphase but not in metaphase; 59 genes in metaphase and anaphase but not in protophase. Conclusion The pattern of DNA methylation could change in the process of 16HBE induced by GMA.

Methylation Profile Difference in Human High-Metastatic Large Cell Lung Cancer Cell Lines with Different Metastatic Potential

Background and Objective Invasion and metastasis is one malignant phenotype of lung cancer and cause the death of lung cancer patients. Present evidence has proved that invasion

A novel method for fabricating polydimethylsiloxane microfluidic chip master molds

We proposed a novel method of fabricating polydimethylsiloxane (PDMS) microfluidic chip polymer master molds in this paper. The method mainly includes two steps. First, a stainless steel slice was laser etched to form a metal model. Then, the organic solution of poly(methyl methacrylate) (PMMA) was casted onto the metal model to fabricate the PMMA master which subsequently would be used to fabricate PDMS chips. We systematically researched different laser parameters influencing the surface status of microchannels and obtained optimized etching parameters. We investigated and optimized the organic solution composition of PMMA while casting chip masters, and developed a method to form fine polymer masters using two different viscosity solutions to cast the model in turn, and studied the repeatable replication. Then, we investigated physical performance of this chip and evaluated the practicability by analyzing Rhodamine B. Compared with present methods, the proposed method does not need photolithography on photoresistant and chemical etching. The entire fabricating progress is simple, fast, low-cost and can be controlled easily. Only several minutes are required to make a metal model, 3 hours for a PMMA master, and one day for PDMS chips.

Active Methyl Cycle and Transfer Related Gene Expression in Response to Drought Stress in Rice Leaves

Three rice varieties, Zhonghan 3, Shanyou 63 and Aizizhan, were used as materials in detecting differential active methyl cycle and transfer related gene expression in response to drought stress. The experiment was performed by gene chip and mRNA differential display technologies under the conditions of drought simulated with 10% PEG6000 solution. The results indicated that the methyl cycle could be activated in the leaves of Zhonghan 3 and Shanyou 63 but inhibited in the leaves of Aizizhan under drought stress. Furthermore, drought stress could induce the expression of a large number of methyltransferase genes, especially the transcription of Rubisco protein methylation related genes, which are beneficial for prevention of Rubisco protein oxidation and degradation, and drought stress could inhibit the transcription of DNA methyltransferase genes and histone methyltransferase genes. This result confirmed that the active methyl cycle and transfer related genes were involved in rice drought resistance.

Phosphate-Methylated Oligonucleotides Past, Present and Future

At the moment, we see a great interest for application of Anti Sense Oligonucleotides (ASOs) in order to regulate the expression of genes related to certain diseases. These nucleotides obtained a number of fascinating properties by means of chemical manipulation of natural DNA and RNA under conservation of Watson-Crick base-pairing. About 35 years ago for our research in this field, we selected synthetically (short) phosphate-methylated DNA and RNA. It was concluded that there is an exclusive selection in hybridization affinity with natural DNA and RNA. These (bio)chemical and physical-chemical properties are extensively published. ASOs have found their way in public health as is clearly shown in the treatment of (progressive) neurological diseases. We focus specifically on the past, present and future of the phosphate-methylated oligonucleotides, illustrated with different research studies in chemistry and biophysics. A new field of application of modified DNAs is based on interactive improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing phosphate-methylated DNA as probe.

冠心病不稳定型心绞痛气虚血瘀证“病”与“证”DNA甲基化差异表达谱分析

目的 探讨冠心病(CHD)所致不稳定型心绞痛气虚血瘀证患者外周血DNA甲基化位点/区域的差异表达谱及潜在分子机制,为冠心病病证结合研究提供科学依据。方法 根据预先确定的纳入和排除标准,将研究对象分为两组,即CHD诱发的不稳定型心绞痛组(G组)和健康对照组(J组),进行“病”分析,而不稳定型心绞痛患者进一步分为气虚血瘀证组(病例组)和非气虚血瘀证组(对照组)进行“证”分析。收集研究对象的一般资料和临床信息,空腹抽取外周静脉血,采用850K甲基化芯片检测各组DNA甲基化差异表达谱。使用Ch AMP软件(V 2.14.0)进行差异甲基化数据分析,阈值为校正后P <0.01。采用基因本体论(GO)和京都基因组百科全书(KEGG)数据库对相关映射基因进行功能和通路富集分析。结果 G组和J组比较,得出代表“病”的差异甲基化表达谱,共筛选出263个差异甲基化位点(DMPs),其中包括cg05845204、cg08906898等191个高甲基化位点和cg26919182、cg13149459等72个低甲基化位点。这些位点主要映射到148个基因,包括RNA结合基序蛋白39(RBM39)、乙酰辅酶A酰基转移酶2(ACAA2)、蛋白磷酸酶1调节亚基12B(PPP1R12B)和双特异性酪氨酸磷酸化调节激酶2(DYRK2)。GO功能富集分析结果显示DMPs基因主要富集于染色体蛋白质定位、细胞形态发生调控、钙介导信号负向调控等方面。KEGG通路分析结果提示这些基因主要富集于脂肪酸代谢和内吞途径。此外,共鉴定出23个代表“病”的差异甲基化区域(DMRs),并识别出跨膜蛋白232(TMEM232)、核糖体蛋白P1(RPLP1)、过氧化物酶体发生因子10(PEX10)和叉头蛋白N3(FOXN3)等重叠基因,GO功能主要富集于Ras蛋白信号转导的负向调节和小GTP酶介导的信号转导、负向调节等方面。病例组与对照组比较获得代表“证”的差异甲基化表达谱,共筛选出1 703个“证”相关DMPs,包括cg05573767等444个甲基化升高位点和1 259个甲基化降低位点,例如cg19938535和cg03893872。这些位点映射到1 108个基因,例如核糖体蛋白S6激酶A2(RPS6KA2)、亮氨酸重复序列16A(LRRC16A)和刺猬酰基转移酶(HHAT)。GO功能富集分析,差异甲基化位点相关基因主要富集于跨膜受体蛋白丝氨酸/苏氨酸激酶信号通路、轴突发生等生物学功能。KEGG通路富集分析结果提示Rap1信号通路、5’-单磷酸腺苷激活蛋白激酶(AMPK)等信号通路参与了证候发展。研究共筛选出21个“证”相关DMRs,包括22个重叠基因,如粘蛋白4(MUC4)、三素修复核酸外切酶1(TREX1)和LIM同源盒6(LHX6)。GO富集分析发现主要参与正向调节跨膜转运、调节脂肪酸代谢和铜离子结合等分子功能。结论 本研究揭示了冠心病不稳定心绞痛气虚血瘀证患者DMPs和DMRs的甲基化特征,脂肪酸代谢、Rap1信号通路和其他分子功能的潜在表观遗传调控参与了冠心病“病”和“证”的发展。

基因芯片在畜禽遗传育种中的应用及展望

基因芯片是一种通过DNA双链或DNA-RNA互补杂交检测特定DNA序列的高通量技术,其中SNP基因分型芯片已经广泛用于畜禽的遗传育种工作,在牛(Bos taurus)、猪(Sus scrofa)、羊(Caprinae)、鸡(Gallus gallus)等畜禽中取得了重大成就。但是在实际生产中使用的基因组选择仅利用了基因组信息,无法完全解释复杂性状的分子遗传基础,限制了基因组选择的准确性。随着表观遗传学研究的不断深入、商用甲基化芯片的推出、表观基因组关联分析(epigenome-wide association study,EWAS)的提出,DNA甲基化已被广泛用于解释遗传与表型的因果关系。未来,有望开发专门针对畜禽的甲基化芯片,通过EWAS探索与畜禽经济性状显著相关的甲基化位点,深化对复杂性状因果变异的理解。结合甲基化芯片与SNP芯片捕获畜禽表观基因组和基因组信息,更准确地解读遗传变异,提高基因组选择的准确性,推动畜禽分子遗传育种工作的精细化发展。本文综述了SNP芯片在畜禽上的应用,并对甲基化芯片在畜禽上的应用进行了展望,以期为基因芯片在动物育种中的进一步应用提供借鉴和参考。

小剂量X射线对小鼠全基因组CpG岛甲基化水平的影响

目的探讨小剂量X射线(LDR)暴露对小鼠启动子CpG岛甲基化水平的影响。方法 BALB/c雄性小鼠20只按随机数字表法均分为2组:对照组和分次0.5 Gy(0.05 Gy/d×10 d)照射组。末次照射后2 h摘除眼球取血。采用Roche-NimbleGen小鼠甲基化DNA免疫共沉淀-芯片(MeDIP-chip),分析小鼠全血基因组启动子CpG岛甲基化改变,并利用MeDIP-qPCR(甲基化DNA免疫共沉淀—实时定量PCR)方法检测目的基因启动子区域DNA甲基化水平。结果与对照组相比较,分次照射组全血基因组共筛选出811个基因启动子区甲基化水平存在显著差异(P<0.05)。这些基因涉及几乎所有主要生物学过程。经MeDIP-qPCR验证,挑选出的8个基因(Rad23b、Tdg、Ccnd1、Ddit3、Llgl1、Rasl11a、Tbx2、Slc6a15)的甲基化水平与芯片结果一致。结论 LDR诱导特定基因的CpG岛发生高甲基化,可能参与LDR损伤的分子机制。

溴甲烷对松木片中松材线虫的熏蒸作用

采用二次正交旋转组合设计,在室内条件下研究了溴甲烷剂量、熏蒸处理时间和处理温度对松木片中松材线虫熏蒸效果的影响。结果表明,溴甲烷剂量是影响熏蒸效果的最主要因子;14、18℃和22℃条件下处理24h,松材线虫死亡率达99.9%时的溴甲烷浓度分别为17.92、16.07、14.87g/m3;14、18、22℃条件下,松材线虫死亡率达99.9%时,溴甲烷浓度和时间乘积,即CT(99.9)分别为192.52、174.705、166.229g.h/m3。溴甲烷可用于口岸松木片中松材线虫的除害处理。

微流控芯片检测肺癌患者血浆中p16基因异常甲基化在临床应用的评价

肺癌是常见的恶性肿瘤之一 ,其死亡率和发病率在世界范围的肿瘤性疾病中均居高不下 .p16基因启动子区异常甲基化被认为是肺癌发生中的一起早期事件 .为了提高检测异常甲基化方法的灵敏度及特异性 ,利用微流控芯片检测p16基因的异常甲基化 ,通过对肺癌患者血浆标本的检测 ,使病人血浆中的p16基因甲基化的异常改变可能成为辅助肺癌早期诊断和高危人群筛选的分子标记物 。

哈萨克族原发性高血压病患者全血DNA甲基化位点筛查及异常甲基化谱的构建

目的应用基因芯片技术筛选哈萨克族原发性高血压病(EH)患者异常DNA甲基化位点,初步构建哈萨克族EH异常甲基化谱,为深入研究哈萨克族EH的发生机制提供理论依据。方法 2014年6月—2015年10月,在新疆乌鲁木齐县随机抽取哈萨克族牧民或半农牧民3 042例为调研对象,从其中的1 487例EH患者中随机选取3例作为EH组,并选取年龄匹配的3例健康者作为对照组。采用Illumina Human Methylation 450K Bead Chip芯片对两组受试者的全血进行全基因组DNA甲基化检测,筛选DNA异常甲基化位点,采用Gene Ontology(GO)富集分析和Pathway分析了解异常甲基化基因的功能。结果 EH组和对照组间存在427个差异甲基化位点,其中高甲基化位点148个,低甲基化位点279个;这些位点位于不同的染色体上,其中以1号和6号染色体最多,Y染色体未见差异甲基化位点;GO富集分析结果显示,差异甲基化基因主要参与糖蛋白生物合成与代谢、血管发育、脉管系统发展、胶原蛋白结合等生物学过程;Pathway分析结果显示,差异甲基化基因主要参与了硫酸软骨素生物合成、胰岛素信号通路、胞吞等。结论发生EH时DNA甲基化状态发生改变,多条差异甲基化基因与EH相关通路有关,但本研究仅是哈萨克族EH全基因组甲基化谱的开端,异常甲基化基因作为发生EH的候选分子标志物,若要获得更全面、更精确的结果,还需要进一步扩大样本量进行分析及验证。

氢醌对人骨髓单个核细胞TOPO Ⅱα表达的影响及其可能机制

目的:观察苯代谢物氢醌(hydroquinone,HQ)对人骨髓单个核细胞拓扑异构酶Ⅱα(TOPOⅡα)表达的影响,探讨TOPOⅡα在HQ所致造血毒性中的作用及其调控机制。方法:50μmol/L HQ培养10h的正常人骨髓单个核细胞设为实验组,等体积灭菌蒸馏水培养10h为对照组。Western blot测定TOPOⅡα含量的变化,RT-PCR测定TOPOⅡαmRNA表达水平的改变,ChIP技术观察HQ对骨髓单个核细胞TOPOⅡα启动子组蛋白乙酰化、甲基化水平的影响。结果:①与对照组相比,人骨髓单个核细胞50μmol/L HQ处理10h后,TOPOⅡα含量、TOPOⅡαmRNA水平明显下降,差异有显著性(P<0.01);②TOPOⅡα含量降低伴随着TOPOⅡα启动子组蛋白H4乙酰化、组蛋白H3K4甲基化水平的明显降低(P<0.01)、组蛋白H3K9甲基化水平升高(P<0.05),不伴有组蛋白H3乙酰化水平的改变(P>0.05)。结论:HQ能抑制造血细胞TOPOⅡα的表达;组蛋白化学修饰可能在苯代谢物所致的造血毒性中发挥一定的作用。

TOPO Ⅱα启动子调控因子组蛋白甲基化修饰在苯中毒致造血毒性中的作用

目的:研究拓扑异构酶(TOPO)Ⅱα启动子调控因子(SP1、ATF-2、SP3、NF-YA、NF-M、P53、CMYB、C-JUN、ICBP90)组蛋白甲基化修饰在苯中毒致造血毒性中的作用。方法:25例临床慢性苯中毒患者骨髓单个核细胞为病例组,25例正常人骨髓单个核细胞为对照组,染色质免疫沉淀分析(Ch IP)技术探讨TOPOⅡα启动子各调控因子组蛋白甲基化水平的变化,RT-PCR法测定TOPO Ⅱα启动子各调控因子m RNA表达水平的改变。结果:1与对照组比较,病例组TOPO Ⅱα启动子调控因子NF-M、C-JUN组蛋白H3K4甲基化水平下降,差异有统计学意义(P<0.05),SP1、ATF-2、SP3、NF-YA、P53、C-MYB、ICBP90组蛋白H3K4甲基化水平无明显改变(P>0.05);SP1、NF-M组蛋白H3K9甲基化水平升高,差异有统计学意义(P<0.05或P<0.01),而ATF-2、SP3、NF-YA、P53、C-MYB、C-JUN、ICBP90组蛋白H3K9甲基化水平无明显改变(P>0.05)。2与对照组比较,病例组TOPO Ⅱα启动子调控因子SP1、NF-YA、C-MYB、NF-M及C-JUN m RNA表达水平降低,差异有统计学意义(P<0.05或P<0.01),ATF-2、ICBP90 m RNA表达水平不变(P>0.05),SP3、P53 m RNA表达水平则升高(P<0.05或P<0.01)。结论:1慢性苯中毒TOPO Ⅱα启动子调控因子组蛋白化学修饰水平的改变伴随着调控因子m RNA水平的变化。2TOPO Ⅱα启动子调控因子组蛋白甲基化修饰在苯中毒所致的造血毒性中发挥一定的作用。

异硫氰酸苯己酯降低U266细胞株p16基因甲基化的研究

本研究探讨异硫氰酸苯己酯对p16基因高甲基化的多发性骨髓瘤U266细胞株是否有去甲基化的作用。用异硫氰酸苯己酯0、5、10μmol/L孵育U266细胞株,提取DNA备用。用亚硫酸氢盐处理正常人基因组DNA,并以此为模板进行PCR扩增。设计1组特殊荧光标记探针以构建1种检测p16基因启动子区3个CpG位点甲基化改变的芯片,特殊荧光标记探针包括1对非甲基化探针和甲基化探针,检测相邻的3个CpG位点甲基化的程度。采用基因芯片的方法,将完全未甲基化的DNA和完全甲基化的DNA混合后制成标准曲线,将U266细胞株样本处理后点在芯片上与标准曲线进行比较后得出定量检测的结果。结果表明:芯片上探针的可重复性和精确性很好,0、5、10μmol/L异硫氰酸苯己酯处理后的U266细胞株p16基因的甲基化程度分别为78.2%、61.7%、54.8%。结论:异硫氰酸苯己酯可以降低U266细胞株p16基因甲基化的程度。

PDMS微流控芯片加工技术研究

对PDMS微流控芯片的制作流程、封装方法和结构特征进行了探讨,并提出了相应的解决方案。

甲基化芯片检测电离辐射诱导人外周血淋巴细胞DNA甲基化水平的变化

目的:筛选正常人细胞基因组中电离辐射后甲基化水平发生变化的基因,为在其中寻找新型辐射损伤标志物奠定研究基础。方法:60Coγ射线照射人外周血全血,照射剂量为0、0.5和2.0 Gy。利用Illumina 450K芯片检测外周血淋巴细胞基因组DNA甲基化水平的变化,筛选甲基化水平差异的基因,利用GO功能富集分析基因的分子功能和参与的生物学途径。结果:0.5和2.0 Gyγ射线照射下人外周血淋巴细胞基因组DNA甲基化水平显著上调的基因有1311个,显著下调的基因286个。GO功能富集分析表明,按富集度大小排列,差异基因在生物途径水平上排在首位的是“细胞过程”(富集度=5.86),在细胞定位水平上排在首位的是“细胞”(富集度=7.48),在分子功能水平上排在首位的是“结合”(富集度=5.27)。进一步细分后得到差异基因显著富集在与转录调控及转录因子活性相关的功能上,与以往的研究结果认为DNA甲基化参与转录调控和基因表达相一致;甲基化水平差异基因还显著富集在核苷连接(GO:0001882)上,并在其中寻找到与DNA修复相关的基因RAD50、RAD54L和INIP,以及与维持染色体结构稳定相关的基因HIST1H4K、SMC1B。结论:正常人外周血淋巴细胞基因甲基化水平受电离辐射影响,可进一步研究将这些与DNA修复、维持染色体结构稳定相关的基因甲基化水平作为辐射损伤标志物的可行性。

应用全基因组甲基化芯片筛选结直肠腺瘤基因启动子区甲基化标志物

目的应用全基因组甲基化芯片筛选结直肠腺瘤基因启动子区甲基化标志物。方法选择2013年1月至2014年12月在广西壮族自治区人民医院及广西医科大学第一附属医院住院的进展期结直肠腺瘤患者9例(腺瘤组),另选择经肠镜排除炎症性肠疾病、癌前病变及癌的患者20例作为对照组。腺瘤组于内镜下切除结直肠腺瘤标本(直径≥2 cm),对照组取结直肠黏膜标本(结肠黏膜13例,直肠黏膜7例)。提取组织DNA,应用全基因组甲基化芯片进行甲基化检测,通过生物信息学分析筛选差异甲基化基因启动子区甲基化标志物。结果以Δβ≥|0.4|,在基因启动子区共发现1870个差异甲基化标志物,1524个为高甲基化标志物,346个为低甲基化标志物,其中包含929个启动子差异甲基化基因。GO分析结果显示,启动子差异甲基化基因主要在化学性突触传递、神经系统发育、细胞外基质组织、细胞外基质结构成分、RNA聚合酶Ⅱ调控区序列特异性DNA结合、序列特异性DNA结合、质膜、蛋白质类细胞外基质、细胞膜等注释条目中出现富集。KEGG_Pathyway分析显示,启动子差异甲基化基因主要参与了神经活性的配体-受体相互作用、尼古丁成瘾、钙信号通路等信号转导通路。结论应用全基因组甲基化芯片筛选结直肠腺瘤甲基化标志物有助于探讨结直肠腺瘤的发病机制,为疾病的早期诊断提供线索。

表达谱芯片与DNA甲基化芯片综合分析探索鼻咽癌发生、发展的分子靶标

目的综合分析表达谱芯片与DNA甲基化芯片,探索鼻咽癌发生、发展的分子靶标与潜在治疗靶点。方法在GEO公共数据库下载编号为GSE64634的表达谱芯片数据以及编号为GSE52068的DNA甲基化芯片数据;利用R语言的相关工具包对表达谱芯片进行差异表达分析,对DNA甲基化芯片进行差异甲基化位点分析;利用DAVID数据库对筛选出的差异表达基因进行基因功能分析和信号通路分析;利用实时荧光定量PCR和甲基化特异性PCR进一步验证生物信息学分析的结果。结果表达谱芯片分析获得筛选出2 327个差异表达基因,其中1 777个基因表达下调,550个基因表达上调;DNA甲基化芯片分析获得2 321个差异甲基化位点,其中2 228个低甲基化位点,93个高甲基化位点;对表达谱芯片和DNA甲基化芯片进行综合分析,找到4个表达水平升高且甲基化修饰水平降低的基因,并利用定量PCR、表达谱芯片和DNA甲基化芯片成功验证该结果。结论综合分析表达谱芯片和DNA甲基化芯片,筛选出4个与鼻咽癌发生、发展相关的分子靶标和潜在的治疗靶点。