人乌头酸脱羧酶1基因及其编码蛋白的生物信息学分析

目的 对人乌头酸脱羧酶1(aconitate decarboxylase 1,ACOD1)基因的转录调控因素及其编码蛋白的分子功能进行分析。方法 采用多种生物信息学软件对人ACOD1基因启动子区域转录因子结合位点、甲基化CpG位点、单核苷酸多态性（singlenucleotide polymorphism,SNP）位点进行预测,对人ACOD1基因编码的蛋白进行GO(Gene Onotology)蛋白功能注释分析及蛋白互作网络分析。结果 人ACOD1基因启动子区域存在3个启动子序列,17个转录因子结合位点,无甲基化CpG位点,并存在12个SNP位点;人ACOD1蛋白具有多种分子功能并参与多种生物过程,可能与白细胞介素-1β(interleukin-1 beta,IL-1β)、C-C基序趋化因子-4(C-C motif chemokine-4,CCL-4)、干扰素调节因子1(interferon regulatory factor 1,IRF1)和C-X-C基序趋化因子10(C-X-C motif chemokine 10,CXCL10)等蛋白具有相互作用关系。结论 本文为进一步研究人AC...     

原发性高血压11β-HSD2基因甲基化的研究进展

11β-类固醇脱氢酶2(11 beta-hydroxysteroid dehydrogenase type 2,11β-HSD2)基因甲基化的表达与原发性高血压(essential hypertension,EH)的发病机制紧密相关。同时,EH中不同血浆肾素水平的11β-HSD2基因启动子区甲基化的表达水平不同。国内外研究表明,11β-HSD2基因启动子区甲基化水平的发生参与EH的发生机制。     

白细胞介素-6基因启动子区-572位点多态性与EV71感染疾病严重程度的相关性分析

目的探讨白细胞介素-6(Interleukin-6,IL-6)基因启动子区-572位点单核苷酸多态性(single nucleotide polymorphism,SNP)与儿童肠道病毒71型(enterovirus 71,EV71)感染疾病严重程度的相关性。方法提取87份EV71重症感染患者和57份无症状隐性感染儿童血液样本基因组DNA,进行PCR扩增及测序,检测IL-6基因启动子区-572位点多态性,利用Logistic回归分析基因型频率。结果 EV71重症感染患者IL-6基因型频率与对照组相比,差异有统计学意义(OR=2.605,95%CI:1.117~6.074,P=0.027)。IL-6多态性与EV71感染严重程度有关(OR=2.411,95%CI:1.236~4.706,P=0.010)。结论 IL-6多态性与中国儿童EV71重症感染可能具有相关性,对确定影响疾病严重程度的宿主遗传因素和增强治疗效果具有重要意义。     

儿童急性淋巴细胞白血病CD34~+CD38~-群细胞在NOD/SCID与NSG小鼠体内增殖能力的比较

目的比较儿童急性淋巴细胞白血病(acute lymphoblastic leukemia,ALL)CD34+CD38-细胞群在非肥胖糖尿病(non obese diabetic,NOD)/重症联合免疫缺陷(severe combined immunodeficient,SCID)小鼠与在NOD/SCID基础上敲除IL-2Rγ链的小鼠(NOD scid gamma,NSG)体内的复制能力。方法采用免疫磁珠法分选ALL患儿骨髓CD34+CD38-细胞(作为阳性细胞)及其他各群细胞(CD34+CD38+、CD34-CD38-、CD34-CD38+,作为阴性对照细胞),经尾静脉分别注射于NOD/SCID或NSG小鼠体内,104个/只,连续监测小鼠的发病情况并记录小鼠生存时间;进行小鼠外周血白血病细胞计数及外周血、骨髓血涂片检查;将濒死小鼠处死后,取肝、脾组织,进行病理学检查。结果 CD34+CD38-细胞注射入小鼠体内后,NOD/SCID小鼠的存活期为56¹50 d,NSG小鼠的存活期为13150 d,NSG小鼠的存活期为13¹20 d;NOD/SCID小鼠外周血白细胞数量缓慢升高直至死亡或处死,NSG小鼠外周血白细胞数量从第7天开始升高,至75 d左右达高峰;NOD/SCID小鼠和NSG小鼠分别于注射CD34+CD38-细胞3周和2周后外周血涂片可见白血病细胞,NSG小鼠外周血涂片发现更多的蓝染幼稚细胞;NSG小鼠骨髓涂片中发现更多的圆形、外源整齐、蓝色深染的幼稚细胞;NSG小鼠脾脏组织中有明显的炎细胞团块,且组织疏松,NOD/SCID小鼠脾脏组织中未发现明显的炎细胞团块,且组织相对致密,两种小鼠的肝脏组织中均未发现明显的炎细胞团块和组织结构改变。结论 ALL CD34+C38-细胞在NOD/SCID小鼠及NSG小鼠体内均能增殖复制出白血病,NSG小鼠复制白血病的时间更短,恶性程度更高,且白血病的复制过程更接近人类白血病的病理过程。本实验为白血病发病机理的研究和临床用药的评价提供了更好的载体。     

多发性骨髓瘤p16基因甲基化与其预后的相关性

目的探讨多发性骨髓瘤p16基因甲基化与其预后之间的相关性。方法采用甲基化敏感的限制性内切酶联合聚合酶链反应方法(REP法),检测52名多发性骨髓瘤(MM)患者骨髓细胞p16基因第1外显子的甲基化状态,并检测存在p16基因第1外显子甲基化的MM患者p16基因的mRNA转录水平。对这52名患者的临床资料及预后因素进行分析。结果52名MM患者中,p16基因启动子区第1外显子甲基化率为53.84%(28/52)。存在p16基因第1外显子甲基化的MM患者中,p16基因mRNA阴性率为75%。p16基因甲基化患者的预后不良因素明显高于非甲基化患者。结论多发性骨髓瘤p16基因甲基化与其预后有关。     

Msi2对急性髓系白血病THP-1细胞体外增殖能力的影响

目的观察Musashi(Msi)2对急性髓系白血病(acute myelogenous leukemia,AML)THP-1细胞体外增殖能力的影响,初步探讨Msi2在AML中可能的调控作用。方法利用RNA干扰技术沉默Msi2表达,实时荧光定量PCR法检测Msi2、cyclin D1、p21、cdk2基因mRNA转录水平;Western blot法检测Msi2蛋白表达水平;生长曲线观察THP-1细胞体外增殖能力;流式细胞仪检测细胞周期进程。结果与scramble组及空白对照组相比,Msi2-siRNA组细胞cyclin D1、cdk2基因mRNA转录水平明显降低(P0.05),p21基因mRNA转录水平明显增高(P0.05),Msi2 mRNA转录及蛋白表达水平均明显降低(P0.05);Msi2-siRNA组细胞体外增殖能力降低(P0.01);G1期细胞比例明显增高(P0.05),S期细胞比例显著降低(P0.05)。结论 Msi2可能通过调控细胞周期进程,促进白血病细胞恶性增殖,在AML的发生发展中发挥重要作用。     

IL-27p28基因多态性与中国朝鲜族人群哮喘的相关性

目的探讨IL-27p28基因启动子区g.-964 T>C、第2外显子区g.2905T>G和第4外显子区g.4730T>C 3个位点的多态性与中国朝鲜族人群哮喘的相关性。方法选取32例中国朝鲜族哮喘患者(病例组)和93名中国朝鲜族正常人(对照组)作为观察对象,采用PCR法扩增IL-27p28基因g.-964 T>C、g.2905T>G和g.4730T>C位点基因片段,单碱基延伸法进行基因分型,分析病例组和对照组的基因型频率及等位基因频率。结果病例组和对照组的基因型分布均符合Hardy-weinberg平衡定律(P>0.05)。g.-964位点的基因型频率及等位基因频率在病例组和对照组之间的分布差异有统计学意义(P<0.001);g.2905和g.4730位点的基因频率及等位基因频率在病例组和对照组之间的分布差异无统计学意义(P>0.05)。结论 IL-27p28基因多态位点g.-964 T>C可能与朝鲜族人群哮喘易感性相关,g.2905T>G和g.4730T>C多态性与朝鲜族人群哮喘未发现有相关性。     

hTERT核心启动子调控HSV-TK自杀基因系统诱导肿瘤细胞的凋亡

目的观察人端粒酶催化亚单位(hTERT)启动子调控逆转录病毒介导的单纯疱疹病毒-胸苷激酶基因/丙氧鸟苷(HSV-TK/GCV)自杀基因系统诱导肿瘤细胞凋亡的效果。方法采用PCR方法扩增胸苷激酶(TK)基因,并构建由hTERT核心启动子调控的胸苷激酶重组逆转录病毒载体pLNC-hTERTp-TK,转染包装细胞PT67,G418筛选阳性细胞克隆,检测病毒滴度,PCR法检测转染细胞中的TK基因。将获得的重组逆转录病毒感染人肺癌细胞株A549、肝癌细胞株SMMC-7721、宫颈癌细胞株HeLa和正常人成纤维细胞系WI-38,G418筛选阳性克隆,MTT法检测重组逆转录病毒对肿瘤细胞的生长抑制作用,流式细胞仪检测肿瘤细胞的凋亡。结果重组逆转录病毒载体pLNC-hTERTp-TK经双酶切,可见1402bp的目的基因片段,表明质粒构建正确。从转染的PT67细胞中扩增出1131bp的基因片段,与TK基因大小一致。在成功转染重组逆转录病毒的PT67细胞中,病毒滴度最高者可达7.8×105CFU/ml。重组逆转录病毒感染的3种肿瘤细胞的增殖均受到抑制,且生长抑制率随着GCV浓度的增加而升高,经较低浓度GCV(10μg/ml)处理的3种肿瘤细胞,凋亡率分别可达37.06%、34.88%和33.59%。结论由hTERT启动子调控的逆转录病毒介导的HSV-TK/GCV自杀基因系统可诱导肿瘤细胞凋亡,且具有较强的特异性和高效性。     

原发高血压患者11β-HSD2基因甲基化表达的相关性研究

目的研究原发性高血压(essential hypertension,EH)患者11β-类固醇脱氢酶2(11 beta-hydroxysteroid dehydrogenase type 2,11β-HSD2)基因甲基化表达,探讨原发性高血压的发病机制。方法选取2018年1月至2019年6月在佳木斯医学院第一附属医院未经治疗的EH患者71例为EH组,选取我院体检的50例健康志愿者为正常对照组,应用甲基化特异性PCR (methylation specific PCR,MSP)方法检测所有入选研究对象11β-HSD2基因甲基化表达。结果 EH组和对照组11β-HSD2基因甲基化表达比较,差异具有统计学意义(P0.05)。结论 11β-HSD2基因甲基化参与EH的发生。     

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.     

The Significance of MGMT Promoter Methylation Status in Diffuse Glioma

A single-institution observational study with 43 newly diagnosed diffuse gliomas defined the isocitrate dehydrogenase 1 and 2 (IDH1/2) gene mutation status and evaluated the prognostic relevance of the methylation status of the epigenetic marker O⁶-methylguanine-DNA methyltransferase (MGMT). Younger patients (<50 years) with surgically resected glioma and temozolomide (TMZ) adjuvant chemotherapy were associated with better prognosis, consistent with other studies. The methylation status depends on the chosen method and the cut-off value determination. Methylation-specific PCR (MSP) established the methylation status for 36 glioma patients (19 (52.8%) positively methylated and 17 (47.2%) unmethylated) without relevancy for the overall survival (OS) (p = 0.33). On the other side, real-time methylation-specific PCR (qMSP) revealed 23 tumor samples (54%) that were positively methylated without association with OS (p = 0.15). A combined MSP analysis, which included the homogenous cohort of 24 patients (>50 years with surgical resection and IDH1/2-wildtype diffuse glioma), distinguished 10 (41.6%) methylated samples from 14 (58.4%) unmethylated samples. Finally, significant correlation between OS and methylation status was noticed (p ≈ 0.05). The OS of the hypermethylated group was 9.6 ± 1.77 months, whereas the OS of the unmethylated group was 5.43 ± 1.04 months. Our study recognized the MGMT promoter methylation status as a positive prognostic factor within the described homogenous cohort, although further verification in a larger population of diffuse gliomas is required.     

Promoter Methylation Changes in KRT17: A Novel Epigenetic Marker for Wool Production in Angora Rabbit

Wool production is an important economic trait of Angora rabbits. Exploring molecular markers related to wool production is one of the essentials of Angora rabbits’ breeding. KRT17 (Keratin 17) is an important gene of hair follicle development, which must be explored for genetic/epigenetic variation to assess its effect on wool production. Based on the effective wool production data of 217 Angora rabbits, the high and low yield groups were screened with 1.5 standard deviations of the population mean. The full-length sequence of KRT17 was obtained by rapid amplification of cDNA ends technology, and the polymorphism was analyzed in the promoter, exon, and intron regions by direct sequencing. KRT17, SP1 over-expression plasmids, and siRNA were constructed and transfected into dermal papilla cells. The mRNA expressions of relevant genes were analyzed by RT-qPCR. The methylation level of the KRT17 promoter was determined by Bisulfite Sequencing PCR. Dual-luciferase system, site-directed mutagenesis, and electrophoretic mobility shift assays were used to analyze the binding relationship between SP1 and the promoter of KRT17. The structure map of KRT17 was drawn, and no SNPs were found in the promoter, exon, and intron, indicating a relatively conserved structure of KRT17. Expression of KRT17 was significantly higher in cutaneous tissues than in other tissues and was significantly upregulated in the high-yield group compared to the low-yield group (p < 0.05). Furthermore, the overall high methylation levels of KRT17 CpG I and CpG III showed significant association with low wool yield; the methylation levels of 5 CpG locus (CpG I site 4 and CpG III site 2–5) were significantly different between the high and low yield groups (p < 0.05). The methylation levels of 3 CpG locus (CpG I site 4 and CpG III site 4, 14) showed a significant correlation with KRT17 expression (p < 0.05). Overall, CpG III site 4 significantly affects wool production and KRT17 expressions (p < 0.05). This site promotes SP1 binding to the KRT17 promoter region (CGCTACGCCC) to positively regulate the KRT17 expression. KRT17 CpG III site 4 can be used as candidate epigenetic markers for the breeding of high wool-producing Angora rabbits.     

Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes

Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.     

Differential DNA Methylation in Relation to Age and Health Risks of Obesity

The aim of this study was to evaluate whether genome-wide levels of DNA methylation are associated with age and the health risks of obesity (HRO); defined according to BMI categories as “Low HRO” (overweight and class 1 obesity) versus “High HRO” (class 2 and class 3 obesity). Anthropometric measurements were assessed in a subsample of 48 volunteers from the Metabolic Syndrome Reduction in Navarra (RESMENA) study and 24 women from another independent study, Effects of Lipoic Acid and Eicosapentaenoic Acid in Human Obesity (OBEPALIP study). In the pooled population; the methylation levels of 55 CpG sites were significantly associated with age after Benjamini-Hochberg correction. In addition, DNA methylation of three CpG sites located in ELOVL2; HOXC4 and PI4KB were further negatively associated with their mRNA levels. Although no differentially methylated CpG sites were identified in relation to HRO after multiple testing correction; several nominally significant CpG sites were identified in genes related to insulin signaling; energy and lipid metabolism. Moreover, statistically significant associations between BMI or mRNA levels and two HRO-related CpG sites located in GPR133 and ITGB5 are reported. As a conclusion, these findings from two Spanish cohorts add knowledge about the important role of DNA methylation in the age-related regulation of gene expression. In addition; a relevant influence of age on DNA methylation in white blood cells was found, as well as, on a trend level, novel associations between DNA methylation and obesity.