虎奶菇菌丝体和菌核基因组甲基化分析

以虎奶菇[Pleurotus tuber-regium (Fr.) Singer]菌丝体和菌核为材料,利用MSAP技术分析其甲基化水平,并对甲基化差异片段进行回收测序,探究虎奶菇生长与DNA全基因组甲基化的关系.结果 表明,菌丝体和菌核的DNA甲基化率分别为7.94％和9.54％,其中半甲基化率分别为4.50％和4.0...     

叶籽银杏DNA甲基化水平与模式变异的研究

以萌动期与展叶期的叶籽银杏和银杏为试材,采用基于DNA甲基化敏感扩增多态性分析（Methylation-sensitive amplification polymorphism,MSAP）方法,在全基因组水平上探究叶籽银杏、银杏不同发育期DNA序列中CCGG位点的甲基化水平及模式变化特征。萌动期选用22对引物,在叶籽银杏和银杏中检测到扩增位点为498和384个,甲基化位点为237和165个,其总甲基化率分别为47.6%和42.4%;展叶期选用40对引物,在叶籽银杏有叶生胚珠（YZ2）、无叶生胚珠（YC）及银杏（CK）叶片中检测到扩增位点767、600及367个,甲基化位点分别为370、244及152个,其总甲基化率分别为48.3%、40.5%及41.5%。进一步对不同发育期叶籽银杏、银杏DNA甲基化模式的变化特征进行分析,结果显示：萌动期、展叶期叶籽银杏与银杏相比均有超过半数的位点（52.1%、54.6%及64.2%）DNA甲基化模式发生多态性变化,萌动期叶籽银杏相对于银杏其变化趋势以超甲基化为主;展叶期叶籽银杏有叶生胚珠相对于叶籽银杏无叶生胚珠及银杏甲基化的变化趋势以超甲基化为主,叶籽银杏有叶生胚珠相对于银杏DNA甲基化模式变异幅度更大,超甲基化水平更高,显示出叶籽银杏基因组独特的DNA甲基化特征。     

离体条件下5 - 氮杂胞嘧啶核苷对菊花DNA甲基化和表型性状的影响

 采用离体处理的方法, 初步研究了5 - 氮杂胞嘧啶核苷(5-azaC) 对菊花的影响。结果表明, 500μmol·L - 1以上时有致死效应, 100μmol·L - 1以上时能抑制离体材料的生长发育, 而各浓度对菊花的丛生芽均具有抑制作用, 且这种抑制作用具有时间累加效应和剂量累计效应。10μmol·L - 1以下时使菊花开花时间有不同程度的提早, 这一效应具有稳定性, 并可通过营养繁殖进行遗传。MSAP技术分析表明,处理材料基因组DNA甲基化水平明显降低, 在去处理后的继代过程中仍有部分位点保持低甲基化状态。     

春化对白菜DNA 甲基化、GA含量及蛋白质的影响

 以普通白菜‘油冬儿’为试材, 研究了低温对白菜开花的诱导效应, 并分析了萌动种子和幼苗春化诱导后DNA 甲基化水平、赤霉素(GA) 含量和蛋白质种类的变化。结果表明, 不论萌动种子还是幼苗, 4 ℃低温处理30 d 均可完成春化。春化处理后, 两处理植株茎尖组织DNA 甲基化水平都较对照下降;但GA 含量明显上升, 为对照的2～3 倍; 低温诱导植株产生了一种分子量为58 kD 的特异蛋白质, 同时也使一种蛋白质消失。说明低温可能通过降低DNA 甲基化水平或增加GA 含量而诱导植物开花。     

矮生观赏杉木DNA甲基化的水平与模式分析

为探讨杉木矮化变异与DNA甲基化的关系,以矮生观赏杉木与野生杉木为试验材料,采用基于DNA甲基化敏感扩增多态性分析（Methylation-sensitive amplification polymorphism,MSAP）方法,研究其DNA 序列CCGG 位点的甲基化水平及模式变化特征。应用20个引物组合,在矮生观赏杉木和野生杉木的叶片DNA中均检测出745个CCGG位点,其中甲基化位点数分别为508个和505个,分别占总扩增位点数的68.17%和67.83%;在矮生观赏杉木与野生杉木木质部DNA中分别检测到742个和737个CCGG位点,其中甲基化位点数分别为471个与498个,分别占总扩增位点数的63.52%和67.51%,差异达极显著水平（P < 0.01）。与野生型相比,矮生观赏杉木叶片和木质部DNA甲基化模式发生了一定变化,在叶片DNA中,去甲基化率为17.81%,明显高于超甲基化率15.44%;在木质部DNA中,去甲基化率17.25%,也明显高于超甲基化率14.65%。通过甲基化序列的初步克隆及比对分析发现,矮生观赏杉木中参与MAPK级联途径的蛋白磷酸酶IBR5基因启动子区域的甲基化水平上升。因此推测,植物激素信号转导及其调控基因的甲基化变化可能是矮生观赏杉木形成的原因之一。     

5–氮杂胞苷对白菜幼苗DNA 甲基化和耐热性的影响

 研究了不同浓度5–氮杂胞苷（5-azaC）处理白菜‘夏帝’和‘冬赏味’品种的种子,对其幼苗甲基化和耐热性的影响。结果表明：不同浓度5-azaC 溶液处理可显著降低幼苗DNA 甲基化水平,植株形态发育未见异常。0.1 ~ 0.2 mol · L^-1 5-azaC 处理可减缓幼苗在高温胁迫下的生长量、POD 活性和蛋白质含量的降低幅度,同时降低MDA 含量和细胞膜透性。     

Methylation and mRNA expression of TIG1 gene in esophageal squamous-cell carcinoma tissues

AIM: To investigate the expression and promoter methylation of tazarotene-induced gene-1 (TIG1) in esophageal squamous-cell carcinoma (ESCC) tissues. METHODS: The methods of methylation-specific PCR and real-time fluorescence quantitative PCR were applied to examine the methylation and mRNA expression of TIG1, respectively, in 43 cases of ESCC tissues, 20 cases of paracancerous tissues and 15 cases of normal tissues. RESULTS: The frequency of promoter methylation of TIG1 gene in ESCC tissues was 25.6% (11/43), which was significantly higher than that in the paracancerous tissues (5.0%, 1/20) and normal tissues (0/20). The hypermethylation of TIG1 gene in these tissues had no correlation with sex, age and clinical stage of the patients. However, it was correlated with the pathological stage (P<0.01) and lymph node metastasis (P<0.05). The mRNA expression of TIG1 in ESCC tissues was significantly lower than that in paracancerous tissues (P<0.05) and normal tissues (P<0.01). However, the expression level of TIG1 mRNA in methylated tissues was significantly lower than that in unmethylated tissues (P<0.01). CONCLUSION: Promoter methylation may be an important mechanism of TIG1 gene inactivation in ESCC, which was related to lymph node metastasis and TNM stage of esophageal carcinoma.     

Noninvasive prenatal diagnosis of β-thalassemia by enriching cell-free fetal DNA in materal plasma

AIM: To establish a kind of simple and efficient method for cell-free fetal DNA (cff-DNA) enrichment and to investigate its range of applications and the advantages and disadvantages.METHODS: (1) The single nucleotide polymorphisms(SNPs), which linked to paternal β-thalassemia mutations, were screened. We analyzed the contact between the SNPs in β- thalassemia gene (HBBgene) and haploid type by the Haploview software, and then selected these close SNPs which have higher heterozygosity with the HBBgene. (2) We selected 4 cases of different β-thalassemia mutations with their husband, and then we used TT-FAST-COLD-PCR to enrich the IVS-II-654 mutations in maternal plasma. If the IVS-II-654 mutation was not detected, we detected the SNP which linked to the IVS-II-654 mutation. Similarly, we used TT-FULL-COLD-PCR to enrich the CD41-42 mutations in the maternal plasma. At the same time, we used the conventional PCR to enrich CD41-42 mutation and IVS-II-654 mutation in the maternal plasma.RESULTS: (1) Nine cases of the SNP (rs7480526) linked to the mutation at IVS-II-654 in HBBgene, and 11 cases of the SNP (rs10768683) linked to the mutation at CD41-42 in HBBgene were detected. (2) We detected 1 case who inherited the paternal β-thalassemia mutation (IVS-II-654). We did not directly detect patermal IVS-II-654 mutation in maternal plasma, but detected the SNP linked to the IVS-II-654 mutation in the other case and had 100% detection, and 2 cases inherited the paternal β-thalassemia mutations (CD41-42) in the maternal plasma by TT-FULL-COLD-PCR and had 100% detection. However, we detected nothing by conventional PCR. CONCLUSION: TT-COLD-PCR is applicable to enrich cell-free fetal DNA in maternal plasma and is a method in the field of noninvasive prenatal diagnosis.     

Advances in modulation of microRNA and DNA methylation in congenital heart diseases

Aberrations in microRNA (miRNA) expression and DNA methylation are causal factors for congenital heart diseases (CHD), which belongs to epigenetic mechanisms. Complex modulation of miRNA on DNA methylation is a critical regulator of gene expression, leading to disease development. The aim of this review is to provide recent progress in the regulation of miRNA and DNA methylation in CHD.     

Advances on studies of miR-21 in vascular endothelial function and angiogenesis

MicroRNAs (miRNAs)are a class of non-coding, endogenous, single-stranded small RNA molecules composed of 19~25 nucleotides. miRNAs are widely involved in the process of human life activities. Recent studies have shown that part of miRNAs regulate the vascular endothelial function and angiogenesis. High expression of miRNA-21 is found to play important roles in the cell proliferation, cell apoptosis, cell growth and death of vascular endothelial cells. This review will focus on the recent progress related to miRNAs in vascular endothelial function and angiogenesis, providing a new insight in cardiovascular disease prevention, clinical diagnosis, prognosis and target therapeutics.     

Effects of c-fos antisense oligoneuleotide and p21 genetic transfection on the intimal proliferation of venous autografts in rabbits

AIM: To investigate the effects of c-fos antisense oligoneuleotide and p21 genetic transfection on the intimal proliferation of venous autografts. METHODS: The external jugule veins were autografted into common carotid arteries in the same side in 20 New Zealand rabbits, which were divided evenly into experimental and control group randomly. The transplanted veins of experimental group were immersed in the adenovirus-mediated p21 gene solution for 15 minutes just before anastomosis and coated with c-fos antisense oligoneucleotide glue gel just after anastomosis, while the control was only treated with empty vector. The transplanted vascular sample were taken at 2 weeks after operation. The intimal thickness (IT), degree of restenosis (DR), expression of proliferating cell nuclear antigen (PCNA), quantity of VSMC were determined by immunohischemistry. RESULTS: The IT, DR and expression of PCNA, VSMC were decreased, compared to control group. CONCLUSION: Transfection of c-fos antisense oligoneuleotide and p21 gene inhibits the intimal proliferation of venous antografs.     

Correlation of gene modification between histone H3 lysine 4 trimethylation and DNA methylation in IgA nephropathy patients

AIM: To investigate the correlation of gene modification between histone H3 lysine 4 (H3K4) trimethylation and DNA methylation in IgA nephropathy patients. METHODS: H3K4 trimethylation variations were analyzed in peripheral blood mononuclear cells (PBMCs) from 40 IgAN patients and 40 healthy controls by the method of chromatin immunoprecipitation linked to microarrays (ChIP-chip). ChIP real-time PCR was used to validate the microarray results. Quantitative real-time PCR (qRT-PCR) was employed to analyze mRNA expression. DNA methylation in 4 selected positive genes was analyzed by the method of methylated DNA immunoprecipitation-quantitative PCR (MeDIP-qPCR). RESULTS: IgAN patients displayed higher H3K4 trimethylation level and lower DNA methylation level than those of the healthy controls. There were significant differences in DNA methylation and H3K4 trimethylation of 4 selected genes between IgAN patients and the healthy controls (P<0.05). CONCLUSION: Our studies indicate that significant alterations of H3K4 trimethylation and DNA methylation exist in IgAN patients. There is a correlation of gene modification between DNA mathylation and histone H3 lysine 4 trimethylation in IgAN patients.     

Effects of FGF-21 and Nrf2 on BLM-induced pulmonary fibrosis in mice

AIM:To investigate the effect of fibroblast growth factor-21 (FGF-21) on bleomycin (BLM)-induced inflammatory response and oxidative stress in the lung, and to further explore the molecular mechanism of FGF-21 against pulmonary fibrosis. METHODS:The lung fibrosis model was induced by BLM intratracheal instillation. A total of 40 mice were randomly divided into control group, BLM group, FGF-21 (1, 2 and 5 mg/kg)+BLM groups. Western blot was used to detected the protein expression of collagen I, fibronectin and nuclear factor E2-related factor 2 (Nrf2). The reactive oxygen species (ROS) production was measured by DCFH-DA staining. The levels of inflammatory cytokines were measured by ELISA. The content of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), and the content of hydroxyproline (HYP) were detected by commercially available assay kits. RESULTS:Treatment with FGF-21 notably attenuated BLM-induced the expression levels of inflammatory mediators tumor necrosis factor-α, interleukin-1β and interleukin-6 in the lung tissue. In addition, FGF-21 treatment remarkably reduced the generation of ROS and the content of MDA trigged by BLM, accompanied with the enhanced activity of anti-oxidative enzymes SOD and GPx (P<0.05). Furthermore, treatment with FGF-21 obviously reduced the extracellular matrix (ECM) accumulation by suppressing the expression of collagen I and fibronectin induced by BLM, accompanied with the decreases in the levels of TGF-β1 and HYP. Silencing of Nrf2 expression abolished the protective effect of FGF-21. CONCLUSION:FGF-21 relieves BLM-induced pulmonary fibrosis by reducing the inflammatory response, mitigating oxidative damage and decreasing the ECM deposition via Nrf2 activation, thus providing the basis for the therapeutic effect of FGF-21 on the lung fibrosis.     

Association between plasma level of microRNA-21 and pulmonary hypertension in heart failure patients with preserved ejection fraction

AIM: To investigate the correlation between microRNA-21 (miR-21) and pulmonary hypertension(PH) in the patients with heart failure with preserved ejection fraction (HFpEF). METHODS: From January 2014 to February 2016, 102 HFpEF patients presenting to Jiangxi Provincial People's Hospital were enrolled in this study. The patients were divided into PH-HFPEF group (n=36, PASP ≥ 50 mmHg) and HFpEF group (n=40, PASP<50 mmHg). Another 36 age-and sex-matched subjects served as healthy controls. The plasma level of miR-21 and its correlations with clinic data were examined, and multivariate logistic regression analysis was performed to identify the independent predictors of PH-HFpEF. Receiver operating characteristic (ROC) curve was analyzed, and the area under the curve (AUC) was calculated. The sensitivity and specificity for PH-HFPEF diagnosis were also determined. RESULTS: Age, plasma endothelin-1 (ET-1), interleukin-6 (IL-6), brain natriuretic peptide (BNP) and left atrial diameter (LAD) were significantly higher in PH-HFPEF group than those in HFPEF group (P<0.05). The level of miR-21 was significantly increased in PH-HFPEF group compared with HFpEF group and healthy control group. In addition, the miR-21 level was correlated with PASP (r=0.267, P=0.000), plasma IL-6 (r=0.302, P=0.013) and left ventricular mass index (LVMI) (r=0.515, P=0.036). Plasma IL-6 was positively correlated with plasma ET-1 (r=0.622, P=0.002). PASP was positively correlated with plasma IL-6 (r=0.36, P=0.023), ET-1 (r=0.76, P=0.004), BNP (r=0.43,P=0.031), and LAD (r=0.39, P=0.044). ROC curve showed that the AUC of miR-21 was 0.80. Multivariate logistic regression analysis showed that miR-21, LAD and BNP were the independent predictors of PH-HFpEF. CONCLUSION: Increased plasma miR-21 level is positively correlated to PH-HFpEF, and is an independent predictor of PH-HFpEF, suggesting that plasma miR-21 might be a useful new biomarker for predicting PH in HFpEF patients.     

Effect of alpha-lipoic acid on expression of miR-21/Smad7 and fibrosis in kidney of diabetic rats

AIM: To investigate the protective effect of alpha-lipoic acid (ALA) on the kidney of the rats with diabetes mellitus (DM), and to discuss the mechanism. METHODS: The DM rats were divided into normal control (NC) group, DM group and ALA group. After treated with ALA for 6 weeks, the rats were sacrificed to detect the relevant biochemical parameters, and the pathological changes of the kidney tissues were observed by HE staining and Masson staining. The protein levels of transforming growth factor-β1 (TGF-1), p-Smad2/3, Smad7, collagen I and collagen Ⅲ were determined by Western blot. In addition, the expression of microRNA-21 (miR-21) was detected by RT-qPCR. RESULTS: Compared with NC group, the kidney weight/body weight, blood glucose (BG), total cholesterol, triglyceride and 24-h urine protein were remarkably increased in DM group (P<0.05). The pathological observation of the kidney tissues showed fibrosis changes in DM group. The level of Smad7 was reduced in DM group, while the levels of TGF-β1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were increased (P<0.05). After treatment with ALA for 6 weeks, all the relevant biochemical parameters were reduced except BG, and the renal fibrosis lesions were obviously alleviated. Compared with DM group, the levels of TGF-1, p-Smad2/3, collagen I, collagen Ⅲ and miR-21 in the kidney tissues were reduced in ALA group, while the level of Smad7 was increased (P<0.05).CONCLUSION: ALA may prevent the development of renal fibrosis in rats through restraining the expression of TGF-β1 and miR-21, increasing the levels of Smad7 protein, and reducing the deposition of extra cellular matrix.     

Promoter methylation status and demethylation-induced expression of SFRP genes in human acute leukemia cell lines

AIM: To investigate the relationship between promoter hypermethylation of secreted frizzled-related protein (SFRP) genes and acute leukemia (AL),and to study the mechanism how 5-aza-2-deoxycytidine (5-Aza-CdR) reverses the hypermethylation of SFRP genes in human AL cell lines. METHODS：Methylation-specific PCR (MSP) was used to detect the methylation levels of SFRP1, SFRP2, SFRP4 and SFRP5 genes in different human AL cell lines (Molt-4, Jurkat, HL-60 and NB4). The methylation levels of these genes in Jurkat cell line before and after 5-Aza-CdR treatment were also analyzed by MSP. The expression of SFRP1, SFRP2, SFRP4 and SFRP5 mRNA was detected by real-time fluorescence quantitative RT-PCR. The mRNA levels of DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B were analyzed by semiquantitative RT-PCR. RESULTS：None of normal human blood or bone marrow mononuclear cells showed methylation of SFRP genes. Hypermethylation in the promoter regions of SFRP1, SFRP2 and SFRP5 genes was observed in all of the four AL cell lines. SFRP4 gene was totally methylated in NB4, Molt-4 and Jurkat cell lines but partially methylated in HL60 cell line. Treatment with 5-Aza-CdR for 72 h successfully reversed the hypermethylation of SFRP genes, and significantly increased the mRNA expression of SFRP. Moreover, the mRNA expression of DNMT1, DNMT3A and DNMT3B was down-regulated by 5-Aza-CdR in a concentration-dependent manner. CONCLUSION：Methylated SFRP genes may serve as potential independent biomarkers for early detection of AL. 5-Aza-CdR activates SFRP gene expression by demethylation of SFRP genes and down-regulation of DNMT1, DNMT3A and DNMT3B mRNA expression.     

Expression of DNA repair gene ERCC1 and its relationship with PAH-DNA adducts in lung cancer tissues

AIM: To investigate the expression of nucleotide excision repair gene ERCC1 and its relationship with PAH (polycyclic aromatic hydrocarbons)-DNA adducts in lung cancer tissues. METHODS: ERCC1 mRNA expression and the PAH-induced DNA adducts were detected in 150 lung cancer tissues, 120 adjacent lung tissues without cancer cells, 40 benign lung lesions and 40 normal lung tissues. The effects of some exposure factors on the expression of ERCC1 gene and the connection between ERCC1 and PAH-DNA adduct was analyzed. RESULTS: Reduced expression levels of ERCC1 were observed in 46 of 150 (30.7%) lung cancer specimens and 1 of 40 (2.5%) normal lung tissues. Smoking may suppress the expression of ERCC1 gene. The level of PAH-DNA adduct was negatively correlated with the expression of ERCC1 gene, the Spearman coefficient was -0.648, P<0.01. CONCLUSION: ERCC1 is an important nucleotide excision repair gene and may participate in the repair of DNA damage, such as PAH-DNA adduct. Low expression of ERCC1 may play an important role in the development of human lung cancer.     

Interspecific hybridization in vanilla and molecular characterization of hybrids and selfed progenies using RAPD and AFLP markers

Vanilla, Vanilla planifolia Andrews, is native to Mexico and Central America, but is now cultivated in other parts of the tropics. Continuous clonal propagation has resulted in very little variability for crop improvement programmes in vanilla. In this study, an attempt has been made to increase the spectrum of variation by interspecific hybridization with Vanilla aphylla, an Indian species which is tolerant to Fusarium. Interspecific hybrids were successfully produced and morphological characters and molecular profiles revealed the true hybridity of the progenies. Ten seedling progenies of V. planifolia, and four interspecific hybrids obtained from crosses between V. planifolia (female) and V. aphylla (male) using a number of different loci as markers were evaluated and 319 amplified fragment length polymorphisms (AFLPs) and 83 random amplified polymorphic DNAs (RAPDs) loci were marked. The profiles indicate similarity between the parents, selfed progenies and interspecific hybrids and that all the progenies tested were variable when compared to each other, which can be exploited for crop improvement in vanilla. This is the first report in vanilla, indicating that RAPD and AFLP profiles coupled with morphological characters can be utilized to assess the variability and hybrid nature of genotypes and of successful interspecific hybridization and production of hybrids between V. planifolia and V. aphylla.