miR-375 is downregulated by promoter hypermethylation in MIN6 insulinoma cells.

Epigenetics may affect the susceptibility for type 2 diabetes mellitus (T2DM). Aberrant DNA methylation patterns are nowadays recognized as a key epigenetic hallmark of T2DM. Previously, our studies have shown that the hypomethylation of human miR-375 promoter may contribute to the pathogenesis of T2DM. However, no comprehensive study defines the miR-375 promoter methylation patterns present in the established pancreatic ß cell line. To address this matter, we have analyzed the DNA methylation profile of insulinoma MIN6 cells by MassARRAY spectrometry and employed the DNA demethylating drug 5-aza-2'-deoxycytidine (5-aza-CdR) to treat MIN6 cells to explore the methylation patterns of the mmu-miR-375. The expression of mmu-miR-375 in mRNA level was measured by quantitative RT-PCR (qRT-PCR). Methylation analysis reveals that MIN6 cells display hypermethylation at the mmu-miR-375 promoter. Following the decreased methylation of mmu-miR-375, the relative expression of mmu-miR-375 increased gradually after 5-Aza-CdR treatment. In addition, we find that there was an inverse correlation between DNA methylation levels and transcription level of mmu-miR-375. In summary, this is the first report for analyzing mmu-miR-375 promoter methylation using MALDI-TOF MS technology and our results indicate that promoter hypermethylation of the mmu-miR-375 is a common event in MIN6 cells.

Analysis of PTEN expression and promoter methylation in Uyghur patients with mild type 2 diabetes mellitus.

Phosphatase and tension homolog deleted on chromosome 10 (PTEN) was considered as a promising target in type 2 diabetes mellitus (T2DM) because of its negative effects on insulin resistance. Alteration in DNA methylation is thought to play a role in the pathogenesis of T2DM. The aim of the present study was to quantitatively evaluate the promoter methylation of PTEN in Uyghur patients with mild T2DM. We evaluated methylation levels in 21 CpG sites from -2515 bp to -2186 bp relative to the translation initiation site in 55 cases of T2DM and 50 cases of normal glucose tolerance (NGT) using the MassARRAY spectrometry. In addition, PTEN mRNA and protein levels were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blotting to determine whether DNA methylation alterations were responsible for PTEN expression. Compared with NGT groups, the PTEN mRNA expression was significantly higher in Uyghur patients with mild T2DM groups. We also showed that PTEN protein expression was upregulated in Uyghur patients with mild T2DM groups, but the level of protein kinase B (AKT) was downregulated. PTEN methylation in T2DM patients was significantly lower than that in NGT groups. In addition, 2 CpG units demonstrated a significant difference between the NGT and Uyghur patients with mild T2DM groups. Furthermore, there was a negative association between promoter methylation and PTEN expression. Together, these findings suggest that epigenetic inactivation of PTEN plays an important role in Uyghur patients with mild T2DM. The aberrant methylation of CpG sites within the PTEN promoter may serve as a potential candidate biomarker for T2DM in the Uyghur population.

Epigenetic regulation of microRNA-375 and its role as DNA epigenetic marker of type 2 diabetes mellitus in Chinese Han population.

Epigenetics may affect the susceptibility for type 2 diabetes mellitus (T2DM). Previously, our studies have shown that the hypomethylation of human miR-375 promoter may contribute to the pathogenesis of T2DM. However, the methylation pattern of miR-375 promoter in T2DM is not yet fully understood. In this study, the DNA methylation status of the different region of miR-375 promoter in Chinese Han population with T2DM were explored. 100 Han patients with T2DM and 100 Han healthy controls with normal glucose tolerance (NGT) were collected. Then the transcription level of pre-miR-375 and mature miR-375 were examined using quantitative real-time PCR and the methylation status of 27 CpG sites in the miR-375 promoter was determined by MassARRAY Spectrometry. The relative expression of mature miR-375 was shown as fold difference relative to miR-16 (3.0-fold, P=0.0260) and pre-miR-375 was markedly unregulated (2.6-fold, P=0.0415) in Han T2DM samples. Aberrant methylation was significantly higher within the amplicon of the miR-375 promoter in T2DMs than in NGTs, an average of 10.27% and 7.24% (P=0.0004; Figure 3A), respectively. Further, one CpG unit (CpG_26.27) was significantly hypermethylated in T2DM samples compared with NGT. Together, our results highlights for the first time that aberrant hypermethylation is a common event in Han T2DM, suggesting that the aberrant methylation of the CpG sites within miR-375 promoter may serve as a potential candidate biomarker for T2DM in the Chinese Han population.

Association of plasma Fetuin-A and clinical characteristics in patients with new-onset type 2 diabetes mellitus.

CONTEXT: Fetuin-A is an abundant plasma protein known to inhibit insulin signaling and pathologic calcification, has emerged as a promising candidate biomarker for diabetes risk. OBJECTIVE: The objective of this study was to investigate the relationships between plasma Fetuin-A level with clinical characteristics in patients with new-onset type 2 diabetes mellitus (nT2DM). SUBJECTS AND METHODS: Plasma Fetuin-A levels, and clinical characteristics were assessed in 100 patients with nT2DM and 100 normal glucose tolerance (NGT). RESULTS: nT2DM subjects had significantly higher Fetuin-A levels than NGT subjects (368.5 ± 15.6 vs 152.7 ± 7.1 mg/ml, P < 0.01). In the Pearson's correlation coefficients, Fetuin-A levels and clinical parameters. Fetuin-A was positively correlated with HOMA-insulin resistance index (HOMA-IR), carotid intima media thickness(CIMT), HbA1c, triglyceride (TG), Low-density lipoprotein cholesterol (LDL-C), body mass index (BMI), systolic blood pressure (SBP), fasting plasma glucose (FBG) and 2 h post-glucose load blood glucose (2 h OGTT) (P < 0.05 and P < 0.01), but negatively with fasting plasma insulin (FINS), 2 h plasma insulin after glucose overload (PINS), High-density lipoprotein cholesterol (HDL-C) and HOMA-beta-cell insulin secretion index (HOMA-IS) (P < 0.05 and P < 0.01). However, no significant relationships were observed between plasma Fetuin-A levels and estimated glomerular filtration rate (eGFR), age and gender in nT2DM subjects. In a multiple linear regression analysis, Fetuin-A levels were independently associated with FBG, 2 h OGTT, HOMA-IS, TG, and CIMT (R(2) = 0.6760). CIMT were negatively associated with FINS and HDL-C (r = -0.33, P = 0.008; r = -0.31, P = 0.01, respectively) in the Pearson's analyses. Moreover, they were positively associated with HOMA-IR (r = 0.28, P = 0.03). It showed significant correlations of plasma CIMT with FINS, PINS and HOMA-IR (R(2) = 0.6760). CONCLUSIONS: Our study suggests that the plasma Fetuin-A levels may be associated with macroangiopathies in nT2DM patients. Therefore, detecting early plasma Fetuin-A levels nT2DM provides an opportunity to intervene of carotid artery disease in diabetic patients and giving timely treatment for the prevention of diabetic vascular complications.

Association between fetuin-A levels with insulin resistance and carotid intima-media thickness in patients with new-onset type 2 diabetes mellitus.

Fetuin-A, which is known to inhibit insulin signaling and pathological calcification, has emerged as a diabetes risk biomarker. In the present study, the association between the fetuin-A levels with insulin resistance (IR) and carotid intima-media thickness (CIMT) was investigated in patients with new-onset type 2 diabetes mellitus (nT2DM). A total of 100 patients with nT2DM (nT2DM group) and 100 normal glucose tolerance (NGT group) controls were evaluated. The serum fetuin-A level was measured by a commercial solid-phase ELISA kit. The estimate of IR was calculated by homeostasis model assessment (HOMA-IR). CIMT was measured by B-mode ultrasound. The association between the serum fetuin-A levels and the metabolic parameters was also analyzed. The serum fetuin-A levels were increased significantly in the nT2DM group compared to the NGT group (368.5±15.6 mg/ml vs. 152.7±7.1 mg/ml, P<0.01). Fetuin-A was positively correlated with HOMA-IR, CIMT, glycated hemoglobin, triglyceride, low-density lipoprotein cholesterol, body mass index, systolic blood pressure, fasting blood glucose and 2 h post-glucose load blood glucose (P<0.05 and P<0.01), but negatively correlated with fasting plasma insulin, 2 h plasma insulin after glucose overload, high-density lipoprotein cholesterol and HOMA-ß-cell insulin secretion index (P<0.05 and P<0.01). To the best of our knowledge, the study demonstrated for the first time that there is a significant association between the serum fetuin-A levels with IR and CIMT in nT2DM. These results indicate that serum fetuin-A levels can be used as independent markers in the diagnosis of macroangiopathies in nT2DM.

Analysis of PTEN methylation patterns in soft tissue sarcomas by MassARRAY spectrometry.

Soft tissue sarcomas (STSs) are a rare and fascinating group of diseases that can be subdivided into specific reciprocal translocations in STSs (SRTSs) and nonspecific reciprocal translocations in STSs (NRTSs). PTEN mutations are rare in STSs, suggesting that PTEN expression may be lost by alternative mechanisms such as methylation. In order to reveal whether aberrant PTEN methylation occurs in STSs, MassARRAY Spectrometry was carried to detect methylation patterns of PTEN in STSs. We evaluated methylation levels in 41 CpG sites from -2,515 to -2,186 bp (amplicon A) and -1,786 to -1,416 bp (amplicon B) relative to the translation initiation site in 110 different cases (46 cases of SRTSs, 40 cases of NRTSs, and 24 cases of normal controls). In addition, immunohistochemistry (IHC) was used to detect the loss of PTEN to determine whether PTEN alterations were responsible for decreased PTEN expression. Our data showed that expression of PTEN was diminished in 49 (57%) STSs, whereas the remaining cases (43%) were classified as high expression. Our previous results found that only 2 of 86 cases (2.3%) had a PTEN mutation suggesting that PTEN may be mainly downregulated in STSs by methylation, but not by mutation of PTEN itself. We observed that amplicon A was hypermethylated in STSs with low PTEN expression, whereas normal controls had low methylation levels (P<0.0001), which was not present in amplicon B (P>0.05), nor were there significant differences in the methylation levels in PTEN between SRTS and NRTS cases. The majority of individual CpG units within two amplicons was demonstrated to be hypermethylated. These findings indicate that PTEN hypermethylation is a common event in STSs suggesting that the inactivation of PTEN may be due to hypermethylation in the promoter of PTEN. The aberrant methylation of the CpG sites within PTEN promoter may serve as a potential candidate biomarker for STSs.

Association between polymorphisms of the E-selectin gene, hepatitis B virus DNA copies and preS1 antigen in patients with chronic hepatitis B infection.

The aim of this study was to investigate the relationship between E-selectin +G98T, +A561C polymorphisms and the levels of hepatitis B virus (HBV) DNA and preS1 antigen (preS1Ag) in patients with chronic hepatitis B (CHB) infection. Polymorphisms of the E-selectin gene in 150 CHB patients and 150 healthy controls of two different nationalities were detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Real-time quantitative PCR was used to detect the levels of HBV DNA. preS1Ag and five items of hepatitis B were detected by enzyme-linked immunosorbent assay. Two genotypes, GG (94%, 96%) and GT (6%, 4%) of the E-selectin +G98T polymorphism, and AA (78.67%, 80.67%) and AC (21.33%, 19.33%) of the +A561C polymorphism, were found in these patients. There were also significant differences in the two nationalities in the genotypic frequencies in +A561C polymorphisms between patients and healthy subjects (χ2=5.489, χ2=5.653; P<0.05). In the patients studied, the relative risk of suffering from CHB in genotype AC was 2.122 and 2.313-fold higher for the two nationalities, respectively, than that of the AA genotype (OR=2.122, 95% CI 1.121-4.019; OR=2.313, 95% CI 1.002-5.360). There was also significant over-representation in the C allele frequency between the two groups (χ2=5.000, χ2=5.30; P<0.05), and the levels of HBV DNA and preS1Ag in the AC genotype patients were higher than those in the AA genotype (P<0.01 and P<0.05). E-selectin +A561C and +G98T polymorphisms were present in the populations studied. Therefore, there is a correlation between E-selectin +A561C polymorphisms and CHB. Allele C may be one of the predisposing factors, and mutation of this locus may impact the virus copy number.