Bio-responsive Au-miR-183 inhibitor enhances immunotherapy in hepatocellular carcinoma by inducing immunogenic cell death.

The treatment of advanced hepatocellular carcinoma (HCC) is limited, and immunotherapy is the current research focus of multi-disciplinary collaborative comprehensive treatment of HCC. Herein, we constructed a bio-responsive Au-miR-183 inhibitor (Au@miR-183i) delivery system targeting liver cancer stem cells (LCSCs), and adopted the strategy of combining αPD-L1 immunotherapy. The multifunctional Au@miR-183i nanocomplexes (NCs), which self-assemble based on the tumor microenvironment, consume NADPH and H2O2, leading to redox homeostasis disturbance, ROS accumulation, regulation of the LCSC niche, and induction of stemness regression. Moreover, self-assembled Au@miR-183i NCs specifically target the delivery of miR-183i to LCSCs, triggering the immunogenic cell death (ICD) effect, promoting the maturation of dendritic cells, inducing infiltration of CD8+ T cells, and facilitating the transformation of 'cold' tumors into 'hot' tumors. More importantly, consistent with the results in vitro, Au@miR-183i NCs demonstrated effective tumor targeting and strong ICD induction in vivo, assisted in enhancing αPD-L1 immunotherapy, and activated a robust systemic anti-tumor immune response in tumor-bearing mouse models. Overall, we provide a simple and universal therapeutic strategy by constructing a multifunctional bio-responsive Au@miR-183i NCs delivery system with LCSC targeting capability. Furthermore, nanocomplex-based ICD inducers have great promise in enhancing anti-tumor immunity and the PD-1/PD-L1 blocking efficacy in HCC, which provides a theoretical basis for effectively eliminating LCSCs and achieving a high-efficiency synergistic treatment strategy for HCC.

Empagliflozin improves mitochondrial dysfunction in diabetic cardiomyopathy by modulating ketone body metabolism and oxidative stress.

Ketone bodies are considered as an alternative energy source for diabetic cardiomyopathy (DCM) and can improve the energy supply of the heart muscle, suggesting that it may be an important area of research and development as a therapeutic target for DCM. Cumulative cardiovascular trials have shown that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce cardiovascular events in diabetic populations. Whether SGLT2 inhibitors improve DCM by enhancing ketone body metabolism remains and whether they help prevent oxidative damage remains to be clarified. Here, we present the combined results of nine GSE datasets for diabetic cardiomyopathy (GSE215979, GSE161931, GSE145294, GSE161052, GSE173384, GSE123975, GSE161827, GSE210612, and GSE5606). We found significant up-regulated gene 3-hydroxymethylglutaryl CoA synthetase 2 (HMGCS2) and down-regulated gene 3-hydroxybutyrate dehydrogenase (BDH1) and 3-oxoacid CoA-transferase1 (OXCT1), respectively. Based on the analysis of the constructed protein interaction network, it was found that HMGCS2 was in the core position of the interaction network. In addition, Gene ontology (GO) enrichment analysis mainly focused on redox process, acyl-CoA metabolic process, catalytic activity, redox enzyme activity and mitochondria. The activity of HMGCS2 in DCM heart was increased, while the expression of ketolysis enzymes BDH1 and OXCT1 was inhibited. In vivo, Empagliflozin (Emp) treated DCM group significantly decreased ventricular weight, myocardial cell cross-sectional area, and myocardial fibrosis. In addition, Emp further promoted the activity of BDH1 and OXCT1, increased the utilization of ketone bodies, further promoted the activity of HMGCS2 in DCM, and increased the synthesis of ketone bodies, prevented mitochondrial breakage and dysfunction, increased myocardial ATP to provide sufficient energy, inhibited oxidative stress and apoptosis of cardiac cells ex vivo, and improved the myocardial dysfunction of DCM. Emp can improve mitochondrial dysfunction in diabetic cardiomyopathy by regulating ketone body metabolism and oxidative stress. These findings provide a theoretical basis for evaluating Emp as a treatment for DCM.

Proteomic Analysis of Serum Lysine Acetylation in Uyghur Patients With T2DM.

Lysine acetylation is a reversible modification process after protein translation, which plays a key regulatory role in various metabolic diseases such as diabetes. The prevalence of type 2 diabetes mellitus (T2DM) in the Uyghur population is high, but the acetylation status of proteomics in Uyghur with T2DM is still unclear. Herein, we performed a quantitative proteomic study of lysine acetylation in T2DM patients using Tandem Mass Tags (TMTs) labeling, acetylation enrichment techniques, and high-resolution liquid chromatography-tandem mass spectrometry. We quantified 422 acetylation sites on 120 proteins, of which 347 sites of 103 proteins contained quantitative information. Compared with the control, we found that a total of eight acetylated sites within proteins were significantly differentially expressed with three upregulated and five downregulated, including histones H4 and H3.3C. Meanwhile, we completed bioinformatics analysis, including protein annotation, functional classification, functional enrichment, and cluster analysis, based on functional enrichment. In addition, the mRNA (ApoB-100, histones H4 and H3.3C) and protein (histones H4 and H3.3C) levels were verified through 60 samples. Besides, we also performed histone H4 chromatin immunoprecipitation analysis at the level of INS-1 cells. These could be potentially useful markers for the prediction of prediabetes and also provided a basis for the pathogenesis of T2DM.

Intelligent Bio-Responsive Fluorescent Au-shRNA Complexes for Regulated Autophagy and Effective Cancer Bioimaging and Therapeutics.

The long non-coding RNA (lncRNA) MALAT1 acts as an oncogene. RNA interference (RNAi) is an effective method to control the expression of specific genes and can be used for the treatment of tumors, but an effective and safe carrier system is a significant obstacle to gene therapy. Herein, we explored the possibility of constructing an in situ bio-responsive self-assembled fluorescent gold-short hairpin RNA nanocomplex (Au-shRNA NCs) delivery system by co-incubating gold and MALAT1-shRNA for precise hepatocellular carcinoma (HCC) imaging and treatment. Due to the characteristics of the cancer microenvironment, Au-shRNA NCs self-assembled in HCC cells (HepG2) but did not occur in control cells (L02) under the same conditions. The in situ bio-responsive self-assembled Au-shRNA NCs delivery system can realize cancer cell bioimaging and promote cell uptake and endosomal escape mechanism, thereby realizing effective transfection. They effectively silenced target gene MALAT1, and with the downregulation of MALAT1, we found that several molecules involved in autophagic flux were also regulated. In vitro and tumor-bearing mouse model experiments demonstrated that the as-prepared fluorescent Au-shRNA NCs can readily realize tumor bioimaging and effectively silence the target gene MALAT1, and those autophagy-related pathway molecules were significantly downregulated, thereby exerting a tumor suppressor efficiency. This raises the possibility of realizing accurate multi-scale bio-imaging from the molecular-level with targeted gene-recognition to cancer cell imaging as well as in vivo tumor tissue imaging for the simultaneous precise cancer therapy.

In situ self-assembly of Au-antimiR-155 nanocomplexes mediates TLR3-dependent apoptosis in hepatocellular carcinoma cells.

MicroRNA 155 (miRNA-155) is frequently dysregulated in hepatocellular carcinoma (HCC) and other cancer types. Toll-like receptor 3 (TLR3), a putative miR-155 target, plays a key role in liver pathophysiology, and its downregulation in HCC cells is associated with apoptosis evasion and poor outcomes. Herein, we examined the ability of in situ self-assembled Au-antimiR-155 nanocomplexes (Au-antimiRNA NCs) to activate TLR3 signaling in HCC cells. Gene expression analysis confirmed an inverse relationship between miR-155 and TLR3 expression in HCC samples, and marked upregulation of miR-155 was observed in HCC cells but not in normal L02 hepatocytes. RNA immunoprecipitation confirmed physical interaction between miR-155 and TLR3, while negative regulation of TLR3 expression by miR-155 was demonstrated by luciferase reporter assays. Au-antimiR-155 NCs were self-assembled within HepG2 HCC cells, but not within control L02 cells. They efficiently silenced miR-155, thereby inhibiting proliferation and migration and inducing apoptosis in HepG2 cells. Molecular analyses suggested these effects are secondary to TLR3 signaling mediating NF-κB transcription, caspase-8 activation, and interleukin-1ß (IL-1ß) release. Our results provide a basis for future studies examining the in vivo applicability of this novel Au-antimiRNA NCs delivery system to halt HCC progression by activating pro-apoptotic TLR3 signaling.

Identification of candidate lncRNAs and circRNAs regulating WNT3/ß-catenin signaling in essential hypertension.

Mounting evidence suggests that noncoding RNAs (ncRNAs) contribute to the pathogenesis of cardiovascular diseases. However, their role in essential hypertension (EH) is still unclear. We therefore identified differentially expressed long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in EH patients from a high-risk population group and constructed a competing endogenous RNA regulatory network that predicts interactions of potential diagnostic and therapeutic relevance between specific lncRNA/circRNA-microRNA-mRNA triplets. Our analysis identified two lncRNAs, transmembrane protein 183A pseudogene (LOC646616) and leucine aminopeptidase 3 pseudogene 2 (LAP3P2), and two circRNAs, hsa_circ_0039388 and hsa_circ_0038648, that are highly co-expressed with both wingless-type MMTV integration site family member 3 (WNT3) and calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2) mRNAs and also share common microRNA binding sites with these two transcripts. We also confirmed that a mutually regulated network composed of LOC646616/microRNA-637/WNT3 controls WNT3 expression and influences viability and invasive properties in human arterial smooth muscle cells in vitro. These findings highlight a novel ncRNA-based regulatory mechanism potentially driving WNT/ß-catenin activation in EH, and suggest that the identified ncRNAs may represent useful biomarkers and therapeutic targets for this condition.

Bio responsive self-assembly of Au-miRNAs for targeted cancer theranostics.

BACKGROUND: MicroRNA (miRNA) therapeutics are a promising approach to cancer treatment. However, this method faces considerable challenges to achieve tissue-specific, efficient, and safe delivery of miRNAs in vivo. METHODS: Herein, we developed a miRNA delivery system based on the in situ self-assembly of Au-miRNA nanocomplexes (Au-miRNA NCs). Within the cancer microenvironment, we constructed in situ self-assembled Au-miRNA NCs by coincubating gold salt and tumor suppressor mimics, such as let-7a, miRNA-34a, and miRNA-200a. FINDINGS: The in vitro experiments demonstrated that characteristic in situ self-assembled Au-miRNA NCs were present in cancer cells and can be taken up to inhibit the proliferation of cancer cells effectively. Most importantly, as proven in subcutaneous tumor treatment models, Au-miRNA NCs were especially useful for accurate target imaging and tumor suppression, with significantly enhanced antitumor effects for combination therapy. INTERPRETATION: These observations highlight that a new strategy for the in situ biosynthesis of Au-let-7a NCs, Au-miR-34a NCs, and Au-miR-200a NCs is feasible, and this may assist in the delivery of more miRNA to tumor cells for cancer treatment. This work opens up new opportunities for the development of miRNA tumor therapy strategies. FUNDING: National Natural Science Foundation of China (91753106); Primary Research & Development Plan of Jiangsu Province (BE2019716); National Key Research and Development Program of China (2017YFA0205300).

In situ self-assembling Au-DNA complexes for targeted cancer bioimaging and inhibition.

Cancer remains one of the most challenging diseases to treat. For accurate cancer diagnosis and targeted therapy, it is important to assess the localization of the affected area of cancers. The general approaches for cancer diagnostics include pathological assessments and imaging. However, these methods only generally assess the tumor area. In this study, by taking advantage of the unique microenvironment of cancers, we effectively utilize in situ self-assembled biosynthetic fluorescent gold nanocluster-DNA (GNC-DNA) complexes to facilitate safe and targeted cancer theranostics. In in vitro and in vivo tumor models, our self-assembling biosynthetic approach allowed for precise bioimaging and inhibited cancer growth after one injection of DNA and gold precursors. These results demonstrate that in situ bioresponsive self-assembling GNC-PTEN (phosphatase and tensin homolog) complexes could be an effective noninvasive technique for accurate cancer bioimaging and treatment, thus providing a safe and promising cancer theranostics platform for cancer therapy.

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.

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.

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.

Relationship between the single nucleotide polymorphisms of ß2-adrenergic receptor 5'-regulatory region and essential hypertension in Chinese Kazakh ethnic minority group.

OBJECTIVE: To study the correlation of ß2-AR gene 5'-regulatory region SNPs and essential hypertension (EH) in Chinese Kazakh ethnic minority group. METHODS: The Sequenom MassArray(®) SNP detection technology was used to detect ß2-AR gene 5'-regulatory region SNPs in 150 Xinjiang Kazakh EH patients and 150 controls. Biochemical analyzer was used to detect lipid and other related biochemical parameters. SHEsis and other software were used to analyze linkage disequilibrium and haplotype. RESULTS: Six loci rs205304 (-1023G/A), rs17108803 (-893T/G), rs12654778 (-654G/A), rs11168070 (-468C/G), rs11959427 (-367C/T) and rs2895795 (-1429T/A) polymorphisms of ß2-AR gene 5'-regulatory region were found in the Xinjiang Kazakh populations. While, there was no significant difference between EH group and NH in genotypes and allele frequency of rs2053044, rs12654778, rs2895795, rs17108803 and rs11959427 (P>0.05). However; significant differences were detected of rs11168070 genotypes and allele frequency in two groups (P<0.05). Analysis of the linkage disequilibrium and haplotype in Kazakh population, there is a strong linkage disequilibrium of rs11168070, rs2053044, rs2895795 gene polymorphism in the EH group, and rs11168070, rs12654778, rs17108803 gene polymorphism in controls. Frequency of haplotype GTCCAT, GACTGT and ATGCGT in EH group was higher (P<0.05), while frequency of ATCTGT, ATGTGT, GTCCGT, GTCTAT, GACCAT and GTCTGT in the EH group was significantly lower than the control (P<0.05). CONCLUSIONS: ß2-AR gene 5'-regulatory region of rs11168070, rs2053044, rs17108803, rs12654778, rs11959427 and rs2895795 genetic polymorphism exists in Kazakh. Among them, rs11168070 locus genotype and allele frequency distribution in the two groups are significant differences. In six polymorphic loci, there is a strong linkage disequilibrium, which haplotypes GTCCAT, GACTGT, ATGCGT are risk factors of EH, and the ATCTGT, ATGTGT, GTCCGT, GTCTAT, GACCAT, GTCTGT are protective factors.

Correlation between polymorphisms of the E-selectin gene, hepatitis B virus DNA copies, pre-S1 antigen and clinical outcomes during chronic hepatitis B.

The aim of this study was to investigate the relationships between E-selectin +G98T, +A561C polymorphisms and different progression in Hepatitis B virus (HBV) infection Xinjiang Han population, also to determine the HBV DNA copies and pre-S1 antigen (preS1Ag) in this population. Polymorphisms of the E-selectin gene in 200 chronic HBV infection (61 cases of chronic HBV carriers, chronic hepatitis B 75, liver cirrhosis 43, liver cancer 21) and 200 healthy controls 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. Liver fibrosis using chemiluminescence, biochemical markers using Roche 7600 automatic biochemical analyzer. E-selectin +A561C polymorphism of A/C genotype and C allele frequency in chronic hepatitis B (CHB) and cirrhosis (LC) group were compared with the control group had significant difference (P < 0.05). The risk of CHB and LC, AC genotype were 2.09, 2.33 times of the AA genotype. In group of CHB, the levels of HBV DNA and preS1Ag in the AC genotype patients were higher than those in the AA genotype (P < 0.05). However, there were no significant differences in comparison of liver function and liver fibrosis index in different genotypes of CHB and LC group. +A561C and +G98T linkage disequilibrium analysis showed: D' = 0.632, r(2) = 0.202, haplotype analysis showed that the G-A haplotype OR = 0.507, G-C haplotype OR = 1.973. E-selectin +A561C polymorphism may have some correlation with the occurrence of CHB and LC, and allele C may be one of the predisposing factors. AC polymorphism may affect HBV replication in CHB, but may not play an important and direct effect on liver injury and liver fibrosis after HBV infection. There were some linkage of +A561C and +G98T, G-C haplotype may be a risk factor for chronic HBV infection.

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.

Association between Hcy levels and the CBS844ins68 and MTHFR C677T polymorphisms with essential hypertension.

The aim of the present study was to investigate the association between the homocysteine (Hcy) levels and polymorphisms of the CBS844ins68 and MTHFR C677T genes in essential hypertension (EH). The effects of the MTHFR C677T and CBS844ins68 haploid genotypes and the combined genotypes on EH and levels of Hcy were further explored. The polymorphisms of CBS844ins68 and MTHFR C677T genes in 200 EH and 200 normal tensive (NT) patients were detected using polymerase chain reaction-restriction fragment length polymorphism and analysis of the distribution of genotypes. An automated biochemical analyzer was used to measure the plasma Hcy levels and the clinical biochemistry data. The plasma Hcy levels in EH were significantly higher than those of the NT group (P<0.05). There were no significant differences (P>0.05) between males and females. Two genotypes, deletion/deletion (DD) and deletion/insertion (DI), of the CBS844ins68 polymorphism were found in two groups with no clear differences in two genotypes and allele frequency distribution (P>0.05). There were significant differences in the three genotype frequencies (χ2=6.658, χ2=4.410, P<0.05) for MTHFR C677T locus genotypes CC, CT and TT. The Hcy levels in genotypes DD and DI had no significant differences (P>0.05) and the CT and TT types were significantly higher compared to the CC genotype (P<0.05). The CC/DD combined genotype in the two groups was significantly different (P<0.05), and the odds ratio (OR), 0.569 showed that the CC/DD genotype may be a protective factor of hypertension. In the two groups, the Hcy levels for combined genotypes CC/DD, CT/DD, TT/DD and TT/DI were significantly different (P<0.05). The SHEsis software analysis linkage disequilibrium coefficient=0.216, indicates that there is probably a weak linkage for MTHFR C677T and CBS844ins68. Haplotype analysis suggested that the C-D haplotype was negatively correlated with EH (OR, 0.727) and that there was a positive correlation between T-D haplotype and EH (OR, 1.376). MTHFR C677T and CBS844ins68 polymorphisms were present in the populations studied and the CBS844ins68 homozygous mutation was not present. Therefore, there is a correlation between the polymorphisms of the MTHFR C677T gene and EH, and allele T may be one of the predisposing factors. MTHFR C677T and CBS844ins68 may exist with a certain linkage and the T-D haplotype may be a risk factor for EH.

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.