The FOXO1 Gene-Obesity Interaction Increases the Risk of Type 2 Diabetes Mellitus in a Chinese Han Population.

Here, we aimed to study the effect of the forkhead box O1-insulin receptor substrate 2 (FOXO1-IRS2) gene interaction and the FOXO1 and IRS2 genes-environment interaction for the risk of type 2 diabetes mellitus (T2DM) in a Chinese Han population. We genotyped 7 polymorphism sites of FOXO1 gene and IRS2 gene in 780 unrelated Chinese Han people (474 cases of T2DM, 306 cases of healthy control). The risk of T2DM in individuals with AA genotype for rs7986407 and CC genotype for rs4581585 in FOXO1 gene was 2.092 and 2.57 times higher than that with GG genotype (odds ratio [OR] = 2.092; 95% confidence interval [CI] = 1.178-3.731; P = 0.011) and TT genotype (OR = 2.571; 95% CI = 1.404-4.695; P = 0.002), respectively. The risk of T2DM in individuals with GG genotype for Gly1057Asp in IRS2 gene was 1.42 times higher than that with AA genotype (OR = 1.422; 95% CI = 1.037-1.949; P = 0.029). The other 4 single nucleotide polymorphisms (SNPs) had no significant association with T2DM (P > 0.05). Multifactor dimensionality reduction (MDR) analysis showed that the interaction between SNPs rs7986407 and rs4325426 in FOXO1 gene and waist was the best model confirmed by interaction analysis, closely associating with T2DM. There was an increased risk for T2DM in the case of non-obesity with genotype combined AA/CC, AA/AC or AG/AA for rs7986407 and rs4325426, and obesity with genotype AA for rs7986407 or AA for rs4325426 (OR = 3.976; 95% CI = 1.156-13.675; P value from sign test [P(sign)] = 0.025; P value from permutation test [P(perm)] = 0.000-0.001). Together, this study indicates an association of FOXO1 and IRS2 gene polymorphisms with T2DM in Chinese Han population, supporting FOXO1-obesity interaction as a key factor for the risk of T2DM.

A SNaPshot assay for the rapid and simple detection of hepatitis B virus genotypes.

A simple technique for the identification of common genotypes of the hepatitis B virus (HBV) remains to be identified. The present study was conducted to establish such a methodology. Four plasmids of genotypes A­D and 123 clinical serum specimens of HBV­infected patients were genotyped. HBV genotypes would be detected successfully when the HBV genotype reached a viral load of 1 x 103 copies/ml or the BC genotype mixed samples reached a 5% level. The lower limit of detection of HBV DNA in serum specimens was determined to be 2.14x102 IU/ml. The assay sensitivity and specificity were 100% and the consistency was demonstrated to reach as high as 90.24 and 100% compared with that of the DNA sequencing and cloning. The frequencies of the genotypes B, C, BC, BD and BCD were found to be 65.0, 23.6, 7.3, 3.3 and 0.8%, respectively. The accuracy of detection of the mixed infections was also higher using the rapid and simple SNaPshot method compared with that achieved with the DNA sequencing methods. The results of the present study indicated that the SNaPshot technique accurately distinguishes the HBV genotypes A­D and is able to be readily applied as a monitoring tool in HBV prognosis and treatment.

Correlation between hepatitis B virus protein and microRNA processor Drosha in cells expressing HBV.

Drosha regulates the biogenesis of microRNAs (miRNAs) and plays an essential role in the regulation of gene expression. Infection with hepatitis B virus (HBV) causes chronic hepatitis and liver cirrhosis. It is also a major risk factor for hepatocellular carcinoma. Emerging evidence suggests that HBV alters miRNA expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. We therefore examined how HBV affected the production of miRNAs. We found that Drosha mRNA and protein expression were downregulated in cells expressing the HBV genome. This was associated with a reduction in the activity of the Drosha gene promoter. Gene silencing of HBx by RNA interference significantly restored the expression of Drosha. In conclusion, our data show that HBV could downregulate Drosha expression by inhibiting promoter activity, and the transcription factors SP1 and AP-2α may be involved in this process. This provides a new understanding of the mechanism of HBV-induced miRNAs dysregulation.

Hepatitis C virus core protein activates Wnt/ß-catenin signaling through multiple regulation of upstream molecules in the SMMC-7721 cell line.

The core protein of hepatitis C virus (HCV) has been implicated in HCV-induced liver pathogenesis. Previous data have shown that the HCV core protein has pleiotropic functions, including transcriptional regulation of a number of cellular genes, although the mechanism of gene regulation remains unclear. Wnt/ß-catenin signaling is also involved in hepatocellular carcinoma (HCC) tumorigenesis. To elucidate the molecular mechanism of HCV pathogenesis, we examined whether HCV core protein activates Wnt/ß-catenin signaling in the hepatoma cell line SMMC-7721. The effects of core protein on Wnt/ß-catenin signaling cascades were investigated by luciferase reporter gene assay, immunofluorescence, western blot and RT-PCR analysis. Here, we demonstrate that HCV core protein plays an essential role in activating ß-catenin/Tcf-4-dependent transcriptional activity and increases active ß-catenin expression and nuclear accumulation in SMMC-7721 cells. An RT-PCR assay indicated that core protein upregulates gene expression of canonical Wnt ligands, such as Wnt2, Wnt3, Wnt3a, Wnt8b, Wnt10a, Wnt10b, frizzled receptors Fzd1, 2, 5, 6, 7, 9, and LRP5/6 co-receptors. However, Wnt antagonists SFRP3, 5 and Dkk1 were moderately repressed. Furthermore, ectopic expression of core protein markedly promoted cell proliferation. The soluble Fzd molecule FrzB or the ß-catenin inhibitor siBC efficiently blocked cell growth stimulation by the core gene. Our present findings demonstrate that the HCV core protein activates canonical Wnt signaling through tight regulation of several important molecules upstream of ß-catenin and presumably results in promotion of cell proliferation in the SMMC-7721 cell line. Taken together, these data suggested that the core protein may be directly involved in Wnt/ß-catenin-mediated liver pathogenesis.

Hepatitis B virus protein up-regulated HLJ1 expression via the transcription factor YY1 in human hepatocarcinoma cells.

The hepatitis B virus (HBV) protein plays a major role in hepatocellular carcinoma (HCC) development. However, its contribution to tumor invasion and metastasis has not been established so far. HLJ1 was recently cloned and classified as a member of the heat shock protein 40 family (Hsp40/DnaJ) which is abundantly expressed in HBV-related tumors, might be involved in tumor progression. In this study, the role of HBV in activation of HLJ1 was investigated. In HepG2 cells with transit or stable expression of HBV, HLJ1 expression was activated by HBV. The activity assay of HLJ1 promoter revealed that HBV up-regulated HLJ1 expression through the transcription factor YY1 sites within the HLJ1 promoter. YY1 expression was significantly up-regulated by HBV in a concentration-dependent manner. Knockdown of YY1 expression could partially reduce the HBV-induced HLJ1 activation which indicated that YY1 would be involved in HBV-induced HLJ1 expression. In conclusion, our data showed that HBV could promote HLJ1 expression by up-regulating the transcription factor YY1, and this provided a new insight of the mechanism of HBV induction in tumor metastasis.

Hepatitis B virus regulates Raf1 expression in HepG2.2.15 cells by enhancing its promoter activity.

Raf1 kinase is a central component of many signaling pathways that are involved in normal cell growth and oncogenic transformation. The expression of Raf1 is significantly increased in hepatocellular carcinoma (HCC). HBV is a major risk factor for HCC. HBx protein can increase the expression of Raf1; however, the mechanism of how HBV regulates Raf1 expression is still unknown. In this study, we showed the Raf1 expression was significantly higher in HepG2.2.15 cells than that in HepG2 cells in vitro. HBV could up-regulate Raf1 expression by enhancing the activity of its promoter in a dose-dependent manner, and HBs and HBx may be involved in this process. After silencing HBs and HBx by using RNA interference, the expression of Raf1 in HepG2.2.15 cells could be significantly inhibited. These results might provide useful information for understanding the mechanism of HCC induced by HBV infection.