Increased P16 DNA Methylation in Mouse Thymic Lymphoma Induced by Irradiation

DNA methylation is an important part of epigenetics. In this study, we examined the methylation state of two CpG islands in the promoter of the p16 gene in radiation-induced thymic lymphoma samples. The mRNA and protein levels of P16 were significantly reduced in radiation-induced thymic lymphoma tissue samples. Twenty-three CpG sites of the CpG islands in the p16 promoter region were detected, and the methylation percentages of −71, −63, −239, −29, −38, −40, −23, 46 CpG sites were significantly higher in radiation-induced thymic lymphoma tissue samples than those in matched non-irradiated thymus tissue samples. This study provides new evidence for the methylation state of p16 in the radiation-induced thymic lymphoma samples, which suggests that the methylation of these CpG sites in the p16 promoter may reduce its expression in the thymic lymphoma after irradiation.     

Transactivation of MicroRNA-320 by MicroRNA-383 Regulates Granulosa Cell Functions by Targeting E2F1 and SF-1 Proteins

Our previous studies have shown that microRNA-320 (miR-320) is one of the most down-regulated microRNAs (miRNA) in mouse ovarian granulosa cells (GCs) after TGF-β1 treatment. However, the underlying mechanisms of miR-320 involved in GC function during follicular development remain unknown. In this study, we found that pregnant mare serum gonadotropin treatment resulted in the suppression of miR-320 expression in a time-dependent manner. miR-320 was mainly expressed in GCs and oocytes of mouse ovarian follicles in follicular development. Overexpression of miR-320 inhibited estradiol synthesis and proliferation of GCs through targeting E2F1 and SF-1. E2F1/SF-1 mediated miR-320-induced suppression of GC proliferation and of GC steroidogenesis. FSH down-regulated the expression of miR-320 and regulated the function of miR-320 in mouse GCs. miR-383 promoted the expression of miR-320 and enhanced miR-320-mediated suppression of GC proliferation. Injection of miR-320 into the ovaries of mice partially promoted the production of testosterone and progesterone but inhibited estradiol release in vivo. Moreover, the expression of miR-320 and miR-383 was up-regulated in the follicular fluid of polycystic ovarian syndrome patients, although the expression of E2F1 and SF-1 was down-regulated in GCs. These data demonstrated that miR-320 regulates the proliferation and steroid production by targeting E2F1 and SF-1 in the follicular development. Understanding the regulation of miRNA biogenesis and function in the follicular development will potentiate the usefulness of miRNA in the treatment of reproduction and some steroid-related disorders.     

Regulation of the Follistatin Gene by RSPO-LGR4 Signaling via Activation of the WNT/β-Catenin Pathway in Skeletal Myogenesis

WNT signaling plays multiple roles in skeletal myogenesis during gestation and postnatal stages. The R-spondin (RSPO) family of secreted proteins and their cognate receptors, members of leucine-rich repeat-containing G protein-coupled receptor (LGR) family, have emerged as new regulatory components of the WNT signaling pathway. We previously showed that RSPO2 promoted myogenic differentiation via activation of WNT/β-catenin signaling in mouse myoblast C2C12 cells in vitro. However, the molecular mechanism by which RSPO2 regulates myogenic differentiation is unknown. Herein, we show that depletion of the LGR4 receptor severely disrupts myogenic differentiation and significantly diminishes the response to RSPO2 in C2C12 cells, showing a requirement of LGR4 in RSPO signaling during myogenic differentiation. We identify the transforming growth factor β (TGF-β) antagonist follistatin (Fst) as a key mediator of RSPO-LGR4 signaling in myogenic differentiation. We further demonstrate that Fst is a direct target of the WNT/β-catenin pathway. Activation and inactivation of β-catenin induced and inhibited Fst expression, respectively, in both C2C12 cells and mouse embryos. Specific TCF/LEF1 binding sites within the promoter and intron 1 region of the Fst gene were required for RSPO2 and WNT/β-catenin-induced Fst expression. This study uncovers a molecular cross talk between WNT/β-catenin and TGF-β signaling pivotal in myogenic differentiation.     

DNA methylation regulates sclerostin (SOST) expression in osteoarthritic chondrocytes by bone morphogenetic protein 2 (BMP-2) induced changes in Smads binding affinity to the CpG region of SOST promoter

IntroductionSclerostin (SOST), a soluble antagonist of Wnt signaling, is expressed in chondrocytes and contributes to chondrocytes’ hypertrophic differentiation; however its role in osteoarthritis (OA) pathogenesis is not well known. Based on our previous findings on the interaction between Wnt/β-catenin pathway and BMP-2 in OA, we aimed to investigate the role of DNA methylation and BMP-2 on SOST’s expression in OA chondrocytes.MethodsSOST mRNA and protein expression levels were investigated using real-time polymerase chain reaction (PCR) and Western blot, respectively. The methylation status of SOST promoter was analysed using methylation-specific PCR (MSP), quantitative methylation-specific PCR (qMSP) and bisulfite sequencing analysis. The effect of BMP-2 and 5’-Aza-2-deoxycytidine (5-AzadC) on SOST’s expression levels were investigated and Smad1/5/8 binding to SOST promoter was assessed by Chromatin Immunoprecipitation (ChΙP).ResultsWe observed that SOST’s expression was upregulated in OA chondrocytes compared to normal. Moreover, we found that the CpG region of SOST promoter was hypomethylated in OA chondrocytes and 5-AzadC treatment in normal chondrocytes resulted in decreased SOST methylation, whereas its expression was upregulated. BMP-2 treatment in 5-AzadC-treated normal chondrocytes resulted in SOST upregulation, which was mediated through Smad 1/5/8 binding on the CpG region of SOST promoter.ConclusionsWe report novel findings that DNA methylation regulates SOST’s expression in OA, by changing Smad 1/5/8 binding affinity to SOST promoter, providing evidence that changes in DNA methylation pattern could underlie changes in genes’ expression observed in OA.     

Berberine reduces methylation of the MTTP promoter and alleviates fatty liver induced by a high-fat diet in rats

High-calorie food leads to nonalcoholic fatty liver disease (NAFLD) through dysregulation of genes involved in lipid metabolism, but the precise mechanism remains unclear. DNA methylation represents one of the mechanisms that contributes to dysregulation of gene expression via interaction with environmental factors. Berberine can alleviate fatty liver in db/db and ob/ob mice. Here, we investigated whether DNA methylation is involved in the pathogenesis of NAFLD induced by a high-fat diet (HFD) and whether berberine improves NAFLD through influencing the methylation status of promoters of key genes. HFD markedly decreased the mRNA levels encoding CPT-1α, MTTP, and LDLR in the liver. In parallel, DNA methylation levels in the MTTP promoter of rats with NAFLD were elevated in the liver. Interestingly, berberine reversed the downregulated expression of these genes and selectively inhibited HFD-induced increase in the methylation of MTTP. Consistently, berberine increased hepatic triglyceride (TG) export and ameliorated HFD-induced fatty liver. Furthermore, a close negative correlation was observed between the MTTP expression and its DNA methylation (at sites −113 and −20). These data indicate that DNA methylation of the MTTP promoter likely contributes to its downregulation during HFD-induced NAFLD and, further, that berberine can partially counteract the HFD-elicited dysregulation of MTTP by reversing the methylation state of its promoter, leading to reduced hepatic fat content.     

Genome-wide methylation and expression differences in HPV(+) and HPV(?) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis

Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(−) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(−) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(−) cell lines. Differentially methylated loci between HPV(+) and HPV(−) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(−) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(−) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(−) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.Key words: epigenetics, human papillomavirus, HNSCC, DNA methylation, squamous cell carcinoma, gene expression, microarrays, illumina infinium humanmethylation27 beadarray