Background: Osteoarthritis is a chronic musculoskeletal disease characterized by age-related gradual thinning and a high risk in females. Recent studies have shown that DNA methylation plays important roles in osteoarthritis. However, the genome-wide pattern of methylation in enhancers in osteoarthritis remains unclear. Methods: To explore the function of enhancers in osteoarthritis, we quantified CpG methylation in human enhancers based on a public dataset that included methylation profiles of 470,870 CpG probes in 108 samples from patients with hip and knee osteoarthritis and hip tissues from healthy individuals. Combining various bioinformatics analysis tools, we systematically analyzed aberrant DNA methylation of the enhancers throughout the genome in knee osteoarthritis and hip osteoarthritis. Results: We identified 16,816 differentially methylated CpGs, and nearly half (8111) of them were from enhancers, suggesting major DNA methylation changes in both types of osteoarthritis in the enhancer regions. A detailed analysis of hip osteoarthritis identified 2426 differentially methylated CpGs in enhancers between male and female patients, and 84.5% of them were hypomethylated in female patients and enriched in phenotypes related to hip osteoarthritis in females. Next, we explored the enhancer methylation dynamics among patients with knee osteoarthritis and identified 280 differentially methylated enhancer CpGs that were enriched in the human phenotypes and disease ontologies related to osteoarthritis. Finally, a comparison of enhancer methylation between knee osteoarthritis and hip osteoarthritis revealed organ source-dependent differences in enhancer methylation. Conclusion: Our findings indicate that aberrant methylation of enhancers is related to osteoarthritis phenotypes, and a comprehensive atlas of enhancer methylation is useful for further analysis of the epigenetic regulation of osteoarthritis and the development of clinical drugs for treatment of osteoarthritis.

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Osteoporosis is an age-related disease characterized by reduced bone volume and disturbed bone metabolism. Novel therapies to rescue or prevent reduced bone mass by guiding the differentiation of pluripotent bone marrow stromal cells away from adipocyte differentiation and toward osteoblastic differentiation may serve as a valuable treatment option against osteoporosis. Estrogen has long been recognized as a key effector of bone formation and mineralization, but the exact mechanisms involved remain poorly understood. In the present study, we investigated the role of the estrogen-specific G protein-coupled receptor 30 (GPR30/GPER) using its specific agonist G1 in MC3T3-E1 preosteoblast cells. Our findings demonstrate that expression of GPR30 is upregulated during osteoblast differentiation and that agonism of GPR30 significantly increases some key markers of mineralization including alkaline phosphatase, osteocalcin, osterix, and type I collagen. We also demonstrate that GPR30 agonism upregulates expression of Runx2, which is recognized as an essential transcription factor involved in bone formation. Additionally, through a series of adenosine monophosphate-activated protein kinase (AMPK)inhibition experiments using compound C, we show that the positive effects of GPR30 on mineralization and differentiation of preosteoblasts are mediated through the AMPK/anti-acetyl-CoA carboxylase (ACC) pathway. Taken together, the findings of the present study demonstrate the potential of GPR30 as a novel target for the treatment and prevention of osteoporosis. K E Y W O R D S adenosine monophosphate-activated protein kinase (AMPK), anti-acetyl-CoA carboxylase (ACC), bone metabolism, estrogen receptor, G protein-coupled receptor 30 (GPR30), G1, osteoblast differentiation, osteoporosis, Runx2

This study aims to investigate the role of lncRNA growth arrest-specific transcript 5 (GAS5)/miR-362-5p/suppressor of morphogenesis in the genitalia 1 (SMG1) axis in 131 I-resistance in thyroid cancer (TC). GAS5, miR-362-5p, and SMG1 expression in TC tissues was assessed and the 131 I-resistant TC cells were established, which were treated with altered GAS5, miR-362-5p, and SMG1. The proliferation and apoptosis of 131 I-resistant TC cells were detected, and the expression of Akt/mTOR signaling pathway-related proteins was assessed. Binding relations between GAS5 and miR-362-5p, and miR-362-5p and SMG1 were confirmed. The role of GAS5 in 131 I-resistant TC cell growth in vivo was observed. GAS5 was downregulated and miR-362-5p was upregulated in TC tissues and 131 I-resistant cells. The 131 I-resistant TC cells had enhanced proliferation and repressed apoptosis, and the Akt/mTOR signaling pathway was activated. Overexpressed GAS5 strengthened 131 I sensitivity and suppressed TC cell growth, while upregulated miR-362-5p had an opposite effect. MiR-362-5p upregulation reversed the effect of GAS5, and SMG1 overexpression eliminated the impact of miR-362-5p upregulation on 131 I-resistant TC cells. GAS5 competitively binds to miR-362-5p and SMG1 is targeted by miR-362-5p. GAS5 sponges miR-362-5p to promote sensitivity of TC cells to 131 I by upregulating SMG1 and inactivating Akt/mTOR signaling pathway.