Epigenome‐wide Association of DNA Methylation in Whole Blood With Bone Mineral Density

Genetic and environmental determinants of skeletal phenotypes such as bone mineral density (BMD) may converge through the epigenome, providing a tool to better understand osteoporosis pathophysiology. Because the epigenetics of BMD have been largely unexplored in humans, we performed an epigenome‐wide association study (EWAS) of BMD. We undertook a large‐scale BMD EWAS using the Infinium HumanMethylation450 array to measure site‐specific DNA methylation in up to 5515 European‐descent individuals (N_Discovery = 4614, N_Validation = 901). We associated methylation at multiple cytosine‐phosphate‐guanine (CpG) sites with dual‐energy X‐ray absorptiometry (DXA)‐derived femoral neck and lumbar spine BMD. We performed sex‐combined and stratified analyses, controlling for age, weight, smoking status, estimated white blood cell proportions, and random effects for relatedness and batch effects. A 5% false‐discovery rate was used to identify CpGs associated with BMD. We identified one CpG site, cg23196985, significantly associated with femoral neck BMD in 3232 females (p = 7.9 × 10^?11) and 4614 females and males (p = 3.0 × 10^?8). cg23196985 was not associated with femoral neck BMD in an additional sample of 474 females (p = 0.64) and 901 males and females (p = 0.60). Lack of strong consistent association signal indicates that among the tested probes, no large‐effect epigenetic changes in whole blood associated with BMD, suggesting future epigenomic studies of musculoskeletal traits measure DNA methylation in a different tissue with extended genome coverage. © 2017 American Society for Bone and Mineral Research.     

重组腺病毒介导外源性SOX9在正常关节软骨细胞中诱导软骨基质合成的体外表达

背景：关节软骨损害是临床一种常见的损伤,软骨形成转录因子SOX9是一种调控Ⅱ型胶原合成的关键因子。 目的：观察以表达外源性SOX9的腺病毒体外成功感染关节软骨细胞后对Ⅱ型胶原和蛋白聚糖（Aggrecan）合成的影响,探讨软骨损伤后修复软骨缺损的基因治疗方法。 方法：体外构建腺病毒载体AdSOX9和AdGFP,成功感染培养的人关节软骨细胞,分别以逆转录聚合酶链式反应（RT-PCR）技术检测了病毒感染前后SOX9、II型胶原和蛋白聚糖基因mRNA的表达,同时以免疫组化技术检测了病毒感染前后关节软骨细胞中Ⅱ型胶原的表达。 结果：应用AdEasy腺病毒构建专利技术体外成功构建了能高效表达外源性SOX9的腺病毒AdSOX9和只表达绿色荧光蛋白GFP的腺病毒AdGFP;以腺病毒AdSOX9和AdGFP体外成功感染人关节软骨细胞后48h,未感染对照组和AdGFP感染组,均检测到了Ⅱ型胶原和蛋白聚糖的表达;而AdSOX9感染组的细胞中,检测到了SOX9基因mRNA的表达明显增高,与未感染对照组和AdGFP感染组相比有显著性差异（P〈0．05）,同时,Ⅱ型胶原和蛋白聚糖的表达也较未感染对照组和AdGFP感染组明显增高,差异显著（P〈0．05）。 结论：以外源性SOX9为目的基因的腺病毒介导基因治疗方法,在促进关节软骨细胞Ⅱ型胶原和蛋白聚糖合成方面得出了初步满意的结果,SOX9可能是关节软骨缺损基因治疗研究领域一个新的理想靶点,值得继续深入研究。     

TET3 Mediates Alterations in the Epigenetic Marker 5hmC and Akt pathway in Steroid‐Associated Osteonecrosis

Steroid‐associated osteonecrosis (SAON) is one of the common complications of clinical glucocorticoid (GC) administration, with osteocyte apoptosis appearing as the primary histopathological lesion. However, the precise mechanism underlying SAON remains unknown. Epigenetic modification may be a major cause of SAON. Recently, cumulative research revealed that Ten‐Eleven Translocation (TET) proteins can catalyze the conversion of 5‐methylcytosine (5mC) to 5‐hydroxymethylcytosine (5hmC) and then alter the epigenetic state of DNA. Here, we report that TET3‐5hmC was upregulated in the femoral head tissues of SAON patients and MLO‐Y4 cells with dexamethasone (Dex) treatment. Knockdown of TET3 in MLO‐Y4 cells decreased 5hmC enrichment and rescued Dex‐induced apoptosis. Meanwhile, the local intramedullary injection of TET3 siRNA in Sprague‐Dawley rats abrogated GC‐induced osteocyte apoptosis, histopathological changes, abnormal MRI signals, and bone microstructure declines in the femoral head in vivo. Moreover, a hydroxymethylated DNA immunoprecipitation (hMeDIP)‐chip analysis of Dex‐treated osteocytes revealed 456 different 5hmC‐enriched genes. The Akt pathway was found to mediate the functional effect of Dex‐induced dynamic 5hmC change; this was further verified in clinical samples. The loss of TET3 in MLO‐Y4 cells abrogated Dex‐induced Akt signaling pathway inhibition. Therefore, our data for the first time identify the effect of TET3‐5hmC on the Akt pathway and the necessity of this signaling cascade in SAON, identifying a new potential therapeutic target. © 2016 American Society for Bone and Mineral Research.     

SOX7与SOX9在鉴别前列腺癌与前列腺增生中的作用

目的 探讨SOX7和SOX9在鉴别前列腺癌与前列腺增生中的临床意义。方法 收集147例前列腺癌与28例前列腺增生患者病理组织及临床资料,运用免疫组化实验分析SOX7与SOX9的表达及其作用。结果 SOX7在前列腺癌组织中表现为低表达（P=0.025）,而SOX9为高表达（P=0.043）,SOX7与SOX9呈负相关（r=-0.263,P=0.001）。将SOX7与SOX9免疫组化评分与前列腺癌病理参数相结合进行分析,发现SOX7在血清PSA水平〈10ng/ml（P=0.048）、Gleason评分〈7（P=0.018）、肿瘤TNM分期〈T2a（P〈0.001）和病理分期T2a~T2c（P=0.002）前列腺癌患者中有较高的表达,而SOX9在Gleason评分≥7（P=0.001）和肿瘤TNM分期≥T2a（P=0.005）的前列腺癌患者中有较高的表达,差异有统计学意义。结论 通过检测SOX7和SOX9的水平可鉴别前列腺癌与前列腺增生,并可以初步判断前列腺癌的恶性程度。 更多还原     

尿道下裂患者SOX9基因突变的研究

目的：探讨SOX9基因在尿道下裂发病中的作用。方法：收集96例先天性尿道下裂患者外周静脉血，提取DNA，采用PCR扩增直接测序的方法检测SOX9基因全部外显子序列。结果：在实验组尿道下裂患者和对照组的SOX9基因外显子片段中，我们未发现任何位点的突变。只是在外显子3的3’UTR发现了1个SNPA／C，经分析没有统计学意义。结论：SOX9基因在性别分化的初期起作用，其变异直接影响性剐取向，结果导致两性畸形或性反转，而非单纯性尿道下裂。     

Targeting Cdk11 in osteosarcoma cells using the CRISPR‐cas9 system

Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)‐Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR‐Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U‐2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR‐Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR‐Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:199–207, 2015.