Junlong Wu1, Yanan Du2, Jidong Song3, Xiaoqian Dang3, Kunzheng Wang3, Yan Wen2, Feng Zhang4, Ruiyu Liu5. 1. Department of Orthopedics, the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, PR China; Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, Henan Province, 471009, China. 2. Key Laboratory of Trace Elements and Endemic Diseases, Collaborative Innovation Center of Endemic Disease and Health Promotion for Silk Road Region, School of Public Health, Health Science Center, Xi'an Jiaotong University, Xi'an, PR China. 3. Department of Orthopedics, the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, PR China. 4. Key Laboratory of Trace Elements and Endemic Diseases, Collaborative Innovation Center of Endemic Disease and Health Promotion for Silk Road Region, School of Public Health, Health Science Center, Xi'an Jiaotong University, Xi'an, PR China. Electronic address: fzhxjtu@mail.xjtu.edu.cn. 5. Department of Orthopedics, the Second Affiliated Hospital, Xi'an Jiaotong University, Xi'an, PR China. Electronic address: liuryu@126.com.
Abstract
OBJECTIVE: Recent studies demonstrated a critical role of hip articular cartilage destruction in the development of osteonecrosis of the femoral head (ONFH). The aim of this study was to characterize the genome-wide DNA methylation profile of hip cartilage obtained from patients with ONFH and healthy subjects. METHODS: Hip articular cartilage specimens were collected from 15 ONFH patients (including 11 males and 4 females) and 15 control subjects (including 11 males and 4 females) with femoral neck fracture. The average ages of the ONFH patients and control subjects were 50.27 ± 5.27 years and 61.67 ± 3.38 years, respectively. Genome-wide DNA methylation profiles of 5 ONFH and 5 control cartilages were determined by Illumina HumanMethylation850 array. Differential methylation analysis of DNA methylation profiles were performed by the empirical Bayes moderated t-test of the limma package. Mass spectrograph (MS) analysis of 10 ONFH cartilages and 10 normal cartilages were performed to validate the results of genome-wide DNA methylation profiling. Immunohistochemistry (IHC) of 4 ONFH cartilages and 4 control cartilages were conducted to evaluate the expression levels of proteins encoded by identified differentially methylated genes. t-test was used to assess the significance of protein expression differences between ONFH patients and controls in IHC. RESULTS: We identified a total of 2872 differentially methylated CpG sites, annotated to 480 hypermethylated genes and 1335 hypomethylated genes for ONFH. The results of MS validation were consistent with that of genome-wide DNA methylation profiling. IHC further confirmed the increased protein expression of CARS (mean and 95%CI of superficial zone 59.67% [48.46, 56.14], and deep zone 31% [25.85, 30.61]), PDE4D (superficial zone 50.33% [33.64, 40.68] and deep zone 28.67% [10.81, 36.47]), ADAMTS12 (superficial zone 53.67% [36.01, 40.93] and deep zone 34.67% [22.56, 37.18]), LRP5 (superficial zone 59.63% [27.32, 39.61] and deep zone 22.95% [5.28, 19.29]), RUNX2 (superficial zone 52.58% [11.64, 31.33] and deep zone 35.01% [10.03, 27.44]) in ONFH articular cartilage. CONCLUSION: Our results suggest the implication of DNA methylation alterations in the development of ONFH, and provide novel clues for pathogenetic and therapeutic studies of ONFH.
OBJECTIVE: Recent studies demonstrated a critical role of hip articular cartilage destruction in the development of osteonecrosis of the femoral head (ONFH). The aim of this study was to characterize the genome-wide DNA methylation profile of hip cartilage obtained from patients with ONFH and healthy subjects. METHODS:Hip articular cartilage specimens were collected from 15 ONFH patients (including 11 males and 4 females) and 15 control subjects (including 11 males and 4 females) with femoral neck fracture. The average ages of the ONFH patients and control subjects were 50.27 ± 5.27 years and 61.67 ± 3.38 years, respectively. Genome-wide DNA methylation profiles of 5 ONFH and 5 control cartilages were determined by Illumina HumanMethylation850 array. Differential methylation analysis of DNA methylation profiles were performed by the empirical Bayes moderated t-test of the limma package. Mass spectrograph (MS) analysis of 10 ONFH cartilages and 10 normal cartilages were performed to validate the results of genome-wide DNA methylation profiling. Immunohistochemistry (IHC) of 4 ONFH cartilages and 4 control cartilages were conducted to evaluate the expression levels of proteins encoded by identified differentially methylated genes. t-test was used to assess the significance of protein expression differences between ONFH patients and controls in IHC. RESULTS: We identified a total of 2872 differentially methylated CpG sites, annotated to 480 hypermethylated genes and 1335 hypomethylated genes for ONFH. The results of MS validation were consistent with that of genome-wide DNA methylation profiling. IHC further confirmed the increased protein expression of CARS (mean and 95%CI of superficial zone 59.67% [48.46, 56.14], and deep zone 31% [25.85, 30.61]), PDE4D (superficial zone 50.33% [33.64, 40.68] and deep zone 28.67% [10.81, 36.47]), ADAMTS12 (superficial zone 53.67% [36.01, 40.93] and deep zone 34.67% [22.56, 37.18]), LRP5 (superficial zone 59.63% [27.32, 39.61] and deep zone 22.95% [5.28, 19.29]), RUNX2 (superficial zone 52.58% [11.64, 31.33] and deep zone 35.01% [10.03, 27.44]) in ONFH articular cartilage. CONCLUSION: Our results suggest the implication of DNA methylation alterations in the development of ONFH, and provide novel clues for pathogenetic and therapeutic studies of ONFH.