Tingwen Zhou1, Dong Han2, Junwei Liu1, Jiawei Shi1, Peng Zhu1, Yongjun Wang3, Nianguo Dong4. 1. Department of Cardiovascular Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. 2. Department of Cardiology, Xijing Hospital, Air Force Medical University, Xi'an, and Department of Cardiology, National Clinical Research Center for Geriatric Diseases, Chinese PLA General Hospital, Beijing, China. 3. Department of Cardiovascular Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Electronic address: wangyongjundoc@hotmail.com. 4. Department of Cardiovascular Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Electronic address: dongnianguodoc@hotmail.com.
Abstract
OBJECTIVE: Human aortic valve interstitial cells redifferentiate into an osteoblast-like phenotype, which is the key cellular mechanism of aortic valve calcification. Methyltransferase-like 3, the N6-methyladenosine methylation writer, has emerged as a new layer of epigenetic regulation for osteogenic differentiation of bone mesenchymal stem cells. The current study sought to determine whether methyltransferase-like 3 also plays a role in the osteogenic differentiation of human aortic valve interstitial cells. METHODS: Aortic valves from patients with aortic stenosis (n = 50) and normal controls (n = 50) were subjected to determination of methyltransferase-like 3 expression. Mineralized bone matrix formation was assessed by Alizarin Red staining. The interaction between methyltransferase-like 3 and twist-related protein 1 was confirmed via luciferase reporter and N6-methyladenosine methylated RNA immunoprecipitation quantitative reverse-transcription polymerase chain reaction. RESULTS: Methyltransferase-like 3 was highly expressed in human calcified aortic valves (1.61 ± 0.50) versus normal valves (3.07 ± 0.62; P < .0001). Osteogenic stimulation for 7 days resulted in a 2.15 ± 0.16-fold increase (P < .0001) in methyltransferase-like 3 protein level compared with the control group in human aortic valve interstitial cells. Functionally, methyltransferase-like 3 acted as a positive regulator of osteogenic differentiation of human aortic valve interstitial cells. Mechanistically, methylated RNA immunoprecipitation quantitative reverse-transcription polymerase chain reaction identified twist-related protein 1 as a target of methyltransferase-like 3-mediated m6A modification. Moreover, N6-methyladenosine-mediated twist-related protein 1 mRNA inhibition relied on the m6A binding protein YTH-domain family member 2-dependent pathway. CONCLUSIONS: Methyltransferase-like 3 promotes osteogenic differentiation of human aortic valve interstitial cells by inhibiting twist-related protein 1 through an N6-methyladenosine YTH-domain family member 2-dependent pathway. Our findings provide novel mechanistic insights into a critical role of methyltransferase-like 3 in the aortic valve calcification progression and shed new light on N6-methyladenosine-directed diagnostics and therapeutics in aortic valve calcification.
OBJECTIVE:Human aortic valve interstitial cells redifferentiate into an osteoblast-like phenotype, which is the key cellular mechanism of aortic valve calcification. Methyltransferase-like 3, the N6-methyladenosine methylation writer, has emerged as a new layer of epigenetic regulation for osteogenic differentiation of bone mesenchymal stem cells. The current study sought to determine whether methyltransferase-like 3 also plays a role in the osteogenic differentiation of human aortic valve interstitial cells. METHODS: Aortic valves from patients with aortic stenosis (n = 50) and normal controls (n = 50) were subjected to determination of methyltransferase-like 3 expression. Mineralized bone matrix formation was assessed by Alizarin Red staining. The interaction between methyltransferase-like 3 and twist-related protein 1 was confirmed via luciferase reporter and N6-methyladenosine methylated RNA immunoprecipitation quantitative reverse-transcription polymerase chain reaction. RESULTS:Methyltransferase-like 3 was highly expressed in human calcified aortic valves (1.61 ± 0.50) versus normal valves (3.07 ± 0.62; P < .0001). Osteogenic stimulation for 7 days resulted in a 2.15 ± 0.16-fold increase (P < .0001) in methyltransferase-like 3 protein level compared with the control group in human aortic valve interstitial cells. Functionally, methyltransferase-like 3 acted as a positive regulator of osteogenic differentiation of human aortic valve interstitial cells. Mechanistically, methylated RNA immunoprecipitation quantitative reverse-transcription polymerase chain reaction identified twist-related protein 1 as a target of methyltransferase-like 3-mediated m6A modification. Moreover, N6-methyladenosine-mediated twist-related protein 1 mRNA inhibition relied on the m6A binding protein YTH-domain family member 2-dependent pathway. CONCLUSIONS:Methyltransferase-like 3 promotes osteogenic differentiation of human aortic valve interstitial cells by inhibiting twist-related protein 1 through an N6-methyladenosine YTH-domain family member 2-dependent pathway. Our findings provide novel mechanistic insights into a critical role of methyltransferase-like 3 in the aortic valve calcification progression and shed new light on N6-methyladenosine-directed diagnostics and therapeutics in aortic valve calcification.
Authors: Jun-Yu Chen; Tao Xiong; Ya-Ru Sun; Juan Cong; Jing-Shuai Gong; Lei Peng; Yu-Wang Rong; Zi-Yao Wang; Qing Chang Journal: Ann Transl Med Date: 2022-09