METTL7A通过m6A甲基化调控牙髓干细胞的衰老和成骨/成牙分化

口腔生物医学 ›› 2024, Vol. 15 ›› Issue (1): 26-31.

METTL7A通过m6A甲基化调控牙髓干细胞的衰老和成骨/成牙分化

王宁¹,²,马华瑞³,苏丽⁴,余日月²

1. 首都医科大学附属北京世纪坛医院
2. 首都医科大学附属北京世纪坛医院口腔科
3. 北京市昌平区中医医院口腔科
4. 北京市宣武区中医医院口腔科

收稿日期:2023-05-06 修回日期:2023-11-05 出版日期:2024-02-25 发布日期:2024-03-23
通讯作者: 余日月 E-mail:yu-riyue@163.com

METTL7A regulates cell senescence and osteogenic/odontogenic differentiation of dental pulp stem cells through m6A methylation

1.
2. Department of Stomatology, Beijing Shijitan Hospital, Capital Medical University
3. Department of Stomatology, Beijing Changping District Hospital of Traditional Chinese Medicine
4. Department of Stomatology, Beijing Xuanwu District Hospital of Traditional Chinese Medicine
5. Department of Stomatology, Beijing Shijitan Hospital, Capital Medical University

Received:2023-05-06 Revised:2023-11-05 Online:2024-02-25 Published:2024-03-23

摘要/Abstract

摘要： 目的：探索甲基转移酶样7A（METTL7A）通过N6-甲基腺苷酸（m6A）甲基化对牙髓干细胞（DPSCs）衰老和成骨/成牙分化的影响。方法：利用慢病毒转染DPSCs获得METTL7A稳定敲低的DPSCs，利用逆转录病毒转染DPSCs获得METTL7A稳定过表达的DPSCs，实时荧光定量PCR和Western blot实验检测敲低及过表达效率。在此基础上，利用衰老相关β-半乳糖苷酶（SA-β-gal）染色及定量检测METTL7A是否调控DPSCs的衰老，斑点杂交实验检测METTL7A是否调控DPSCs的m6A甲基化水平。METTL7A过表达组加入m6A甲基化抑制剂环亮氨酸（Cyc）后，利用SA-β-gal染色及定量检测和碱性磷酸酶（ALP）活性实验检测METTL7A通过m6A甲基化调控DPSCs的衰老和成骨/成牙分化。结果：利用慢病毒转染DPSCs，与对照组（Consh）相比，METTL7A基因和蛋白在METTL7A敲低组（METTL7Ash）的表达显著降低（P<0.01），METTL7Ash组的SA-β-gal染色及定量检测发现阳性细胞数量增加（P<0.05）；利用逆转录病毒转染DPSCs，与对照组（Vector）相比，METTL7A在METTL7A过表达（HA-METTL7A）组的表达显著增加（P<0.01），HA-METTL7A组的SA-β-gal染色及定量检测显示阳性细胞数量减少（P<0.01）。此外，相比于对照组，METTL7Ash组DPSCs的m6A甲基化修饰水平显著降低，而过表达METTL7A组DPSCs的m6A甲基化修饰水平明显升高。在此基础上，在METTL7A过表达组加入Cyc，SA-β-gal染色及其定量检测显著增加因METTL7A过表达而减少的阳性细胞数量（P<0.01），同时也显著抑制因METTL7A过表达促进的ALP活性（P<0.05）。结论：METTL7A可能通过调控m6A甲基化水平抑制DPSCs的衰老并促进其成骨/成牙分化。

关键词: METTL7A, m6A甲基化, 牙髓干细胞, 衰老, 成骨/成牙分化

Abstract: Objective:To explore the effect of METTL7A on cell senescence and osteogenic/odontogenic differentiation of DPSCs through m6A methylation. Methods:The lentivirus was used to transfect the DPSCs to obtain the DPSCs of METTL7A knockdown. The retrovirus was used to transfect the DPSCs to obtain the DPSCs of METTL7A overexpression. The knockdown and overexpression efficiency was detected by qRT-PCR and western blot assay. On this basis, SA-b-gal staining and quantification of the SA-b-gal staining was used to detect whether METTL7A regulated the cell senescence of DPSCs. Dot hybridization experiments were conducted to determine whether METTL7A regulated m6A methylation levels of DPSCs. After adding the Cyc to the METTL7A overexpression group, the SA-b-gal staining, the quantification of the SA-b-gal staining and ALP activity confirmed that METTL7A regulated cell senescence and osteogenic/odontogenic differentiation of DPSCs through m6A methylation. Results:The lentivirus was used to transfect the DPSCs. Compared with the Consh group, the gene and protein expression of METTL7A in METTL7A knockdown group (METTL7Ash) was significantly decreased (P<0.01). The quantity of positive cells was increased, which was detected by SA-b-gal staining and quantification of the SA-b-gal staining in METTL7Ash group (P<0.05). The retrovirus was used to transfect the DPSCs. Compared with the control group (Vector), the expression of METTL7A in the METTL7A overexpression group (HA-METTL7A) was significantly increased (P<0.01). The quantity of positive cells detected by SA-b-gal staining and quantification of SA-b-gal staining in the HA-METTL7A group was significantly decreased (P<0.01). In addition, compared with the control group, the m6A methylation modification level of DPSCs in the METTL7Ash group was significantly decreased, while the m6A methylation modification level of DPSCs in the METTL7A overexpression group was significantly increased. After adding the Cyc to the METTL7A overexpression group, the quantity of positive cells detected by SA-b-gal staining and quantification significantly increased compared with the METTL7A overexpression group (P<0.01). After adding the Cyc to the METTL7A overexpression group, the ALP activity was significantly inhibited that promoted by METTL7A overexpression (P<0.05). Conclusions: METTL7A may inhibit the cell senescence and promote osteogenic/odontogenic differentiation of DPSCs by regulating m6A methylation levels.

Key words: METTL7A, m6A methylation, dental pulp stem cell, cell senescence, osteo/odontogenic differentiation

王宁马华瑞苏丽余日月. METTL7A通过m6A甲基化调控牙髓干细胞的衰老和成骨/成牙分化[J]. 口腔生物医学, 2024, 15(1): 26-31.

参考文献 1.

Fu X, Liu G, Halim A, Ju Y, Luo Q, Song, et al. Mesenchymal Stem Cell Migration and Tissue Repair. Cells. 2019;8(8).2. Liu J, Gao J, Liang Z, Gao C, Niu Q, Wu F, et al. Mesenchymal stem cells and their microenvironment. Stem Cell Res Ther. 2022;13(1):429.3. Li J, Wang W, Li M, Liu L. Repair of segmental bone defect using tissue engineered heterogeneous deproteinized bone doped with lithium. Sci Rep. 2021;11(1):4819.4. Duan W, Zou H, Zang N, Ma D, Yang B, Zhu L. Metformin increases bone marrow adipose tissue by promoting mesenchymal stromal cells apoptosis. Aging (Albany NY). 2023;15(2):542-52.5. Wang N, Han X, Yang H, Xia D, Fan Z. miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A. Front Cell Dev Biol. 2021;9:759192.6. Pierce JL, Begun DL, Westendorf JJ, McGee-Lawrence ME. Defining osteoblast and adipocyte lineages in the bone marrow. Bone. 2019;118:2-7.7. Chen Q, Shou P, Zheng C, Jiang M, Cao G, Yang Q, et al. Fate decision of mesenchymal stem cells: adipocytes or osteoblasts? Cell Death Differ. 2016;23(7):1128-39.8. Hu M, Xing L, Zhang L, Liu F, Wang S, Xie Y, et al. NAP1L2 drives mesenchymal stem cell senescence and suppresses osteogenic differentiation. Aging Cell. 2022;21(2):e13551.9. Zhang Y, Gu X, Li D, Cai L, Xu Q. METTL3 Regulates Osteoblast Differentiation and Inflammatory Response via Smad Signaling and MAPK Signaling. Int J Mol Sci. 2019;21(1).10. Chen X, Hua W, Huang X, Chen Y, Zhang J, Li G. Regulatory Role of RNA N6-Methyladenosine Modification in Bone Biology and Osteoporosis. Frontiers In Endocrinology. 2019;10:911.11. Li Z, Wang P, Li J, Xie Z, Cen S, Li M, et al. The N6-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6. Cell Death & Disease. 2021;12(6):578.12. Chen LS, Zhang M, Chen P, Xiong XF, Liu PQ, Wang HB, et al. The m6A demethylase FTO promotes the osteogenesis of mesenchymal stem cells by downregulating PPARG. Acta Pharmacol Sin. 2022;43(5):1311-23.13. Wu Y, Xie L, Wang M, Xiong Q, Guo Y, Liang Y, et al. Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis. Nat Commun. 2018;9(1):4772.14. Jiang X, Liu B, Nie Z, Duan L, Xiong Q, Jin Z, et al. The role of m6A modification in the biological functions and diseases. Signal Transduct Target Ther. 2021;6(1):74.

[1]	符莉娅师梦楠刘玥蒋少云. 牙源性干细胞衰老相关机制的研究进展[J]. 口腔生物医学, 2021, 12(4): 277-280.
[2]	胡钦朝姜徕博彭建敏吴桐程斌夏娟. 利用荧光染料CFSE分选衰老细胞的探究[J]. , 2020, 11(2): 81-85.
[3]	张娟戈杰李光南周培培江宏兵. 颅骨锁骨发育不全患者骨髓基质细胞的增殖、成骨和衰老[J]. , 2015, 6(2): 66-70.