METTL7A通过m6A甲基化调控牙髓干细胞的衰老和成骨/成牙分化

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

目的:探索甲基转移酶样7A(METTL7A)通过N6-甲基腺苷(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Consh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。

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-β-gal staining and quantification of the SA-β-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-β-gal staining,the quantification of the SA-β-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-β-gal staining and quantification of the SA-β-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-β-gal staining and quantification of SA-β-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-β-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.