基于m^(6)A甲基化修饰探讨减味寿胎丸对自然流产滋养细胞黏附功能的影响

Effects of Jianwei Shoutai Pills on the adhesion function of trophoblast in spontaneous abortion based on m6A methylation

目的:探讨自然流产的发生与m^(6)A甲基化修饰的相关性及减味寿胎丸对滋养细胞m^(6)A甲基化修饰的影响。方法:临床研究收集自然流产患者(SA组)及正常妊娠女性(NP组)绒毛组织(各15例),细胞实验以米非司酮诱导损伤的HT R-8/S Vneo滋养细胞为流产细胞模型,分为对照组、模型组、减味寿胎丸组及孕酮组,药物或基础培养基干预24 h。m^(6)A甲基化定量试剂盒检测m^(6)A整体水平,Western Blot法检测METTL3、WTAP、YTHDF2、ALKBH5及FN1蛋白的表达,qRT-PCR检测FN1 mRNA的表达,划痕实验检测各组细胞迁移能力,Transwell实验检测各组细胞侵袭能力。结果:临床研究:与NP组比较,SA组绒毛组织中m^(6)A整体水平降低(P<0.01),ALKBH5表达显著升高(P<0.01),FN1表达显著降低(P<0.01,P<0.05)。细胞实验:与对照组比较,模型组滋养细胞METTL3、WTAP及FN1表达显著降低(P<0.01,P<0.05),ALKBH5表达显著升高(P<0.05),细胞迁移能力和侵袭能力显著下降(P<0.01,P<0.05);与模型组比较,减味寿胎丸组METTL3、WTAP、YTHDF2及FN1表达显著升高(P<0.01,P<0.05),ALKBH5表达降低(P<0.01),细胞迁移能力和侵袭能力显著增强(P<0.01,P<0.05)。结论:自然流产的发生与滋养细胞m^(6)A甲基化修饰缺陷相关,减味寿胎丸治疗自然流产可能与上调滋养细胞中FN1的m^(6)A甲基化修饰,促进滋养细胞黏附功能相关。

Objective:To study the correlation between the occurrence of spontaneous abortion and m^(6)A methylation,as well as the effects of Jianwei Shoutai Pills on the m^(6)A modification in trophoblasts.Methods:Clinical villus originated from spontaneous abortion(SA)women(SA group)and normal pregnant women(NP group)were collected,15 in each one.Trophoblast cell string,HTR-8/SVneo,induced by mifepristone was used as cell model of SA in vitro,which was divided into control group,model group,Jianwei Shoutai Pills group and progesterone group.And cells were treated with culture medium with or without medicine for 24 h.The total m^(6)A level was measured by quantified m^(6)A methylation kits.The expression of METTL3,WTAP,YTHDF2,ALKBH5 and FN1 were measured by Western blot,and the expression of FN1 mRNA was measured by qRT-PCR.The experiments of wound-healing and Transwell were used to assess the function of cell migration and invasion.Results:Clinical research:the total m^(6)A level in the SA group was lower than that in the NP group(P<0.01),with the increased expression of ALKBH5(P<0.01)and decreased expression of FN1(P<0.01,P<0.05).Cell experiment:the expression of METTL3,WTAP and FN1 were lower in the model group than those in the control group(P<0.01,P<0.05),with increased expression of ALKBH5(P<0.05)and declined functions of cell migration and invasion(P<0.01,P<0.05).And the expression of METTL3,WTAP,YTHDF2 and FN1 were higher in the Jianwei Shoutai Pills group than those in the model group(P<0.01,P<0.05),with decreased expression of ALKBH5(P<0.01)and enhanced functions of cell migration and invasion(P<0.01,P<0.05).Conclusion:The occurrence of SA is associated with m^(6)A modification deficiency,and the mechanism of Jianwei Shoutai Pills on SA may be relevant to up-regulating the m^(6)A modification of FN1 and promoting the function of cell adhesion.