低分子肝素通过调控DNMT1介导的启动子甲基化对绒毛外滋养层细胞功能的影响  

The effect of low molecular weight heparin on the function of extravillous trophoblast cells by regulating DNMT1 mediated promoter methylation

作　　者：欧妍 杨纪珂 李嘉慧 OU Yan;YANG Jike;LI Jiahui(Department of Obstetrics and Gynecology,Shunyi Hospital,Beijing Traditional Chinese Medicine Hospital,Beijing 101300,China)

机构地区：[1]北京中医医院顺义医院妇产科,北京101300

出　　处：《中国优生与遗传杂志》2024年第12期2480-2486,共7页Chinese Journal of Birth Health & Heredity

基　　金：北京中医医院顺义医院院级自然科学基金项目(SYYJ-201905)。

摘　　要：目的探究低分子肝素(LMWH)通过调控DNA甲基化转移酶1(DNMT1)介导的启动子甲基化对绒毛外滋养层细胞功能的影响。方法CCK-8实验检测LMWH对HTR-8/SVneo细胞存活率的影响。HTR-8/SVneo细胞分为Control组、Hypoxia组、si-DNMT1组、si-NC组、LMWH组、LMWH+DNMT1组、LMWH+NC组。细胞进行相应转染后,Control组细胞常氧培养48 h,其他各组细胞使用/不使用LMWH(5 IU/mL)低氧培养48 h。CCK-8实验检测细胞存活率;EdU染色检测细胞增殖;划痕实验检测细胞迁移;Transwell实验检测细胞侵袭;甲基化特异性PCR(MSP)检测细胞Ki67、MMP2启动子甲基化水平;RT-qPCR、Western blot检测细胞DNMT1、Ki67、MMP2 mRNA和蛋白水平。结果1、2.5、5 IU/mL LMWH对HTR-8/SVneo细胞存活率无明显影响(P>0.05)。低氧处理后,细胞存活率、EdU阳性率、划痕愈合率降低,侵袭细胞数减少,DNMT1 mRNA和蛋白水平升高,Ki67、MMP2启动子甲基化水平升高,Ki67、MMP2 mRNA和蛋白水平降低(P<0.05);LMWH处理或敲低DNMT1表达后,细胞存活率、EdU阳性率、划痕愈合率升高,侵袭细胞数增多,DNMT1 mRNA和蛋白水平降低,Ki67、MMP2启动子甲基化水平降低,Ki67、MMP2 mRNA和蛋白水平升高(P<0.05);过表达DNMT1能够减弱LMWH对上述指标的影响(P<0.05)。结论LMWH通过调控DNMT1介导的启动子甲基化促进HTR-8/SVneo细胞增殖、迁移和侵袭。Objective To explore the effect of low molecular weight heparin(LMWH)on the function of extravillous trophoblast cells by regulating DNA methyltransferase 1(DNMT1)mediated promoter methylation.Methods CCK-8 experiment was used to detect effect of LMWH on survival rate of HTR-8/SVneo cells.HTR-8/SVneo cells were divided into Control group,Hypoxia group,si-DNMT1 group,si-NC group,LMWH group,LMWH+DNMT1 group,and LMWH+NC group.After corresponding transfection,cells in Control group were cultured under normoxic conditions for 48 h,while cells in other groups were cultured under low oxygen conditions with or without LMWH(5 IU/mL)for 48 h.CCK-8 experiment was used to detect cell survival rate.EdU staining was used to detect cell proliferation.Scratch test was used to detect cell migration.Transwell experiment was used detect cell invasion.Methylation specific PCR(MSP)was used to detect Ki67 and MMP2 promoter methylation levels in cells.RT-qPCR and Western blot were used to detect DNMT1,Ki67,and MMP2 mRNA and protein levels in cells.Results 1,2.5,5 IU/mL LMWH had no significant effect on the survival rate of HTR-8/SVneo cells(P>0.05).After hypoxia treatment,cell survival rate,EdU positivity rate,scratch healing rate were decreased,number of invasive cells was decreased,DNMT1 mRNA and protein levels were increased,Ki67 and MMP2 promoter methylation levels were increased,Ki67 and MMP2 mRNA and protein levels were decreased(P<0.05).After LMWH treatment or knocking down DNMT1 expression,cell survival rate,EdU positivity rate,scratch healing rate were increased,number of invasive cells was increased,DNMT1 mRNA and protein levels were decreased,Ki67 and MMP2 promoter methylation levels were decreased,Ki67 and MMP2 mRNA and protein levels were increased(P<0.05).Overexpression of DNMT1 could weaken the effect of LMWH on the above indicators(P<0.05).Conclusion LMWH promoted HTR-8/SVneo cell proliferation,migration,and invasion by regulating DNMT1 mediated promoter methylation.

关 键 词：子痫前期 低分子肝素 DNMT1 启动子甲基化 绒毛外滋养层细胞 

分 类 号：R714.244[医药卫生—妇产科学]