摘要
目的探讨微小RNA-21(miR-21)靶向Ras GTP酶活化蛋白1(ras GTPase-activating protein 1,RASA1)调节RAS-丝裂原活化蛋白激酶(RAS-mitogen-activated protein kinase,RAS-MAPK)通路对子痫前期(PE)滋养层细胞功能的影响。方法收集2023年1月至12月在湖州市妇幼保健院治疗的40例PE患者和40例正常分娩孕妇的胎盘组织。RT-PCR检测PE患者和正常孕妇胎盘组织中miR-21和RASA1 mRNA表达水平;将滋养层HTR-8/SVneo细胞分为CK组、miR NC组、miR-21 mimic组、miR-21 mimic+oe-NC组、miR-21 mimic+oe-RASA1组;RT-PCR检测各组HTR-8/SVneo细胞中miR-21和RASA1 mRNA表达水平;CCK-8实验检测细胞增殖情况;Transwell实验检测细胞迁移和侵袭情况;流式细胞术检测细胞凋亡率;Western blot检测细胞E-cadherin、N-cadherin、vimentin、Bax、Bcl-2、RASA1及RAS-MAPK通路相关蛋白的表达;验证miR-21与RASA1的靶向关系。结果PE患者胎盘组织中miR-21表达水平显著低于正常孕妇胎盘组织,RASA1 mRNA表达水平显著高于正常孕妇胎盘组织(t值分别为24.393、15.917,P<0.001)。CK组、miR NC组、miR-21 mimic组、miR-21 mimic+oe-NC组、miR-21 mimic+oe-RASA1组的HTR-8/SVneo细胞中miR-21和RASA1 mRNA表达水平比较,差异均有统计学意义(F值分别409.444、633.065,P<0.001);五组HTR-8/SVneo细胞OD 450值(48h、72h)、迁移数、侵袭数、凋亡率比较,差异均有统计学意义(F值分别为32.074、29.428、70.934、103.170、71.849,P<0.001);五组HTR-8/SVneo细胞中E-cadherin、N-cadherin、vimentin、Bax、Bcl-2、RASA1蛋白表达水平比较,差异均有统计学意义(F值分别为50.597、92.860、57.177、48.272、90.235、55.249,P<0.001);五组HTR-8/SVneo细胞中p-Raf/Raf、p-MEK/MEK和p-ERK/ERK蛋白表达水平比较,差异均有统计学意义(F值分别为100.931、87.709、75.244,P<0.001)。miR-21 mimic+RASA1-WT组荧光素酶活性显著低于miR-NC+RASA1-WT组(q=22.570,P<0.05)。结论miR-21可能通过下调RASA1来激活RAS-MAPK通路,进而促进HTR-8/SVneo细胞增殖、迁移和上皮间质转化,降低细胞凋亡率,改善滋养层细胞功能。
Objective To investigate effect of microRNA 21(miR-21)targeting ras GTPase-activating protein 1(RASA1)to regulate RAS-mitogen-activated protein kinase(RAS-MAPK)signal pathway on function of trophoblast cells in preeclampsia(PE).Methods The placental tissues of 40 patients with PE and 40 parturient women with normal delivery who admitted to Huzhou Maternal&Child Health Care Hospital from January to December 2023 were collected.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect expression levels of miR-21 and RASA1 mRNA in the placental tissues of PE patients and normal pregnant women.The human trophoblast cell line 8/SV40 immortalized cells(HTR-8/SVneo)were divided into normal culture group(CK group),miR NC transfection group(miR NC group),miR-21 mimic transfection group(miR-21 mimic group),miR-21 mimic+oe-NC transfection group(miR-21 mimic+oe-NC group)and miR-21 mimic+oe-RASA1 transfection group(miR-21 mimic+oe-RASA1)group,and the RT-qPCR was applied to detect expression levels of miR-21 and RASA1 mRNA in the trophoblast HTR-8/SVneo cells in the five groups.CCK-8 reagent test was applied to detect proliferation of the cells.Transwell experiment was applied to detect migration and invasion of the cells.Flow cytometry was applied to detect rate of cell apoptosis.Western blot was applied to detect expressions of E-cadherin,N-cadherin,vimentin,Bax,Bcl-2,RASA1,and RAS-MAPK pathway related proteins in the cells.The targeting relationship between miR-21 and RASA1 was validated.Results The expression level of miR-21 in PE placental tissues was significantly lower than that in the normal placental tissues,while the expression level of RASA1 mRNA was significantly higher than that in the normal placental tissues(t=24.393 and 15.917 respectively,both P<0.001).The expression levels of miR-21 and RASA1 mRNA in HTR-8/SVneo cells among the CK group,the miR-NC group,the miR-21 mimic group,the miR-21 mimic+oe-NC group and the miR-21 mimic+oe-RASA1 group were compared and showed statistically significant differences(F=409.444 and 633.065 respectively,both P<0.001).There were statistically significant differences in OD450 values(48h,72h),migration number,invasion number and apoptosis rate of HTR-8/SVneo cells among the five groups(F=32.074,29.428,70.934,103.170 and 71.849 respectively,all P<0.001).And also,there were statistically significant differences in E-cadherin,N-cadherin,vimentin,Bax,Bcl-2 and RASA1 among the five HTR-8/SVneo cells groups(F=50.597,92.860,57.177,48.272,90.235 and 55.249 respectively,all P<0.001).The comparison of protein expression levels of p-Raf/Raf,p-MEK/MEK and p-ERK/ERK in the HTR-8/SVneo cells among the five groups showed statistically significant differences(F=100.931,87.709 and 75.244 respectively,all P<0.001).The luciferase activity in the miR-21 mimic+oe-RASA1-wild type transfection group(miR-21 mimic+RASA1-WT group)was significantly lower than that in the miR-NC+RASA1-wild type transfection group(miR-NC+RASA1-WT group)(q=22.570,P<0.001).Conclusion MiR-21 may activate RAS-MAPK pathway by down-regulating RASA1,and then promote proliferation,migration and epithelial-mesenchymal transition of the HTR-8/SVneo cells,thereby,reduce apoptosis rate and improve function of the trophoblast cells.
作者
丁忠英
陆艳
沈伟卫
沈国松
DING Zhongying;LU Yan;SHEN Weiwei;SHEN Guosong(Center of Medical Laboratory,Huzhou Maternity&Child Health Care Hospital,Zhejiang Huzhou 313000,China;Department of Gynecology,Huzhou Maternity&Child Health Care Hospital,Zhejiang Huzhou 313000,China;Department of Obstetrics,Huzhou Maternity&Child Health Care Hospital,Zhejiang Huzhou 313000,China)
出处
《中国妇幼健康研究》
2025年第10期29-37,共9页
Chinese Journal of Woman and Child Health Research
基金
湖州市科技计划公益性应用研究项目(2022GYB07)。
