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miRNA-494调控PI3K/AKT/mTOR通路促进滋养细胞增殖和迁移
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Abstract:
目的：探讨不明原因复发性流产(unexplained recurrent spontaneous abortion, uRSA)小鼠模型的微小RNA (miRNA)-494、PI3K/AKT/mTOR [哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)]的表达和促进胎盘滋养细胞增殖和迁移以及调节子宫和胎盘的炎症反应和免疫功能的作用机制。方法：首先构建了miRNA-494的过表达载体，实验分成miRNA-494模拟物组、模拟物对照组、miRNA-494抑制剂组、抑制剂对照组，采用脂质体2000分别转染miRNA-494模拟物(50 nmol/L)、miRNA-494抑制剂(50 nmol/L)，使用实时荧光定量PCR 技术检测miRNA-494模拟物组、模拟物对照组、miRNA-494抑制剂组、抑制剂对照组的表达水平；采用克隆形成实验检测各组胎盘滋养细胞的增殖能力，transwell法检测各组胎盘滋养层细胞的迁移程度，western blot检测各组胎盘滋养层细胞PI3K、AKT、mTOR的表达水平。结果：miRNA-494转染48 h后，miRNA-494模拟物组滋养细胞的miRNA-494表达水平明显高于模拟物对照组、抑制剂对照组和miRNA-494抑制剂组(P < 0.001)；反之miRNA-494抑制剂组滋养细胞的miRNA-494表达水平明显低于对照组和miRNA-494模拟物组(P < 0.001)；miRNA-494抑制剂组的PI3K/AKT和mTOR表达水平明显低于抑制剂对照组、模拟物对照组和miRNA-494模拟物组(P < 0.001)；miRNA-494抑制剂组的滋养细胞增殖和迁移比例显著低于抑制剂对照组、模拟物对照组和miRNA-494模拟物组(P < 0.01)。结论：miRNA-494通过调节Akt/PI3K/mTOR基因的表达，可以促进胎盘滋养细胞增殖和迁移，并参与调节子宫和胎盘的炎症反应和免疫功能，表明了调节miRNA-494的表达水平可能作为uRSA的预防和治疗的新靶点。
Objective: To investigate the expression of miRNA-494, PI3K/AKT/mTOR [mammalian target of rapamycin, mTOR] in a mouse model of unexplained recurrent spontaneous abortion (uRSA), and their mechanisms in promoting placental trophoblast proliferation and migration, as well as regulating inflammation and immune function in the uterus and placenta. Method: Firstly, an overexpression vector of miRNA-494 was constructed, and the experiment was divided into miRNA-494 mimic group, mimic control group, miRNA-494 inhibitor group, and inhibitor control group. Liposome 2000 was used to transfect miRNA-494 mimic group (50 nmol/L) and miRNA-494 inhibitor group (50 nmol/L), respectively. Real time fluorescence quantitative PCR technology was used to detect miRNA-494 mimic group, mimetic control group, and miRNA-494 inhibitor group The expression level of the inhibitor control group; The proliferation ability of placental trophoblast cells in each group was detected using clone formation experiments, the migration degree of placental trophoblast cells in each group was measured using Transwell method, and the expression levels of PI3K, AKT, and mTOR in placental trophoblast cells in each group were detected using Western blot. Result: After 48 hours of miRNA-494 transfection, the expression level of miRNA-494 in the trophoblast cells of the miRNA-494 mimic group was significantly higher than that of the mimic control group, inhibitor control group, and miRNA-494 inhibitor group (P < 0.001); On the contrary, the expression level of miRNA-494 in trophoblast cells in the miRNA-494 inhibitor group was significantly lower than that in the control group and miRNA-494 mimic group
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