彭涛, 梅雪峰, 李翔. miR-145和PTEN/AKT/mTOR通路在砷致大鼠流产及滋养层细胞损害中的作用[J]. 环境与职业医学, 2022, 39(3): 331-336, 347. DOI: 10.11836/JEOM21322
引用本文: 彭涛, 梅雪峰, 李翔. miR-145和PTEN/AKT/mTOR通路在砷致大鼠流产及滋养层细胞损害中的作用[J]. 环境与职业医学, 2022, 39(3): 331-336, 347. DOI: 10.11836/JEOM21322
PENG Tao, MEI Xuefeng, LI Xiang. Role of miR-145 and PTEN/AKT/mTOR pathway in rat abortion and damage of trophoblast cells induced by arsenic exposure[J]. Journal of Environmental and Occupational Medicine, 2022, 39(3): 331-336, 347. DOI: 10.11836/JEOM21322
Citation: PENG Tao, MEI Xuefeng, LI Xiang. Role of miR-145 and PTEN/AKT/mTOR pathway in rat abortion and damage of trophoblast cells induced by arsenic exposure[J]. Journal of Environmental and Occupational Medicine, 2022, 39(3): 331-336, 347. DOI: 10.11836/JEOM21322

miR-145和PTEN/AKT/mTOR通路在砷致大鼠流产及滋养层细胞损害中的作用

Role of miR-145 and PTEN/AKT/mTOR pathway in rat abortion and damage of trophoblast cells induced by arsenic exposure

  • 摘要: 背景 砷染毒可损害滋养层细胞进而诱导流产,但其机制尚不清楚。

    目的 探讨miR-145和PTEN/AKT/mTOR通路在砷致孕鼠流产及滋养层细胞损害中的作用。

    方法 动物实验:SD孕鼠20只,随机分为正常对照组(生理盐水灌胃)、砷染毒流产组(10.65 mg·kg−1亚砷酸钠溶液灌胃,灌胃体积10 mL·kg−1),每组10只;砷染毒流产组出现流产后(染毒5~6 d),收集两组胎盘组织,实时定量PCR(RT-PCR)检测微小RNA-145(miR-145)、磷酸酶和张力蛋白同源物(PTEN)、蛋白激酶B(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)mRNA的表达水平,蛋白印迹法检测PTEN、AKT、mTOR、p-AKT、p-mTOR蛋白表达水平。细胞实验:将永生化人滋养层细胞系(HTR-8/SVNEO细胞)分为对照组、砷染毒组、miR-145过表达组、砷染毒+miR-145过表达组,各组细胞密度为5×105个·孔−1,砷染毒剂量为20 μmol·L−1,培养环境为37 °C、5%CO2,处理72 h,MTT法检测细胞存活率,结晶紫染色法检测单克隆形成数目,流式细胞法检测凋亡水平,Image J Angiogenesis Analyzer 1.8.0插件测定细胞总血管长度和总血管数目;基因和蛋白的检测指标和方法同“动物实验”。

    结果 (1)动物实验:与正常对照组比较,砷染毒流产组胎盘组织miR-145表达水平升高(P<0.05),PTEN、AKT、mTOR mRNA及其蛋白和p-AKT、p-mTOR蛋白表达水平降低(P<0.05)。(2)细胞实验:与对照组比较,砷染毒组、miR-145过表达组、砷染毒+miR-145过表达组存活率、单克隆形成数目、总血管长度、总血管数目降低,凋亡率升高(P<0.05);与砷染毒组、miR-145过表达组比较,砷染毒+miR-145过表达组存活率、单克隆形成数目、总血管长度、总血管数目降低,凋亡率水平升高(P<0.05)。与对照组比较,砷染毒组、miR-145过表达组、砷染毒+miR-145过表达组miR-145 mRNA表达水平升高(P<0.05),PTEN、AKT、mTOR mRNA及其蛋白和p-AKT、p-mTOR蛋白表达水平降低(P<0.05);与砷染毒组、miR-145过表达组比较,砷染毒+miR-145过表达组miR-145 mRNA表达水平升高(P<0.05),PTEN、AKT、mTOR mRNA及其蛋白和p-AKT、p-mTOR蛋白表达水平降低(P<0.05)。

    结论 miR-145可能与砷染毒所致流产有关,miR-145可抑制滋养层细胞HTR-8/SVNEO增殖、血管形成,促进其凋亡,其机制可能与PTEN/AKT/mTOR通路的抑制有关。

     

    Abstract: Background Exposure to arsenic can damage trophoblast cells and thus induce abortion, but the mechanism is not known.

    Objective To investigate the role of miR-145 and PTEN/AKT/mTOR pathway in arsenic-induced abortion and trophoblast cell damage in rats.

    Methods In the animal experiment, twenty SD pregnant rats were randomly divided into a normal control group (saline gavage) and an arsenic-induced abortion group (10.65 mg·kg−1 sodium arsenite solution was administered by gavage, and the gavage volume was 10 mL·kg−1), with 10 rats in each group. After the miscarriage occurred in the arsenic-induced abortion group (5-6 d after exposure), placental tissues were collected from the two groups. The mRNA expression levels of microRNA-145 (miR-145), phosphatase and tensin homologue (PTEN), kinase B (AKT), mammalian target of rapamycin (mTOR) were detected by real-time quantitative PCR (RT-PCR), and the protein expression levels of PTEN, AKT, mTOR, p-AKT, and p-mTOR were detected by Western blotting. For the in vitro study with immortalized human trophoblast cell line (HTR-8/SVneo cells), a control group, an arsenic exposure group, an miR-145 overexpression group, and an arsenic exposure+miR-145 overexpression group were prepared and cultured for 72 h with 37 °C and 5% CO2, at cell density of 5×105 cells per well, and the arsenic exposure concentration was 20 μmol·L−1. The MTT method was applied to detect cell viability, crystal violet staining to detect the number of monoclonal formation, flow cytometry to detect the level of apoptosis, Image J Angiogenesis Analyzer 1.8.0 plug-in to evaluate total blood vessel length and total blood vessel number; the detection indexes and methods of genes and proteins were the same as "animal experiment".

    Results (1) In the animal experiment, compared with the normal control group, the expression level of miR-145 mRNA in the placenta tissues of the arsenic-induced abortion group was increased (P<0.05), and the expression levels ofPTEN, AKT, mTOR mRNA and proteins, and p-AKT and p-mTOR proteins were decreased (P<0.05). (2) For thein vitro study, compared with the control group, the cell viability rate, number of monoclonal formation, total vessel length, and total vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the arsenic exposure group and the miR-145 overexpression group, the cell viability rate, number of monoclonal formation, total vessel length, and vessel number were decreased, and the apoptosis rate was increased in the arsenic exposure+miR-145 overexpression group (P<0.05). Compared with the control group, the levels ofmiR-145 mRNA in the arsenic exposure group, the miR-145 overexpression group, and the arsenic exposure+miR-145 overexpression group increased (P<0.05), the expression levels ofPTEN, AKT, mTOR mRNA and protein and the expression levels of p-AKT and p-mTOR protein were decreased (P<0.05); compared with the arsenic exposure group and the miR-145 overexpression group, the level ofmiR-145 mRNA in the arsenic exposure+miR-145 overexpression group was increased (P<0.05), and the levels ofPTEN, AKT, mTOR mRNA and protein as well as p-AKT and p-mTOR protein were decreased (P<0.05).

    Conclusion miR-145 might be related to abortion due to arsenic exposure. miR-145 could inhibit the proliferation and angiogenesis of trophoblast HTR-8/SVNEO cells, and promotes their apoptosis; the mechanism may be related to the inhibition of PTEN/AKT/mTOR pathway.

     

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