子痫前期中整合素α5的DNA甲基化水平及其对滋养细胞增殖、迁移及侵袭能力的影响

DNA methylation of ITGA5 in preeclampsia and its effect on proliferation, migration and invasion of trophoblast

目的探究整合素α5(ITGA5)在子痫前期(PE)胎盘中的甲基化水平及其对滋养细胞增殖、迁移及侵袭能力的影响。方法收集2018年7月至2020年7月在我院产科分娩的32例子痫前期患者(PE组)与38例正常妊娠孕产妇(Control组)的胎盘组织,分别应用反转录酶-定量聚合酶连锁反应(RT-qPCR)、免疫印迹(Westernblot)及免疫组织化学染色检测ITGA5在两组孕产妇胎盘组织中的mRNA、蛋白表达及其定位;甲基化特异性PCR(MSP)与亚硫酸盐测序PCR(BSP)检测ITGA5基因甲基化水平在PE组与Control组胎盘中的差异;体外培养滋养细胞系HTR8与JEG-3,应用脂质体转染技术将过表达ITGA5质粒与空载体质粒分别转染入滋养细胞中,并将其记为pcCtrl组与pcITGA5,再次应用RT-qPCR、Western blot试验检测转染效率;分别利用CCK-8增殖试验、划痕试验及Transwell侵袭试验检测过表达ITGA5对滋养细胞的增殖、侵袭及侵袭能力的影响;Westernblot检测过表达ITGA5对滋养细胞中磷脂酰肌醇三激酶(PI3K)的丝氨酸473(Ser473)和蛋白激酶B(AKT)的苏氨酸308(Thr308)位点的磷酸化水平的影响。结果与Control组相比,主要定位于滋养细胞中的ITGA5在PE胎盘组织中的mRNA及蛋白表达显著降低(P<0.05),而ITGA5基因的DNA甲基化水平却显著增加(P<0.05);与HTR-8或JEG-3的pcCtrl组细胞相比,pcITGA5组细胞的增殖、迁移及侵袭能力均显著增强(P<0.05),PI3K、AKT的Ser473和Thr308位点的磷酸化水平也显著升高(P<0.05)。结论本研究表明PE胎盘中ITGA5的启动子区域高甲基化水平导致其在PE胎盘组织中的表达显著降低,而体外过表达ITGA5可能通过激活PI3K/AKT通路促进滋养细胞的增殖、迁移及侵袭能力。

Objective To investigate the methylation level of integrin α 5(ITGA5) in preeclampsia(PE) placenta and its effect on proliferation, migration and invasion of trophoblast. Methods The placental tissues of 32 preeclampsia patients(PE group) and 38 normal pregnant women(control group) were collected from July 2018 to July 2020. RT-qPCR, Western blot and immunohistochemical staining were used to detect the mRNA, protein expression and localization of ITGA5 in placentas of the two groups. Methylation specific PCR(MSP) and sulfite sequencing PCR(BSP) were used to detect the difference of ITGA5 gene methylation level between PE group and control group. HTR8 and JEG-3 trophoblast cell lines were cultured in vitro. The over expressed ITGA5 plasmids were transfected into trophoblast cells by liposome transfection technique.The trophoblast cells were divided into pcCtrl group and pcITGA5 group. RT-qPCR and Western blot were used to detect the transfection efficiency. CCK-8 proliferation test, scratch test and Transwell invasion test were used to detect the effects of overexpression of ITGA5 on the proliferation, invasion and invasion of trophoblasts. Western blot was used to detect the effect of overexpression of ITGA5 on the phosphorylation of Ser473 in PI3K, and Thr308 in AKT. Results Compared with the control group, the mRNA and protein expression of ITGA5 mainly located in trophoblast in PE placenta significantly decreased(P<0.05), while the DNA methylation level of ITGA5 gene significantly increased(P<0.05). Compared with pcCtrl cells of HTR-8 or JEG-3, the proliferation, migration and invasion ability of pcITAG5 cells significantly enhanced(P<0.05), and the phosphorylation levels of Ser473 in PI3K and Thr308 in AKT also significantly increased(P<0.05). Conclusion The DNA hypermethylation level of ITGA5 promoter region in PE placenta leads to a significant decreasing of its expression in PE placenta. Overexpression of ITGA5 in vitro may promote the proliferation, migration and invasion of trophoblast by activating PI3K/AKT pathway.