Zhang Xiaoli, Wang Gang, Li Hui, Jiang Xiangming, Zhao Xiaoyong
Department of Obstetrics and Gynecology, Affiliated Huadu Hospital, Southern Medical University (People's Hospital of Huadu District) Guangzhou 510800, China.
The Third School of Clinical Medicine, Southern Medical University Guangzhou 510800, China.
Am J Transl Res. 2021 May 15;13(5):4103-4119. eCollection 2021.
To investigate the effects of 5-aza-2'-deoxycytidine (5-AZA-DC) on preeclampsia (PE) and functional mechanisms dependent on STAT3.
Trophoblastic cells (HTR8/Svneo, JEG-3, JAR and BeWo) were used to constructed STAT3-overexpressing or -silenced cells. qRT-PCR, Western blot, and FISH were used to detect mRNA and protein expression. GST-pull down, ChIP and dual luciferase reporter were used to prove the association of STAT3 and PTEN or TSC2, LC-MS/MS for proteome, and MeDIP-Seq for transcriptome. CCK-8 and flow cytometry were used to examine cell proliferation and apoptosis. C57BL/6J mice were divided into 4 groups (control, control + 5-AZA-DC, PE and PE + 5-AZA-DC). Systolic blood pressure, 24-h urinary protein, APTT, D-D, PT, ALT, Scr, and BUN were determined. Placental blood flow velocity was detected by Doppler ultrasound, HE staining for kidney injury.
STAT3, PTEN and TSC2 were the dominantly differential expressed genes in preeclampsia. Aberrant STAT3 expression increased DNMT1 levels. STAT3 regulated PTEN promoter activity. STAT3 interacted with PTEN and TSC2. DNMT1 was increased while STAT3, PTEN and TSC2 were decreased by 5-AZA-DC. Cell proliferation was promoted and apoptosis was inhibited by 5-AZA-DC. PE-induced STAT3 down-regulation was restored by 5-AZA-DC. Systolic blood pressure, 24-h urinary protein, APTT, D-D, PT, ALT, Scr and BUN were increased, and velocity of placental blood flow was inhibited in PE compared with control mice, while 5-AZA-DC relived these indicators.
Preeclampsia symptoms was relieved by 5-AZA-DC, suggesting that 5-AZA-DC could be used as a potential drug for epigenetic treatment of preeclampsia.
探讨5-氮杂-2'-脱氧胞苷（5-AZA-DC）对子痫前期（PE）的影响及其依赖信号转导和转录激活因子3（STAT3）的作用机制。
采用滋养层细胞（HTR8/Svneo、JEG-3、JAR和BeWo）构建STAT3过表达或沉默细胞。运用实时定量聚合酶链反应（qRT-PCR）、蛋白质免疫印迹法（Western blot）和荧光原位杂交（FISH）检测mRNA和蛋白质表达。采用谷胱甘肽-S-转移酶下拉实验（GST-pull down）、染色质免疫沉淀实验（ChIP）和双荧光素酶报告基因实验证明STAT3与第10号染色体缺失的磷酸酶和张力蛋白同源物（PTEN）或结节性硬化症复合物2（TSC2）的关联，采用液相色谱-串联质谱（LC-MS/MS）进行蛋白质组学分析，采用甲基化DNA免疫沉淀测序（MeDIP-Seq）进行转录组学分析。运用细胞计数试剂盒-8（CCK-8）和流式细胞术检测细胞增殖和凋亡。将C57BL/6J小鼠分为4组（对照组、对照组+5-AZA-DC组、子痫前期组和子痫前期+5-AZA-DC组）。测定收缩压、24小时尿蛋白、活化部分凝血活酶时间（APTT）、D-二聚体（D-D）、凝血酶原时间（PT）、谷丙转氨酶（ALT）、血清肌酐（Scr）和血尿素氮（BUN）。采用多普勒超声检测胎盘血流速度，采用苏木精-伊红（HE）染色观察肾损伤情况。
STAT3、PTEN和TSC2是子痫前期中差异表达显著的基因。异常的STAT3表达增加了DNA甲基转移酶1（DNMT1）水平。STAT3调节PTEN启动子活性。STAT3与PTEN和TSC2相互作用。5-AZA-DC降低了DNMT1水平，同时使STAT3、PTEN和TSC2水平升高。5-AZA-DC促进细胞增殖并抑制细胞凋亡。5-AZA-DC恢复了子痫前期诱导的STAT3下调。与对照小鼠相比，子痫前期小鼠的收缩压、24小时尿蛋白、APTT、D-D、PT、ALT、Scr和BUN升高，胎盘血流速度受到抑制，而5-AZA-DC缓解了这些指标。
5-AZA-DC缓解了子痫前期症状，表明5-AZA-DC可作为子痫前期表观遗传治疗的潜在药物。
