Wu Zhi-Hong, Wang Yong-Heng, Liu Tai-Hang, Ruan Ling-Ling, Xie You-Long, Li Fang-Fang, Ding Yu-Bin
Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Chongqing 400016, China.
Joint International Research Laboratory of Reproduction and Development of the Ministry of Education of China, Chongqing Medical University, Chongqing 400016, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2022 Sep;53(5):805-814. doi: 10.12182/20220960301.
To explore the effects of hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) on the migration and invasion of HTR-8/SVneo cells, a human trophoblast cell line, and its potential mechanism of action.
Immunofluorescence staining was done to evaluate the expression levels of HADHA in samples of normal villi and recurrent spontaneous abortion (RSA) villi at 6-8 weeks. Lentiviral infection system was used to construct stable HTR-8/SVneo cell lines with overexpression and knockdown. Western blot, qRT-PCR, Wound-healing assay, and Transwell assay were used to determine the effect of HADHA on the migration and invasion of HTR-8/SVneo cells and the expression of relevant genes. Transcriptome sequencing and bioinformatics analysis were done to screen for the potential target genes and signaling pathways regulated by HADHA. The specific molecular mechanism of how HADHA regulates the migration and invasion of HTR-8/SVneo cells was examined by adding the inhibitor of protein kinase B (PKB/AKT).
HADHA was highly expressed in extravillous trophoblasts (EVT) of RSA villus samples as compared with samples from the normal control group. In HTR-8/SVneo cells overexpressing , the expression levels of migration and invasion-related genes, including -, 2, 9, and , were decreased (<0.01,<0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened (<0.05). knockdown increased the expression levels of -, 2, 9, and (<0.01, <0.05), and promoted the migration and invasion of HTR-8/SVneo cells (<0.05). In addition, overexpression decreased the phosphorylation levels of PI3K and AKT (<0.05) and inhibited the PI3K/AKT signaling pathway. knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with knockdown, the migration and invasion of the cells were significantly reduced.
HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway.
探讨α-羟酰基辅酶A脱氢酶（HADHA）对人滋养层细胞系HTR-8/SVneo细胞迁移和侵袭的影响及其潜在作用机制。
采用免疫荧光染色法评估6 - 8周正常绒毛和复发性自然流产（RSA）绒毛样本中HADHA的表达水平。利用慢病毒感染系统构建HADHA过表达和敲低的稳定HTR-8/SVneo细胞系。采用蛋白质免疫印迹法（Western blot）、实时定量聚合酶链反应（qRT-PCR）、划痕实验和Transwell实验，检测HADHA对HTR-8/SVneo细胞迁移和侵袭的影响以及相关基因的表达。进行转录组测序和生物信息学分析，筛选HADHA调控的潜在靶基因和信号通路。通过添加蛋白激酶B（PKB/AKT）抑制剂，研究HADHA调控HTR-8/SVneo细胞迁移和侵袭的具体分子机制。
与正常对照组样本相比，RSA绒毛样本的绒毛外滋养层细胞（EVT）中HADHA高表达。在过表达HADHA的HTR-8/SVneo细胞中，迁移和侵袭相关基因（包括-、2、9和）的表达水平降低（<0.01，<0.05），HTR-8/SVneo细胞的迁移和侵袭能力减弱（<0.05）。敲低HADHA可增加-、2、9和的表达水平（<0.01，<0.05），并促进HTR-8/SVneo细胞的迁移和侵袭（<0.05）。此外，过表达HADHA可降低PI3K和AKT的磷酸化水平（<0.05），并抑制PI3K/AKT信号通路。敲低HADHA可激活PI3K/AKT信号通路。当向敲低HADHA的稳定HTR-8/SVneo细胞系中添加AKT抑制剂MK-2206时，细胞的迁移和侵袭显著减少。
HADHA通过抑制PI3K/AKT信号通路抑制HTR-8/SVneo细胞的迁移和侵袭。
