HADHA通过调节PI3K/AKT信号通路抑制人绒毛膜滋养细胞HTR-8/SVneo迁移与侵袭

吴芝红; 王永恒; 刘太行; 阮灵灵; 谢游龙; 李方方; 丁裕斌

doi:10.12182/20220960301

HADHA通过调节PI3K/AKT信号通路抑制人绒毛膜滋养细胞HTR-8/SVneo迁移与侵袭

HADHA Inhibits the Migration and Invasion of HTR-8/SVneo Cells by Regulating PI3K/AKT Signaling Pathway

摘要

摘要:
目的探讨羟酰基辅酶A脱氢酶α亚基（hydroxyacyl-CoA dehydrogenase alpha subunit, HADHA）对人绒毛膜滋养细胞HTR-8/SVneo迁移和侵袭能力的影响及其潜在作用机制。
方法通过组织免疫荧光染色检测HADHA在6～8周正常早孕绒毛及复发性自然流产绒毛样本中表达水平的差异；慢病毒系统构建HADHA过表达及敲低稳转HTR-8/SVneo细胞系，并采用qRT-PCR、Western blot、Transwell、细胞划痕实验评价HADHA对HTR-8/SVneo细胞迁移侵袭能力和相关基因表达的影响；转录组测序及生物信息学分析筛选HADHA可能调控的靶基因及信号通路；加入蛋白激酶B（protein kinase B, AKT）抑制剂明确HADHA调节HTR-8/SVneo细胞迁移侵袭的具体分子机制。
结果 HADHA在复发性自然流产样本的绒毛外滋养层（extravillous trophoblast, EVT）中较正常对照组中高表达。过表达HADHA的HTR-8/SVneo细胞中迁移侵袭相关基因HLA-G、MMP2、MMP9、NCAD的表达水平降低（P<0.01，P<0.05），且迁移和侵袭能力减弱（P<0.05）；敲低HADHA后，基因HLA-G、MMP2、MMP9、NCAD的表达水平增高（P<0.01，P<0.05），且迁移和侵袭能力增强（P<0.05）。此外，过表达HADHA后p-PI3K、p-AKT水平降低（P<0.05），PI3K/AKT信号通路被抑制；敲低HADHA后PI3K/AKT信号通路被激活。在HADHA敲低稳转细胞系中加入AKT抑制剂MK-2206后，细胞迁移侵袭能力较对照敲低组减弱（P<0.01，P<0.05）。
结论 HADHA通过抑制PI3K/AKT信号通路抑制HTR-8/SVneo细胞的迁移与侵袭。

Abstract:
Objective 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.
Methods 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 HADHA 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).
Results 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 HADHA, the expression levels of migration and invasion-related genes, including HLA-G, MMP2, MMP9, and NCAD, were decreased (P<0.01, P<0.05), and the migration and invasion abilities of HTR-8/SVneo cells were weakened (P<0.05). HADHA knockdown increased the expression levels of HLA-G, MMP2, MMP9, and NCAD (P<0.01, P<0.05), and promoted the migration and invasion of HTR-8/SVneo cells (P<0.05). In addition, HADHA overexpression decreased the phosphorylation levels of PI3K and AKT (P<0.05) and inhibited the PI3K/AKT signaling pathway. HADHA knockdown activated the PI3K/AKT signaling pathway. When MK-2206, an AKT inhibitor, was added to stable HTR-8/SVneo cell lines with HADHA knockdown, the migration and invasion of the cells were significantly reduced.
Conclusion HADHA inhibits the migration and invasion of HTR-8/SVneo cells by inhibiting the PI3K/AKT signaling pathway.

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