生长分化因子G8通过调控性别决定区Y框蛋白2表达影响滋养层细胞增殖、迁移和侵袭的研究

GDF-8 regulates trophoblast cell proliferation, migration, and invasion by modulating SOX2 expression

摘要

目的 探讨生长分化因子G8(GDF-8)在子痫前期(PE)产妇胎盘组织中的表达,并分析GDF-8通过调控性别决定区Y框蛋白2(SOX2)的表达影响滋养层细胞增殖、迁移和侵袭能力的分子机制。方法 收集行剖宫产手术的PE产妇和正常产妇的胎盘组织,qRT-PCR法检测胎盘组织中GDF-8 mRNA表达.体外培养人绒毛膜滋养层细胞系HTR-8/SVneo细胞,将pcDNA-GDF-8、pcDNA-NC1、pcDNA-SOX2、pcDNA-NC2、pcDNA-GDF-8+si-SOX2和pcDNA-GDF-8+si-NC分别转染入细胞,用正常培养、不做转染处理的细胞作为对照.qRT-PCR法检测细胞中GDF-8和SOX2 mRNA的表达水平.CCK-8法检测细胞增殖能力.EdU染色并计数实时增殖的活细胞数占比.划痕实验检测细胞迁移能力.Transwell小室实验测定细胞侵袭能力.Western blot法检测细胞中GDF-8、SOX2、增殖细胞核抗原(PCNA)、Ki67、基质金属蛋白酶2(MMP-2)、MMP-9蛋白的表达水平。结果 与正常产妇比较,PE产妇胎盘组织中GDF-8 mRNA表达水平降低(t=35.174,P<0.001).与pcDNA-NC1组比较,pcDNA-GDF-8组的HTR-8/SVneo细胞增殖能力、EdU染色阳性细胞占比、细胞划痕愈合率以及PCNA、Ki67、MMP-2和MMP-9蛋白相对表达水平均升高,细胞侵袭数目增多(t=21.052、35.174、47.602、19.751、20.519、28.412、25.016、69.453,P<0.001);细胞中SOX2 mRNA和蛋白相对表达水平均升高(t=31.520、26.478,P<0.001).与pcDNA-NC2组比较,pcDNA-SOX2组的HTR-8/SVneo细胞增殖能力、EdU染色阳性细胞占比、细胞划痕愈合率以及PCNA、Ki67、MMP-2和MMP-9蛋白相对表达水平均升高,细胞侵袭数目增多(t=18.025、26.510、32.518、22.690、21.547、24.751、20.957、59.120,P<0.001).与pcDNA-GDF-8+si-NC组比较,pcDNA-GDF-8+si-SOX2组的HTR-8/SVneo细胞增殖能力、EdU染色阳性细胞占比、细胞划痕愈合率以及PCNA、Ki67、MMP-2和MMP-9蛋白相对表达水平均降低,细胞侵袭数目减少(t=13.085、19.250、26.510、15.420、14.862、24.599、26.743、72.061,P<0.001)。结论 PE产妇胎盘组织中GDF-8表达降低,过表达GDF-8可能通过诱导SOX2表达促进滋养层细胞增殖、迁移和侵袭,从而影响PE的发生发展.

Abstract

Objective To investigate the expression of growth differentiation factor-8 (GDF-8) in the placental tissue of patients with preeclampsia (PE) and to explore the molecular mechanism by which GDF-8 regulates the proliferation, migration, and invasion of trophoblast cells by modulating the expression of sex determining region Y-box 2 (SOX2). Methods Placental tissues were collected from PE patients and healthy pregnant women undergoing cesarean section. The mRNA expression level of GDF-8 in placental tissues was detected by quantitative real-time PCR (qRT-PCR). The human chorionic trophoblast cell line HTR-8/SVneo was cultured in vitro. Cells were transfected with pcDNA-GDF-8, pcDNA-NC1, pcDNA-SOX2, pcDNA-NC2, pcDNA-GDF-8+si-SOX2, and pcDNA-GDF-8+si-NC, respectively. Cells cultured under normal conditions without transfection were used as the control. The mRNA expression levels of GDF-8 and SOX2 were detected by qRT-PCR. Cell proliferation was assessed using the CCK-8 assay. The proportion of EdU-positive proliferating cells was calculated. Cell migration ability was evaluated using a scratch assay, and invasion ability was assessed using the Transwell chamber assay. Western blot analysis was used to determine the protein expression levels of GDF-8, SOX2, proliferating cell nuclear antigen (PCNA), Ki67, matrix metalloproteinase-2 (MMP-2), and MMP-9. Results Compared with normal pregnant women, the expression level of GDF-8 mRNA in the placental tissue of PE patients was significantly decreased (t=35.174, P<0.001). Compared with the pcDNA-NC1 group, the HTR-8/SVneo cells in the pcDNA-GDF-8 group showed significantly enhanced proliferation, increased proportion of EdU-positive cells, improved scratch wound healing rate, upregulated expression of PCNA, Ki67, MMP-2, and MMP-9 proteins, and increased number of invading cells (t=21.052, 35.174, 47.602, 19.751, 20.519, 28.412, 25.016 and 69.453, respectively, P<0.001). Both SOX2 mRNA and protein expression levels were also significantly elevated in these cells (t=31.520 and 26.478, respectively, P<0.001). Compared with the pcDNA-NC2 group, the pcDNA-SOX2 group exhibited enhanced cell proliferation, higher proportion of EdU-positive cells, improved scratch wound healing rate, upregulated expression of PCNA, Ki67, MMP-2, and MMP-9 proteins, and increased cell invasion (t=18.025, 26.510, 32.518, 22.690, 21.547, 24.751, 20.957 and 59.120, respectively, P<0.001). In contrast, compared with the pcDNA-GDF-8+si-NC group, cells in the pcDNA-GDF-8+si-SOX2 group showed reduced proliferation, lower proportion of EdU-positive cells, decreased scratch healing rate, downregulated expression of PCNA, Ki67, MMP-2, and MMP-9, and reduced cell invasion (t=13.085, 19.250, 26.510, 15.420, 14.862, 24.599, 26.743 and 72.061, respectively, P<0.001). Conclusion The expression of GDF-8 is decreased in the placental tissue of patients with PE. Overexpression of GDF-8 may promote the proliferation, migration, and invasion of trophoblast cells by inducing SOX2 expression, thereby influencing the occurrence and development of PE.

关键词

子痫前期 / 滋养层细胞 / 生长分化因子G8 / 性别决定区Y框蛋白2 / 增殖 / 迁移 / 侵袭

Key words

preeclampsia / trophoblast cell / GDF-8 / SOX2 / proliferation / migration / invasion

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基金资助

湖南省卫生健康委科研计划项目(B202305016863)