高迁移率族蛋白HMGB3致胎儿生长受限的分子机制研究

HMGB3 is a protein which is found by means of mass-spectrometric technique and is significantly lower expression in the placentas of FGR(fetal growth restriction) than those of normal pregnancy. Our studies have found that overexpression HMGB3 can promote the proliferation and inhibit the apoptosis of placental trophoblastic cells. Further studies have revealed the effect of HMGB3 may be related to the abnormal Wnt/β-catenin signaling pathway which through the protein LEF1. Furthermore, many studies have confirmed that the Wnt/β-catenin signaling pathway is associated with the dysfunction of placental trophoblastic cells, and LEF1 is the transcriptional activator of Wnt/β-catenin signaling pathway.This study intends to demonstrate the effect of HMGB3 on the function of placental trophoblastic cells and Wnt/β-catenin signaling pathway through overexpression and expression silencing of HMGB3, which with the research object of HTR - 8/SVneo trophoblastic cells. Further study will carried out using rescue experiment strategy to demonstration the molecular mechanism of HMGB3 inhibiting the Wnt/β-catenin signaling pathway and inducing the dysfunction of placental trophoblastic cells through interaction with LEF1. With the aid of in vivo animal models, the potential application of HMBG3 in the treatment of FGR will be evaluated. There is not any report about HMGB3 in the FGR, our studies will provide new theoretical basis and targets in the treatment of FGR.

HMGB3是我们借助质谱技术发现的、差异低表达于FGR（胎儿生长受限）胎盘的蛋白。前期研究发现，HMGB3过表达可促进滋养细胞增殖、抑制其凋亡，且可上调Wnt/β-catenin通路；免疫共沉淀发现，Wnt/β-catenin通路的转录激活因子LEF1可与其结合。已知Wnt/β-catenin通路与滋养细胞功能紊乱有关，因此提示HMGB3可能调控LEF1介导的Wnt/β-catenin通路而发挥作用。研究以HTR-8/SVneo滋养细胞为研究对象，从正反两方面论证HMGB3对滋养细胞功能及Wnt/β-catenin通路的影响；以挽救实验策略，系统论证HMGB3与LEF1相互作用、抑制Wnt/β-catenin通路、引起滋养细胞功能紊乱、致FGR发生的机制；借助在体动物模型，评估HMGB3在FGR治疗中的潜在价值。目前未见HMGB3与FGR的功能报道，研究将为FGR防治提供新依据。

胎儿生长受限（FGR）是妊娠的主要并发症之一，可导致围生儿发生严重的近远期疾病。目前已有大量研究发现高迁移率族3（HMGB3）蛋白有助于多种癌症的发展。然而，FGR的发病机制尚不十分清楚，尤其关于HMGB3在FGR发病中的作用还未见报道。通过蛋白质免疫印迹（Western blotting）和实时定量聚合酶链式反应（qRT-PCR），我们检测了6例正常妊娠和5例FGR患者的胎盘组织中HMGB3的表达水平。通过构建HMGB3过表达质粒及干扰片段，转染HTR-8/SVneo滋养层细胞系以实现HMGB3在滋养细胞中的过表达和表达沉默。以CCK8检测、Transwell小室和流式细胞术评估差异表达的HMGB3对滋养细胞功能的影响。结果显示与正常对照组相比，FGR胎盘中HMGB3的表达在蛋白质水平显著降低，而在mRNA水平未见明显改变。当HMGB3蛋白在HTR-8/SVneo滋养层细胞系中过表达时，可观察到滋养细胞的增殖和迁移能力明显增强，同时这些细胞的凋亡能力明显受到抑制。而干扰HMGB3蛋白表达后滋养细胞功能实验呈现相反的结果。同时通过qRT-PCR检测细胞功能相关基因（如PCNA、Ki67、Tp53、Bax、MMP-2、E-cadherin）的表达变化。而质谱分析结果显示HMGB3可能与71个蛋白质发生直接或间接的相互作用。我们选取其中差异倍数最大的蛋白MYH10做进一步研究，通过挽救实验我们发现HMGB3 过表达组的增殖、迁移和抗凋亡能力较对照组有明显增强，而共转染了MYH10干扰片段的滋养细胞的增殖、迁移和抗凋亡能力下降。说明HMGB3可能与MYH10相互作用导致胎盘滋养细胞功能紊乱致FGR的发生。最后我们包载了过表达的HMGB3腺相关病毒，尾静脉注射LPS诱导的大鼠FGR模型，发现过表达HMGB3组的胎鼠体型及体重均有明显改善，说明HMGB3有潜在的FGR治疗价值。