由一种lncRNA调控的HLA-G在子痫前期胎盘中的作用机制研究

The inhibition of expression by HLA-G has been contributed to the pathogenesis of preeclampsia. The lower lever or expression level of HLA-G had been observed, but till now, the cause leading to the phenomena had not been making clear. It had been reported that the genetic variation or gene polymorphisms are closely associated with the lower expression level, but there is no report to study the regulation of expression in HLA-G by HOTAIR and the function of trophoblast cells in preeclampsia. In our previous study, we observed that the positive correlation of the expression of HOTAIR and HLA-G. It had been believed that HOTAIR promotes HLA-G expression via inhibiting miR-152, furthermore the bioinformatics analysis supports that potential interaction between HOTAIR and miR-152, which makes us speculate that HOTAIR down regulate the HLA-G expression level via enhancing miR-152. The lower HLA-G leads to the dysfunction of the trophoblast cells in placeta. In the study, real-time PCR, luciferase assay, loss-of-function and gain-of-function were used to investigate the regulation of HOTAIR on HLA-G via miR-152. The HTR8-SV40 cells were transfected with HOTAIR-plasmid si-HOTAIR. The effect of the inhibited expression and overexpression HOTAIR on HLA-G, including trophoblast invasion, and natural killer cell-mediated cytolysis were assessed by reverse-transcription polymerase chain reaction and western blot analysis, transwell invasion assay and MTT assay,respectively. The research will contribute to further explore potential application of HOTAIR in the treatment of preeclampsia and improving the therapeutic strategies and better prognosis.

HLA-G的低表达在子痫前期的发病机制中占有重要的作用，但引起HLA-G偏低的因素及作用机制仍不清楚。我们已观察到一种lncRNA HOTAIR与 HLA-G的表达呈正相关，并且在子痫前期中低表达。肿瘤细胞的研究支持由miR-152介导了HOTAIR对HLA-G的调控，促使我们推测HOTAIR的低表达调控了HLA-G的低表达，并为miR-152所介导，引起了子痫前期胎盘中滋养细胞的功能改变。因此，本研究拟采用qRT-PCR，荧光报告载体实验，获得性或者丢失功能试验，研究HOTAIR, HLA-G和miR-152的相互调控关系，并阐明其参与子痫前期的作用机制。转染滋养细胞HTR8-SVneo表达载体使其高表达和干扰HLA-G表达，采用MTT比色、Transwell实验、流式细胞术等阐明HOTAIR在子痫前期中的功能。该研究为预防子痫前期的药物开发和制定治疗方案提供了依据。

子痫前期是妊娠特有的疾病，由胎盘的植入所引起，唯一缓解的方法只有娩出胎盘。HLA-G 与母体对半抗原胎儿的妊娠耐受关系非常密切。HOTAIR影响了HLA-G胎盘中的表达，可能参与了子痫前期的致病机制。.主要完成的研究内容包括：.（1）对胎盘组织进行RNA-seq测序分析，在组织中初步研究了HOTAIR、miR-152-3p及HLA-G的相互调控关系。.（2）构建HOTAIR高表达及抑制慢病毒载体，体外转染HTR-8/Svneo细胞后，检测HLA-G蛋白表达情况，以及滋养细胞增殖、迁移、侵袭功能变化，还有EMT相关蛋白变化水平。.（3）体外细胞转染miR-152-3p mimics、inhibitor后检测HLA-G及EMT相关蛋白表达情况，以及滋养细胞迁移、侵袭功能变化。.（4）滋养细胞中过表达及抑制HLA-G后，检测HLA-G表达情况及EMT相关蛋白表达情况、细胞迁移、侵袭功能变化，检测NK细胞对转染过表达及抑制HLA-G载体后的滋养细胞的细胞毒性反应。.（5）通过早孕期尾部一次性注射LPS构建妊娠早期子痫前期SD大鼠模型，在模型大鼠胎盘组织中验证HOTAIR-miR152-HLA-G调控机制、维生素D代谢通路、炎症信号通路、氧化应激水平、血管内皮功能和细胞凋亡以及AKT，MAPK/ERK信号通路相关因子表达的变化。通过干预胎盘中HLA-G的表达观察模型SD大鼠的表型及妊娠结局的改变。.重要的研究结果为：.（1）子痫前期过程中HOTAIR通过miRNA-152-3p靶向HLA-G的调控机制是ceRNA机制。.（2）HOTAIR通过miR-152-3p调控HLA-G的表达促进EMT过程增加滋养细胞的迁移、侵袭功能。.（3）HLA-G高表达后可保护HTR-8/Svneo细胞避免NK细胞的杀伤作用。.（4）建立LPS诱导的孕早期子痫前期SD大鼠模型，通过对大鼠胎盘中的HOTAIR-miR152-HLA-G调控轴可以改善母体子痫前期的表型、妊娠结局和子代认知功能。.本研究从体内外系统阐明了HOTAIR在子痫前期发病机制中的功能和对下游基因的调控机制。.该研究为预防子痫前期的药物开发和制定治疗方案提供了科学的依据和重要的实验基础。