长链非编码RNA LINC00534调控滋养细胞侵袭参与子痫前期发生的作用与机制研究

A long non-coding RNA (lncRNA) named as LINC00534 is identified by our lncRNA microarray. Our previous study indicated that LINC00534 was more highly expressed in preeclampsia placental tissue and conserved in species. Overexpression of LINC00534 inhibits trophoblast invasion and promotes apoptosis. Moreover, bioinformatic analysis and RIP-PCR experiment revealed that LINC00534 could bind with the transcriptional co-activating protein p300/CBP so as to gain an epigenetically regulation potential. And p300/CBP could bind with MUC1 promoter. It is known that MUC1 could inhibit trophoblast invasiveness. Further expression microarray analysis and in vitro validation suggested that overexpression of LINC00534 significantly increased the expression of MUC1. Based on above results, we therefore hypothesized that LINC00534 participates in the pathogenesis of preeclampsia by recruiting p300/CBP-mediated epigenetic regulation of MUC1 expression to suppress trophoblast invasion. In this proposal, firstly, we plan to clarify the function of LINC00534 in vitro (HTR-8/SVneo cell) using overexpression and knockdown methods. Secondly, to demonstrate that LINC00534 could recruit p300/CBP to MUC1 promoter and promote MUC1 transcription in the regulation of preeclampsia, we will perform RIP, RNA pull-down, ChIP, deletion mapping, chromosomal conformation capture analysis and rescue experiments. Thirdly, we will test the function of LINC00534 in inhibiting trophoblast invasion and regulating the occurrence of preeclampsia in rat animal models. The function and mechanism of LINC00534 have not been reported. This study may provide a new target for the prevention and treatment of preeclampsia.

LINC00534是我们前期通过lncRNA芯片技术筛选获得的高表达于子痫前期胎盘组织、物种间保守、功能未知的lncRNA。前期发现：LINC00534过表达抑制滋养细胞侵袭、促进细胞凋亡；生物信息学分析与实验提示，LINC00534可与转录辅激活物p300/CBP结合，而p300/CBP可结合于抑制滋养细胞侵袭的MUC1基因的启动子；表达谱芯片与实验证实LINC00534过表达致MUC1表达显著上调。本研究拟采用过表达/沉默策略，论证LINC00534对滋养细胞功能的影响；应用RIP、RNA pull-down、ChIP、挽救实验等，论证LINC00534招募p300/CBP结合MUC1启动子、促进MUC1转录致子痫前期发生的分子机制；通过在体动物模型，评估LINC00534抑制滋养细胞侵袭参与子痫前期发生的作用。LINC00534功能与机制未见报道，研究可能为子痫前期防治提供新靶标。

子痫前期常可导致严重胎儿生长受限、母亲重要脏器损害、早产等结局，是导致母婴死亡率最高的疾病之一，但子痫前期病因尚不明确。目前，临床上缺乏有效的治疗方法。因此，探讨子痫前期的发生机制，具有重要意义。近年来，lncRNA在子痫前期中的研究亦如火如荼。. lncRNA可与miRNA结合，调节靶基因表达，从而在各种生理过程中发挥作用。LINC00534是通过lncRNA芯片技术筛选获得的高表达于子痫前期胎盘组织的未知的lncRNA。本研究旨在探讨LINC00534在子痫前期中的作用及其可能机制。我们主要检测子痫前期患者胎盘组织中LINC00534 的表达水平，从临床水平系统评估LINC00534 与子痫前期之间的关系；采用过表达/沉默策略，从细胞水平论证LINC00534对滋养细胞功能增殖、迁移和凋亡的影响；应用RNA测序、qRT-PCR和蛋白质印迹，论证LINC00534通过降低miR-494-3p促进PTEN表达导致子痫前期发生的分子机制。主要发现：1）LINC00534在子痫前期胎盘组织中的表达较对照组升高；2）在HTR-8/SVneo滋养细胞中过表达LINC00534，可使细胞增殖被抑制、细胞侵袭（MMP2、MMP9）相关基因显著下调、细胞凋亡（Bcl-2）相关基因显著上调，提示LINC00534可以促进HTR-8/SVneo滋养细胞凋亡，抑制HTR-8/SVneo滋养细胞的增殖和侵袭能力；敲低LINC00534后产生了相反的趋势。3）PI3K/AKT信号通路受LINC00534调控，敲低 LINC00534 可降低PTEN 的表达；LINC00534通过靶向mir-494-3p发挥作用，mir-494-3p通过调节PTEN/PI3K/AKT控制HTR-8/SVneo滋养层细胞的生长和迁移。LINC00534是我们从子痫前期患者的胎盘组织中筛选出的差异lncRNA，迄今，国内外均未见LINC00534参与滋养细胞生物学行为的研究报道，可能为子痫前期防治提供新靶标。