环状RNA circ_0002348参与调控滋养细胞功能及其在子痫前期发病机制中的作用

The dysfunctions of placental trophoblast cell is involved in the occurrence of preeclampsia, though its mechanisms have not been fully explored. Circular RNA (circRNA) is a novel class of non-coding RNA characterized by covalently closed loop structures without 5' and 3' polarities, working as competitive endogenous (ceRNA) to bind to “miRNA sponge”. Our previous study indicated that the expression of circ_0002348 in placental tissue of patients with preeclampsia was significantly elevated than those in healthy women. Meanwhile, increased proliferation and enhanced invasion of HTR-8/SVneo cells was observed in circ_0002348 knockdown groups compared with control group. Therefore, we hypothesize that circ_0002348 acts as a ceRNA binding to miR-377, then regulates the key molecules of PI3K/Akt/mTOR signals pathways, and then affects the function of trophoblast cells, leading to the happening of preeclampsia. The current project aims to investigate the effect of circ_0002348 on the proliferation, apoptosis, migration and invasion of trophoblast cell by cell function test and next to elucidate its underlying molecular mechanism by RNA-seq, RIP and dual-luciferase reporter assay. Finally, we will explore the function of circ_0002348 in vivo by establishing preeclampsia rat model. We hope to provide a theoretical basis for prediction and treatment of preeclampsia.

滋养细胞功能异常介导子痫前期的发生，但其确切机制尚未完全阐明。环状RNA(circRNA)是一类5'末端和3'末端通过反向剪接共价结合的非编码RNA，主要通过ceRNA机制与miRNA结合发挥调节作用。我们前期研究发现circ_0002348在子痫前期胎盘中表达异常升高，且能够调控滋养细胞增殖和迁移功能。基于此，提出假说：circ_0002348通过ceRNA方式结合miR-377并靶向调控PI3K/Akt/mTOR信号通路关键分子的表达，从而影响滋养细胞功能，参与子痫前期发病。本项目拟采用细胞生物学功能实验，探讨circ_0002348对滋养细胞增殖凋亡、迁移侵袭功能的影响；利用高通量测序、RIP和荧光报告实验，揭示circ_0002348调控滋养细胞功能的具体分子机理；构建大鼠子痫前期疾病模型进行circ_0002348的体内功能研究。以期为子痫前期的预测和治疗提供新思路。

背景：子痫前期被认为是一种胎盘源性疾病。胎盘形成过程中滋养层的生物功能障碍与子痫前期有关。越来越多的证据表明，环状RNA（circRNAs）参与调节子痫前期的病理过程。然而，hsa_circ_0002348在子痫前期中的作用和调控机制尚未阐明。.研究内容：本研究通过对胎盘和内皮细胞的circRNA表达谱进行重叠分析以发现了一种功能未知的circRNA，即hsa_circ_0002348，实时定量PCR（qRT-PCR）和原位杂交（ISH）检测其在滋养细胞和胎盘组织中的表达，通过建立脂多糖（LPS）诱导的子痫前期小鼠模型，以确定hsa_circ_0002348在体内的作用，借助RNA免疫沉淀（RIP）、荧光素酶报告物测定、qRT-PCR、westernblot、功能获得和丧失以及挽救实验，以揭示hsa_circ_0002348及其与miR-126-3p和BAK1在调节滋养细胞增殖和凋亡中的作用。另外，通过荧光原位杂交（FISH）和免疫组织化学（IHC）分别检测小鼠和人胎盘中miR-126-3p和BAK1的表达。.重要结果和关键数据：Hsa_circ_0002348在子痫前期胎盘组织中表达显著增加，并与子痫前期患者临床表现的严重程度呈正相关。在LPS诱导的子痫前期小鼠模型中，其过度表达加剧了子痫前期样临床表现。在功能上，发现hsa_circ_0002348抑制滋养细胞增殖并促进凋亡。从机制上讲，hsa_circ_0002348作为内源性miR-126-3p海绵，上调BAK1的表达。此外，hsa_circ_0002348敲低和miR-126-3p过表达都激活了哺乳动物雷帕霉素（mTOR）和ERK1/2信号通路。.科学意义：Hsa_circ_0002348是一种通过miR-126-3p/BAK1轴调节滋养细胞增殖和凋亡的新型调控因子，可能是检测和治疗子痫前期的潜在靶点。