You Guangning, Li Zhe, Li Ling, Xu Chengfang
Department of Gynecology and Obstetrics, The Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, Guangdong Province, PR China.
Heliyon. 2024 May 3;10(9):e30702. doi: 10.1016/j.heliyon.2024.e30702. eCollection 2024 May 15.
Pre-eclampsia (PE) is a syndrome with no specific pathological mechanism and is specific to pregnancy. The combined analysis of proteomics and transcriptomics possesses many benefits for treating this disease. mA modification plays a major role in PE; however, mechanism have not been studied clearly. This study investigated the potential mechanism underlying the role of mA in PE.
Mass spectrometry-based label-free quantitative proteomics and transcriptomics experiments were conducted on the placenta of patients with pre-eclampsia and normal pregnancies, and the two omics were followed by joint analysis. Total m6A modification in placental tissues, HTR8/SVneo cells, and JEG-3 cells was measured by dot blot. The levels of RBM15 and CD82 in tissues and cells were detected using qPCR. The protein levels of G3BP1, RBM15, MMP-2, YTHDF2, and MMP-9 were measured by western blotting. The function, migration, and invasion characteristics of HTR8/SVneo and JEG-3 cells were measured using Transwell assays. SRAMP predicted the m6A modification site in the CD82 mRNA 3'UTR, and this was confirmed using luciferase activity and YTHDF2-RIP.
mA modification was promoted in the PE group, and the RBM15 abundance was increased. Overexpression of RBM15 increased mA modification. However, overexpression of RBM15 suppressed the expression of MMP-2 and MMP-9 and also the migratory and invasive capabilities of HTR8/SVneo and JEG-3 cells. CD82 expression levels were decreased in PE, and CD82 expression was confirmed via qPCR, western blotting and immunofluorescence. Furthermore, RBM15 overexpression reduced CD82 mRNA and protein levels. Luciferase activity and YTHDF2-RIP results verified that overexpression of RBM15 promoted the binding ability between YTHDF2 and the CD82 3'UTR, thereby decreasing CD82 expression. Finally, CD82 overexpression reversed the effect of RBM15 overexpression on the expression of MMP-2 and MMP-9 and on the migratory and invasive capabilities of the cells.
Overexpression of RBM15 hindered the migratory and invasive capabilities of trophoblasts, while concurrently enhancing m6A modification. The potential mechanism was that overexpression of RBM15 promoted the binding capability between YTHDF2 and CD82 3'UTR and decrease the expression of CD82. Thus, this study provides a theoretical basis for the treatment of PE.
子痫前期（PE）是一种无特定病理机制且仅发生于妊娠期间的综合征。蛋白质组学和转录组学的联合分析在治疗该疾病方面具有诸多优势。N6-甲基腺苷（m6A）修饰在子痫前期中起主要作用；然而，其机制尚未得到明确研究。本研究探讨了m6A在子痫前期中发挥作用的潜在机制。
对患有子痫前期的患者和正常妊娠患者的胎盘进行基于质谱的无标记定量蛋白质组学和转录组学实验，并对这两种组学进行联合分析。通过斑点印迹法检测胎盘组织、HTR8/SVneo细胞和JEG-3细胞中的总m6A修饰。使用qPCR检测组织和细胞中RBM15和CD82的水平。通过蛋白质印迹法测定G3BP1、RBM15、MMP-2、YTHDF2和MMP-9的蛋白质水平。使用Transwell实验测定HTR8/SVneo和JEG-3细胞的功能、迁移和侵袭特性。SRAMP预测CD82 mRNA 3'UTR中的m6A修饰位点，并使用荧光素酶活性和YTHDF2-RIP进行验证。
子痫前期组的m6A修饰增强，RBM15丰度增加。RBM15的过表达增加了m6A修饰。然而，RBM15的过表达抑制了MMP-2和MMP-9的表达以及HTR8/SVneo和JEG-3细胞的迁移和侵袭能力。子痫前期中CD82表达水平降低，并通过qPCR、蛋白质印迹法和免疫荧光法进行了确认。此外，RBM15的过表达降低了CD82 mRNA和蛋白质水平。荧光素酶活性和YTHDF2-RIP结果证实，RBM15的过表达促进了YTHDF2与CD82 3'UTR之间的结合能力，从而降低了CD82的表达。最后，CD82的过表达逆转了RBM15过表达对MMP-2和MMP-9表达以及细胞迁移和侵袭能力的影响。
RBM15的过表达阻碍了滋养层细胞的迁移和侵袭能力，同时增强了m6A修饰。潜在机制是RBM15的过表达促进了YTHDF-2与CD82 3'UTR之间的结合能力并降低了CD82的表达。因此，本研究为子痫前期的治疗提供了理论依据。
