METTL3-Mediated m6A RNA Methylation of ZBTB4 Interferes With Trophoblast Invasion and Maybe Involved in RSA

Abstract

N6-methyladenosine (m6A) was the most abundant modification of mRNA and lncRNA in mammalian cells and played an important role in many biological processes. However, whether m6A modification was associated with recurrent spontaneous abortion (RSA) and its roles were still unclear.Methods: Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) was used to study the global m6A modification pattern in RSAs and controls. RNA sequencing (RNA-Seq) was used to study the level of global mRNA in two groups. Real-time quantitative PCR (RT-qPCR) was used to verify the level of mRNA of METTL3 and ZBTB4. MeRIP–qPCR was conducted to test the level of ZBTB4 m6A modification in two groups. In order to further explore whether ZBTB4 was the substrate of METTL3, the HTR-8/SVneo (HTR-8) cell line was selected for the knockdown and overexpression of METTL3. To study whether METTL3 regulated the ZBTB4 expression by recognizing ZBTB4 mRNA m6A motifs in coding sequences (CDS), dual-luciferase reporter assay was conducted. RNA stability assays using actinomycin D were conducted to study the RNA stability of the HTR-8 cell line with METTL3 overexpression and knockdown. To illustrate the role of METTL3 in the invasion of trophoblast, matrigel invasion assays and transwell migration assays were conducted using the HTR-8 cell line with METTL3 overexpression and knockdown.Results: A total of 65 genes were found with significant differences both in m6A modification and mRNA expression. We found m6A methyltransferase METTL3 was significantly down-regulated in the RSA group. Through gene function analysis, RT-qPCR, MeRIP–qPCR validation experiment, knockdown, and overexpression of METTL3 in the HTR-8 cell line, ZBTB4 was selected as one target of METTL3. Furthermore, we clarified that METTL3 regulated the expression of ZBTB4 by recognizing ZBTB4 mRNA m6A motifs in the CDS using the dual-luciferase reporter assay and METTL3 regulated the invasion of trophoblast by altering the stability and expression of ZBTB4 by RNA stability, matrigel invasion, and transwell migration assays.Conclusion: Our study revealed the mechanism by which METTL3 regulated the stability and expression of ZBTB4 and the trophoblast migration ability of RSA. A new perspective was provided for exploring the mechanism of embryonic development in RSA patients.

FTO protein regulates the TGF‐β signalling pathway through RNA N6‐methyladenosine modification to induce unexplained recurrent spontaneous abortion

“…Li et al [14] found that the overexpression of ALKBH5 in embryonic villus tissues of RSA patients resulted in significantly decreased global mRNA m6A methylation, and proliferation and invasion of trophoblasts could be controlled via regulation of the half-life of CY R61 mRNA. Huang et al [15] found that the low expression of METTL3 protein in RSA embryonic villous tissues resulted in reduced m6A modification and stability of ZBTB4 RNA, which in turn affected the invasive capacity of trophoblasts. Zhao et al [16] found that ALKBH5 overexpression in the ESC line caused the hyposecretion of VEGF and subsequently impair macrophage recruitment and M2 differentiation, which could be the potential cause of RSA.…”

“…Huang et al . [15] found that the low expression of METTL3 protein in RSA embryonic villous tissues resulted in reduced m6A modification and stability of ZBTB4 RNA, which in turn affected the invasive capacity of trophoblasts. Zhao et al .…”

DNA methylation regulator-mediated modification patterns and risk of intracranial aneurysm: a multi-omics and epigenome-wide association study integrating machine learning, Mendelian randomization, eQTL and mQTL data

“…It is associated with the cellular response to DNA damage stimuli and negative regulation of RNA polymerase II transcription. Recently, the role of METTL3-mediated RNA methylation of m6ZBTB4 in trophoblastic invasion and recurrent spontaneous abortion (RSA) has been highlighted [ 54 ]. In addition, METTL3-mediated modification of m6ZBTB4 mRNA is also involved in smoking-induced EMT in lung cancer [ 55 ], Recent findings demonstrate a role for ZBTB4 in epigenetic regulation, particularly in DNA methylation, and its dysfunction has been implicated in recurrent spontaneous abortion and smoking-induced epithelial-to-mesenchymal transition in lung cancer.…”

The landscape of implantation and placentation: deciphering the function of dynamic RNA methylation at the maternal-fetal interface

“…Based on stromal gel invasion assay and transwell migration assays with METTL3 over-expressing and knockdown in HTR-8 cell lines, the reduction of METTL3-mediated m6A modification attenuated the attenuation of ZBTB4 and increased the expression of ZBTB4, thereby impairing the invasive ability of trophoblast cells. m6A modification in RNA is crucial for controlling trophoblast invasion ( 167 ). Another m6A methyltransferase, WTAP, promotes trophoblast invasion and metastasis by regulating the stability of HMGN3 mRNA in a m6A-dependent way ( 168 ).…”