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The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of CYR61 mRNA.

ABSTRACT: N⁶-Methyladenosine (m⁶A) is the most prevalent internal modification in mammalian mRNAs. Although m⁶A is important in many biological processes, its roles in the placenta are unclear. Methods: Levels of global mRNA m⁶A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m⁶A RNA methylation quantification, and immunohistochemical methods. Using ALKBH5 overexpression and knockdown methods, we determined the role of ALKBH5 in trophoblast invasion at the maternal interface through trophoblasts and an extravillous explant culture experiments. Furthermore, the regulation of CYR61 by ALKBH5 was explored by RNA-sequencing coupled with methylated RNA immunoprecipitation. Results: We found that the level of global mRNA m⁶A methylation was significantly decreased in placental villous tissue from RM patients, while ALKBH5 expression was specifically unregulated. Furthermore, we demonstrated that ALKBH5 knockdown in human trophoblast promoted trophoblast invasion. Conversely, overexpression of ALKBH5 inhibited cell invasion. ALKBH5 knockdown promoted trophoblast invasion in villous explant culture experiments, while overexpression of ALKBH5 repressed these effects. Furthermore, we clarified that ALKBH5 inhibited trophoblast invasion by regulating CYR61 mRNA stability, and this RNA regulation is m⁶A dependent. Mechanistic analyses showed that decreased ALKBH5 in trophoblast increased the half-life of CYR61 mRNA and promoted steady-state CYR61 mRNA expression levels. Conclusions: We elucidated the functional roles of ALKBH5 and mRNA m⁶A methylation in trophoblast and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns in RM diseases.

SUBMITTER: Li XC

PROVIDER: S-EPMC6587351 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of CYR61 mRNA.

Li Xiao-Cui XC Jin Feng F Wang Bei-Ying BY Yin Xiang-Jie XJ Hong Wei W Tian Fu-Ju FJ

Theranostics 20190531 13

N6-Methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs. Although m6A is important in many biological processes, its roles in the placenta are unclear. Methods: Levels of global mRNA m6A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m6A RNA methylation quantification, and immunohistochemical methods. Using ...[more]

PMID: 31281518

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Project description:ContextDysregulated immune hemostasis occurs in unexplained recurrent spontaneous abortion (URSA). Synthesized by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), hydrogen sulfide (H2S) promotes regulatory T-cell differentiation and regulates immune hemostasis; yet, its role in URSA is elusive.ObjectiveTo determine if H2S plays a role in early pregnancy and if dysregulated H2S signaling results in recurrent spontaneous abortion.DesignFirst trimester placenta villi and decidua were collected from normal and URSA pregnancies. Protein expression was examined by immunohistochemistry and immunoblotting. Human trophoblast HTR8/SVneo and JEG3 cells were treated with H2S donors; HTR8/SVneo cells were transfected with CBS ribonucleic acid interference (RNAi) or complementary deoxyribonucleic acid. Cell migration and invasion were determined by transwell assays; trophoblast transcriptomes were determined by RNA sequencing (RNA-seq). Wild-type, CBS-deficient, and CBA/J × DBA/2 mice were treated with CBS and CSE inhibitors or H2S donors to determine the role of H2S in early pregnancy in vivo.ResultsCBS and CSE proteins showed cell-specific expressions, but only CBS decreased in the villous cytotrophoblast in URSA versus normal participants. H2S donors promoted migration and invasion and MMP-2 and VEGF expression in human placenta trophoblast cells that contain SV40 viral deoxyribonucleic acid sequences (HTR8/SVneo) and human placenta trophoblast cells (JEG3 cells), similar to forced CBS expression in HTR8/SVneo cells. The CBS-responsive transcriptomes in HTR8/SVneo cells contained differentially regulated genes (ie, interleukin-1 receptor and prostaglandin-endoperoxide synthase 2) that are associated with nuclear factor-κB-mediated inflammatory response. In vivo, dysregulated CBS/H2S signaling significantly increased embryonic resorption and decidual T-helper 1/T-helper 2 imbalance in mice, which was partially rescued by H2S donors.ConclusionCBS/H2S signaling maintains early pregnancy, possibly via regulating maternal-fetal interface immune hemostasis, offering opportunities for H2S-based immunotherapies for URSA.

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The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of CYR61 mRNA.

Publications

The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of <i>CYR61</i> mRNA.

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