ALKBH5 modulation of ferroptosis in recurrent miscarriage: implications in cytotrophoblast dysfunction

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Biochemistry, Biophysics and Molecular Biology

Main article text

 

Introduction

Materials & Methods

Patient characteristics

Tissue collection

Prussian blue staining

Cell culture

Transfection protocol

RNA isolation and qRT-PCR

Western blotting

Quantification of m6A in total RNA

Immunohistochemistry (IHC) assay

Immunofluorescent staining

Cell Counting Kit-8 (CCK8) assay

Glutathione disulfide/glutathione (GSSG/GSH) quantification

2′,7′-dichlorfluorescein-diacetate (DCFH-DA) staining

Malonaldehyde (MDA) assay

Analysis of published RNA-sequencing data

Statistical analysis

Results

Iron accumulation in villous trophoblasts of patients with RM

Increased ALKBH5 in cytotrophoblasts of patients with RM

RSL3 increased ALKBH5 during in vitro cell experiments

ALKBH5 attenuated the events leading to ferroptosis

FTL is a functional target of ALKBH5 in trophoblasts

Increased FTL in cytotrophoblasts of patients with RM

Discussion

Conclusions

Supplemental Information

Primers used for qPCR in the present study

DOI: 10.7717/peerj.18227/supp-1

Uncropped western blotting images

DOI: 10.7717/peerj.18227/supp-2

Efficiency of the knockout and overexpression of ALKBH5

(A) qRT-PCR results showing ALKBH5 mRNA levels in HTR8 cells transfected with pLV or pLV-ALKBH5. (B, C) Western blotting results showing ALKBH5 levels in HTR8 cells transfected with pLV or pLV-ALKBH5. (D) qRT-PCR results showing ALKBH5 mRNA levels in HTR8 cells transfected with siNC or siALKBH5. (E, F) Western blotting results showing ALKBH5 levels in HTR8 cells transfected with siNC or siALKBH5. (**p < 0.01).

DOI: 10.7717/peerj.18227/supp-3

Additional Information and Declarations

Competing Interests

The authors declare there are no competing interests.

Author Contributions

Chuanmei Qin performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Jiayi Wu performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Xiaowei Wei performed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Xueqing Liu performed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Yi Lin conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Human Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The study was approved by the Medical Ethics Committee of the International Peace Maternity & Child Health Hospital of China Welfare Institute, Shanghai [(GKLW) 2021-49].

Data Availability

The following information was supplied regarding data availability:

The data is available at figshare: Chuanmei, Qin; Wu, Jiayi; Wei, Xiaowei; Liu, Xueqing; Lin, Yi (2024). Elevated ALKBH5 is associated with ferroptosis-related dysfunctions in cytotrophoblasts through FTL in patients with recurrent miscarriage. figshare. Journal contribution. https://doi.org/10.6084/m9.figshare.24902073.v1.

Funding

This work was supported by the National Natural Science Foundation of China (No. 82471722, No. 82171669, No. 81971403), the Shanghai Jiao Tong University Trans-Med Awards Research (No. 20210201), and the Funds for Outstanding Newcomers, Shanghai Sixth People’s Hospital (No. X-3664). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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