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Integration of single-cell transcriptomics and epigenetic analysis reveals enhancer-controlled TIMP1 as a regulator of ferroptosis in colorectal cancer

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Abstract

Background

Ferroptosis is an iron-dependent non-apoptotic programmed cell death. However, the regulatory mechanism of ferroptosis in colorectal cancer (CRC) is still unclear.

Objective

The aim of this study was to investigate the role and mechanism of enhancer-controlled genes in ferroptosis in CRC.

Methods

Dimensionality reduction and differentially expressed genes (DEGs) identification were conducted using Seurat algorithm based on single-cell RNA sequencing (scRNA-seq) data from the GSE200997 dataset. Ferroptosis-related pathway enrichment analysis was performed using the FerrDb V2 database. Enhancers were identified using HOMER algorithm based on H3K27ac ChIP-seq data from the GSE166254 dataset. Kaplan-Meier Plotter online tool was used to analyze prognosis and gene expression correlation. Transcription factors were predicted using the transcription factor affinity prediction web tool. The binding of enhancer to transcription factor and H3K27ac enrichment were detected by ChIP-qPCR. RSL3 was used to induce ferroptosis in CRC cells. Gene transcription was detected by qRT-PCR. Cell proliferation was detected by CCK8 assay.

Results

Nine cell clusters including T cells, natural killer cells, macrophages, mast cells, epithelial cells, fibroblasts, goblet cells, B cells and dendritic cells were identified in CRC and normal colonic tissue samples. Compared to normal colonic tissue-derived epithelial cells, 1075 DEGs were screened in CRC tissue-derived epithelial cells. Ferroptosis-related pathway enrichment suggested that DEGs were associated with the regulation of ferroptosis. DPEP1, ETV4, CEBPG, TIMP1, DUOX2 and LCN2 were identified as the significantly upregulated genes enriched in the “ferroptosis regulator” term, and their H3K27ac signals were significantly higher in CRC tissues than in normal colonic tissues. Of these, only the expression of TIMP1 predicted a poor prognosis of CRC patients. Transcription factor SPI1 drove TIMP1 transcription by binding to its enhancer. Overexpression of TIMP1 significantly promoted the resistance to ferroptosis induced by RSL3 in CRC cells, which was partially restored by SPI1 knockdown.

Conclusion

Transcription of TIMP1 was driven by transcription factor SPI1 in combination with its enhancer, consequently promoting CRC cells against ferroptosis. The SPI1/TIMP1 axis confers ferroptosis resistance in CRC, and thus has the potential to be the molecular targets for CRC treatment.

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Data Availability

The data generated or analyzed during this study can be obtained by contacting the corresponding author under reasonable request.

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Funding

This study was supported by the Science and Technology Extension Project of Hebei Provincial Health Commission Medical Science Research Fund Program (20220124).

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  1. Department of Gastrointestinal Surgery, Hebei Medical University Third Hospital, Shijiazhuang, Hebei, 050051, China

    Meng Li, Qian-yang Ni & Su-yang Yu

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  1. Meng Li
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Correspondence to Su-yang Yu.

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Li, M., Ni, Qy. & Yu, Sy. Integration of single-cell transcriptomics and epigenetic analysis reveals enhancer-controlled TIMP1 as a regulator of ferroptosis in colorectal cancer. Genes Genom 46, 121–133 (2024). https://doi.org/10.1007/s13258-023-01474-7

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