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.

中文翻译：

---

单细胞转录组学和表观遗传学分析的整合揭示了增强子控制的 TIMP1 作为结直肠癌铁死亡的调节因子

背景

铁死亡是一种铁依赖性非凋亡性程序性细胞死亡。然而，结直肠癌（CRC）铁死亡的调控机制仍不清楚。

客观的

本研究的目的是探讨增强子控制基因在结直肠癌铁死亡中的作用和机制。

方法

使用基于 GSE200997 数据集中的单细胞 RNA 测序 (scRNA-seq) 数据的 Seurat 算法进行降维和差异表达基因 (DEG) 识别。使用 FerrDb V2 数据库进行铁死亡相关通路富集分析。使用基于 GSE166254 数据集中的 H3K27ac ChIP-seq 数据的 HOMER 算法识别增强子。使用Kaplan-Meier Plotter在线工具分析预后和基因表达相关性。使用转录因子亲和力预测网络工具预测转录因子。通过 ChIP-qPCR 检测增强子与转录因子的结合以及 H3K27ac 富集。 RSL3 用于诱导 CRC 细胞铁死亡。通过qRT-PCR检测基因转录。通过CCK8测定检测细胞增殖。

结果

在CRC和正常结肠组织样本中鉴定出9个细胞簇，包括T细胞、自然杀伤细胞、巨噬细胞、肥大细胞、上皮细胞、成纤维细胞、杯状细胞、B细胞和树突状细胞。与正常结肠组织来源的上皮细胞相比，在CRC组织来源的上皮细胞中筛选出1075个DEG。铁死亡相关通路富集表明 DEG 与铁死亡的调节有关。DPEP1、ETV4、CEBPG、TIMP1、DUOX2和LCN2被确定为“铁死亡调节因子”术语中富集的显着上调基因，其H3K27ac信号在CRC组织中显着高于正常结肠组织。其中，只有TIMP1的表达预测CRC患者的不良预后。转录因子 SPI1通过与其增强子结合来驱动TIMP1转录。 TIMP1的过度表达显着促进了CRC细胞对RSL3诱导的铁死亡的抵抗力，而SPI1敲低可部分恢复这种抵抗力。

结论

TIMP1的转录由转录因子 SPI1 与其增强子结合驱动，从而促进 CRC 细胞抵抗铁死亡。 SPI1/TIMP1 轴赋予 CRC 抗铁死亡的能力，因此有可能成为 CRC 治疗的分子靶点。