circRNA hsa_circ_0001819抑制铁死亡并促进结直肠癌细胞恶性表型的作用及潜在机制
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1中南大学湘雅三医院 胃肠外科,湖南 长沙 410013;2中南大学湘雅三医院 基础医学博士后流动站,湖南 长沙 410013;3中南大学湘雅三医院 血液内科,湖南 长沙 410013

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龙飞,中南大学湘雅三医院副研究员,主要从事肿瘤细胞死亡调控机制方面的研究(

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国家自然科学基金资助项目 82303255国家自然科学基金资助项目(82303255)。


Role and potential mechanism of circRNA hsa_circ_0001819 in suppressing ferroptosis and promoting malignant phenotypes in colorectal cancer cells
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1Department of Gastrointestinal Surgery, the Third Xiangya Hospital, Central South University, Changsha 410013, China;2Postdoctoral Research Station of Basic Medicine, the Third Xiangya Hospital, Central South University, Changsha 410013, China;3Department of Hematology, the Third Xiangya Hospital, Central South University, Changsha 410013, China

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    摘要:

    背景与目的 铁死亡是一种以铁依赖性脂质过氧化为特征的调节性细胞死亡方式,在结直肠癌(CRC)治疗中具有潜在应用价值。环状RNA(circRNA)可通过竞争性内源性RNA(ceRNA)机制参与肿瘤发生发展,但其在CRC铁死亡中的作用及调控机制尚不明确。本研究旨在筛选CRC中异常表达的circRNA,探讨hsa_circ_0001819(circ1819)对CRC细胞铁死亡及恶性生物学行为的影响,并初步分析其潜在分子机制。方法 整合分析GSE223001和GSE205643 circRNA微阵列数据集,并利用GSE126094进行验证,筛选CRC中差异表达的circRNA。采用qRT-PCR检测circ1819在CRC组织及细胞中的表达,并通过RNase R消化、放线菌素D处理及核质分离实验验证其环状结构、稳定性和亚细胞定位。采用siRNA敲低circ1819,结合铁死亡诱导剂(RSL3)及铁死亡抑制剂(Lip-1),通过细胞活力、脂质活性氧(ROS)、丙二醛(MDA)及铁死亡相关蛋白检测评价其对CRC细胞铁死亡的影响;采用CCK-8、平板克隆形成及Transwell实验评价其对CRC细胞增殖和迁移的影响。通过AGO2-RNA结合蛋白免疫沉淀(AGO2-RIP)、RNA pulldown实验及生物信息学分析探讨circ1819潜在的ceRNA调控关系。结果 circ1819在CRC组织和细胞中显著高表达,其表达水平与肿瘤直径及T分期相关。circ1819具有典型的circRNA特征,主要定位于细胞质。敲低circ1819可显著增强CRC细胞对RSL3诱导的铁死亡敏感性,表现为细胞活力降低、脂质ROS和MDA水平升高,该效应可被Lip-1部分逆转。circ1819敲低后GPX4的mRNA及蛋白表达显著降低,而SLC7A11和ACSL4表达无明显变化。同时,敲低circ1819可显著抑制CRC细胞增殖、克隆形成及迁移能力。AGO2-RIP和RNA pulldown实验提示circ1819可与miR-618和miR-1231发生相互作用;进一步结合靶基因预测及富集分析,构建了以circ1819为核心的circRNA-miRNA-mRNA潜在调控网络。结论 circ1819在CRC中异常高表达,敲低circ1819可增强CRC细胞铁死亡敏感性并抑制其增殖和迁移,其作用可能与miR-618和miR-1231介导的ceRNA调控及GPX4表达变化有关。circ1819可能是连接CRC恶性进展与铁死亡抵抗的潜在调控分子,但其具体下游靶基因及因果调控关系仍需进一步验证。

    Abstract:

    Background and Aims Ferroptosis is an iron-dependent form of regulated cell death characterized by lipid peroxidation and has potential applications in colorectal cancer (CRC) therapy. Circular RNAs (circRNAs) can regulate tumor progression through competing endogenous RNA (ceRNA) mechanisms; however, their roles and regulatory mechanisms in CRC ferroptosis remain incompletely understood. This study aimed to identify dysregulated circRNAs in CRC, investigate the effects of hsa_circ_0001819 (circ1819) on ferroptosis and malignant phenotypes of CRC cells, and preliminarily explore its potential molecular mechanism.Methods CircRNA microarray datasets GSE223001 and GSE205643 were integrated to identify differentially expressed circRNAs, with GSE126094 used for validation. The expression of circ1819 in CRC tissues and cell lines was examined by qRT-PCR. RNase R digestion, actinomycin D treatment, and nuclear-cytoplasmic fractionation assays were performed to characterize its circular structure, stability, and subcellular localization. Following siRNA-mediated circ1819 knockdown, cell viability, lipid reactive oxygen species (ROS), malondialdehyde (MDA), and ferroptosis-related proteins were assessed in the presence of the ferroptosis inducer RSL3 and inhibitor Liproxstatin-1 (Lip-1). CCK-8, colony formation, and Transwell assays were used to evaluate cell proliferation and migration. AGO2 RNA immunoprecipitation (AGO2-RIP), RNA pulldown, and bioinformatics analyses were performed to investigate the potential ceRNA regulatory mechanism of circ1819.Results Circ1819 was significantly upregulated in CRC tissues and cell lines, and its expression was associated with tumor diameter and T stage. Circ1819 exhibited typical characteristics of a circular RNA and was predominantly localized in the cytoplasm. Circ1819 knockdown significantly increased the sensitivity of CRC cells to RSL3-induced ferroptosis, as evidenced by reduced cell viability and increased lipid ROS and MDA levels; these effects were partially reversed by Lip-1. Circ1819 knockdown significantly reduced GPX4 mRNA and protein expression, whereas SLC7A11 and ACSL4 expression remained unchanged. Moreover, circ1819 knockdown significantly inhibited CRC cell proliferation, colony formation, and migration. AGO2-RIP and RNA pulldown assays suggested interactions between circ1819 and miR-618 or miR-1231. Combined with target prediction and enrichment analyses, a potential circ1819-centered circRNA-miRNA-mRNA regulatory network was constructed.Conclusion Circ1819 is aberrantly upregulated in CRC, and its knockdown enhances ferroptosis sensitivity while suppressing proliferation and migration of CRC cells. These effects may be associated with ceRNA regulation involving miR-618 and miR-1231 and alterations in GPX4 expression. Circ1819 may represent a potential regulatory link between malignant progression and ferroptosis resistance in CRC, although its downstream targets and causal regulatory relationships require further validation.

    图1 circ1819在CRC中的表达特征 A:火山图显示circRNA微阵列数据集GSE223001和GSE205643整合分析后获得的差异表达circRNA,红点所示为上调的6个circRNA;B:circ1819在GSE126094验证数据集中的表达;C:qRT-PCR检测circ1819在24对CRC组织和癌旁组织中的表达差异;D:qRT-PCR检测circ1819在肿瘤直径<4 cm患者和肿瘤直径≥4 cm患者中的表达差异;E:qRT-PCR检测circ1819在肿瘤浸润T1~T2期患者和肿瘤浸润T3~T4期患者中的表达差异;F:qRT-PCR检测circ1819在人正常结肠上皮细胞系(FHC)和4株人CRC细胞系中的表达差异Fig.1 Expression characteristics of circ1819 in CRC A: Volcano plot showing differentially expressed circRNAs identified by integrated analysis of the GSE223001 and GSE205643 circRNA microarray datasets, with red dots indicating the six upregulated circRNAs; B: Expression of circ1819 in the GSE126094 validation dataset; C: qRT-PCR analysis of circ1819 expression in 24 pairs of CRC and matched adjacent normal tissues; D: qRT-PCR analysis of circ1819 expression in patients with tumor diameter <4 cm and ≥4 cm; E: qRT-PCR analysis of circ1819 expression in patients with T1-T2 and T3-T4 tumors; F: qRT-PCR analysis of circ1819 expression in one human normal colonic epithelial cell line (FHC) and four CRC cell lines
    图2 circ1819的成环验证、稳定性检测和亚细胞定位 A:使用circPrimer 2.0设计的circ1819 DP示意图;B:使用circPrimer 2.0设计的circ1819 CP示意图;C:使用DP和CP分别对CRC细胞(HCT116和SW480)的cDNA和gDNA进行qRT-PCR检测,观察circ1819的扩增情况;D:RNase R处理(10 U/μg,30 min)后,qRT-PCR检测CRC细胞中circ1819和线性UBR5(UBR5 mRNA)的表达;E:放线菌素D(5 μg/mL)处理不同时间点后,qRT-PCR检测CRC细胞中circ1819和线性UBR5(UBR5 mRNA)的表达;F:细胞质与细胞核RNA分离实验显示circ1819在HCT116和SW480细胞中的亚细胞定位,GAPDH和U6分别作为细胞质和细胞核的阳性对照Fig.2 Validation of the circular structure, stability, and subcellular localization of circ1819 A:Schematic diagram of DP designed for circ1819 using circPrimer 2.0; B: Schematic diagram of CP designed for circ1819 using circPrimer 2.0; C: qRT-PCR amplification of circ1819 in HCT116 and SW480 cells using DP and CP with cDNA and gDNA as templates; D: qRT-PCR analysis of circ1819 and linear UBR5 mRNA expression after RNase R treatment (10 U/μg, 30 min); E: qRT-PCR analysis of circ1819 and linear UBR5 mRNA expression at the indicated time points after actinomycin D treatment (5 μg/mL); F: Subcellular localization of circ1819 in HCT116 and SW480 cells determined by nuclear-cytoplasmic RNA fractionation, using GAPDH and U6 as cytoplasmic and nuclear positive controls, respectively
    图3 敲低circ1819对CRC细胞铁死亡的影响 A:qRT-PCR检测si-circ1819在HCT116和SW480细胞中的敲低效率;B:细胞活力检测实验观察敲低circ1819对RSL3诱导的CRC细胞(HCT116和SW480)铁死亡的影响;C:敲低circ1819后HCT116和SW480细胞内脂质ROS水平检测;D:敲低circ1819后HCT116和SW480细胞内MDA水平检测;E:qRT-PCR检测circ1819敲低后GPX4、SLC7A11、ACSL4的mRNA水平变化;F:Western blot检测circ1819敲低后上述基因的蛋白水平变化Fig.3 Effect of circ1819 knockdown on ferroptosis in colorectal cancer cells A: qRT-PCR analysis of circ1819 knockdown efficiency in HCT116 and SW480 cells; B: Cell viability assays evaluating the effect of circ1819 knockdown on RSL3-induced ferroptosis in HCT116 and SW480 cells; C: Detection of lipid ROS levels in HCT116 and SW480 cells following circ1819 knockdown; D: Detection of MDA levels in HCT116 and SW480 cells following circ1819 knockdown; E: qRT-PCR analysis of GPX4, SLC7A11, and ACSL4 mRNA expression following circ1819 knockdown; F: Western blot analysis of GPX4, SLC7A11, and ACSL4 protein expression following circ1819 knockdown
    图4 敲低circ1819对CRC细胞增殖和迁移的影响 A:CCK-8实验观察敲低circ1819对HCT116和SW480细胞增殖能力的影响;B:平板克隆形成实验观察敲低circ1819对HCT116和SW480细胞克隆形成能力的影响;C:Transwell实验观察敲低circ1819对HCT116和SW480细胞迁移能力的影响Fig.4 Effect of circ1819 knockdown on proliferation and migration of colorectal cancer cells A: CCK-8 assay evaluating the effect of circ1819 knockdown on the proliferation of HCT116 and SW480 cells; B: Colony formation assays evaluating the effect of circ1819 knockdown on the colony-forming ability of HCT116 and SW480 cells; C: Transwell assay evaluating the effect of circ1819 knockdown on the migration of HCT116 and SW480 cells
    图5 以circ1819为核心的circRNA-miRNA-mRNA调控网络的构建 A:circInteractome在线网站预测circ1819与miRNA结合蛋白AGO2的相互作用;B:qRT-PCR检测CRC细胞裂解液经AGO2-RIP实验后产物中circ1819的富集情况;C:维恩图显示两个在线网站(circInteractome和circBank)预测与circ1819相互作用的候选miRNA的交集;D:qRT-PCR显示在CRC细胞中进行circ1819 pulldown实验后RNA的富集情况;E:基于R语言igraph和ggraph包构建的以circ1819为核心的circRNA-miRNA-mRNA调控网络;F:GO富集分析的气泡图显示circ1819的靶基因可能参与的生物学过程;G:KEGG富集分析的气泡图显示circ1819的靶基因可能参与的信号通路Fig.5 Construction of a potential circ1819-centered circRNA-miRNA-mRNA regulatory network A: Prediction of the interaction between circ1819 and AGO2 using the circInteractome online database; B: qRT-PCR analysis of circ1819 enrichment in AGO2-RIP products from colorectal cancer cell lysates; C: Venn diagram showing the overlap of candidate miRNAs predicted to interact with circ1819 by circInteractome and circBank; D: qRT-PCR analysis of RNA enrichment following circ1819 pulldown in colorectal cancer cells; E: circ1819-centered circRNA-miRNA-mRNA regulatory network constructed using the igraph and ggraph packages in R; F: Bubble plot showing the biological processes potentially associated with circ1819 target genes based on GO enrichment analysis; G: Bubble plot showing the signaling pathways potentially associated with circ1819 target genes based on KEGG enrichment analysis
    表 1 引物序列Table 1 Primer sequences
    表 2 在CRC组织中上调circRNA的注释信息Table 2 Annotation of upregulated circRNAs in colorectal cancer tissues
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龙飞,梁妮,郑勋日,叶永鑫,陈淼. circRNA hsa_circ_0001819抑制铁死亡并促进结直肠癌细胞恶性表型的作用及潜在机制[J].中国普通外科杂志,2026,35(8):1607-1619.
DOI:10.7659/j. issn.1005-6947.260287

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  • 收稿日期:2026-05-22
  • 最后修改日期:2026-08-10
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  • 在线发布日期: 2026-09-29
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