Targeting HCG18 counteracts ferroptosis resistance via blocking the miR-30a-5p/RRM2/GSS pathway in hepatocellular carcinoma

Abstract

Background: Finding effective strategies and novel targets for reversing drug resistance is one of the major frontiers in hepatocellular carcinoma (HCC) research. Ferroptosis is participate in the malignant progression and drug resistance of HCC. However, the underlying molecular mechanisms remail largely uninvestigated. Methods: HCC cell lines and xenografted nude mice were used as experimental models. Biological functions were investigated by various molecular biology experiments. An HCC population was used to reveal clinical significance. Results: In our study, HCG18 and RRM2 was found to be associated with unfavorable prognosis. HCG18 regulates RRM2 expression through competitively binding to miR-30a-5p, consequently impacting ferroptosis. RRM2 directly regulated GSS to increase GSH synthesis. The colony formation assay demonstrated that overexpression of HCG18 inhibited erastin-induced cell death. In addition, in vivo experiments have also confirmed that HCG18 can inhibit ferroptosis by regulating the expression of RRM2, thereby promoting HCC proliferation. Conclusion: Our study discovered a novel lncRNA HCG18, as a "switch-like" molecule of the axis of miR-30a-5p/RRM2/GSS, confers resistance to ferroptosis and holds promise as a potential target for ferroptosis-dependent therapy.

Keywords: GSS; HCG18; RRM2; ferroptosis therapy; hepatocellular carcinoma.

Figure 1

Identification of HCG18-miR-30a-5p-RRM2 axis in HCC. a (Left) Venn map showed the overlapping mRNAs in significantly differentially expressed mRNAs from GSE69164, GSE77509, TCGA-LIHC and ferroptosis-related mRNAs from FerrDb. (Right) Intersection gene list and gene expression. b GO and c KEGG enrichment analysis of ferroptosis-related differentially expressed genes in HCC. d Venn map showed miR-30a-5p was the only overlapping miRNA between significantly differentially expressed miRNAs in GSE76903 and predicted miRNAs in miTarBase. e Venn map showed the overlapping lncRNAs interacting with miR-30a-5p were predicted by LncBase and starBase. f Construction of ceRNA regulatory network based on 5 lncRNAs, a miR-30a-5p and a mRNA. g Kaplan-Meier survival analysis of HCG18, SNHG16, RRM2 and miR-30a-5p in HCC. h Pearson correlation analysis of RRM2-HCG18 and RRM2-SNHG16. i The potential regulatory relationship between HCG18, miR-30a-5p and RRM2.

Figure 2

Clinical significance of HCG18 and RRM2. a TCGA database was used to analyze the expression levels of HCG18 and RRM2 in HCC tissues and normal liver tissues. b TCGA database was used to analyze the correlation between the expression of HCG18 and RRM2 and tumor stage. c-d The relative expression of HCG18 and RRM2 in HCC tissues and adjacent normal tissues of 40 cases. e-j The relative expression of HCG18 and RRM2 in different tumor stages, age and gender of 150 cases. k-l Kaplan-Meier survival analysis of HCG18 and RRM2 in 150 HCC cases. The scale bar is 100µm and the magnification scale bar is 20µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Figure 3

Knockdown of HCG18 promotes ferroptosis in HCC cells. a-b CCK-8 assay was used to measure the cell viability, c-d flow cytometry was used to measure lipid ROS level, e-f iron detection assay was used to detected intracellular Fe²⁺, and g-h GSH detection assay was used to measure intracellular GSH concentration in control and siHCG18 HCC cells treated with erastin or RSL3 for 24 h. Control group represents non-treated cells. The scale bar is 100µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Figure 4

Overexpression of HCG18 inhibits ferroptosis in HCC cells. a-b CCK-8 assay was used to measure the cell viability, c-d flow cytometry was used to measure lipid ROS level, e-f iron detection assay was used to detected intracellular Fe²⁺, and g-h GSH detection assay was used to measure intracellular GSH concentration in vector and HCG18 HCC cells treated with erastin or RSL3 for 24 h. Control group represents non-treated cells. The scale bar is 100µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Figure 5

HCG18 regulates ferroptosis by impacting RRM2. a-b CCK-8 assay was used to measure the cell viability, c-d flow cytometry was used to measure lipid ROS level, e-f iron detection assay was used to detected intracellular Fe²⁺, and g-h GSH detection assay was used to measure intracellular GSH concentration in HCC cells transfected with indicated constructs and treated with erastin or RSL3 for 24 h. Control group represents non-transfected cells. The scale bar is 100µm. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6

miR-30a-5p is involved in the regulation of RRM2 by HCG18. a StarBase database was used to predict the potential binding site for miR-30a-5p within HCG18 and RRM2. b Construction of wild type and mutant sequences of HCG18 and RRM2 3' UTR. c The relative expression levels of miR-30a-5p in siNC and siHCG18 HCC cells. d The relative expression levels of miR-30a-5p in vector, HCG18 and HCG18-MT HCC cells. e Luciferase reporter assay was used to valid the regulatory relationship between HCG18 and miR-30a-5p in HCC cells. f Luciferase reporter assay was used to valid the regulatory relationship between RRM2 and miR-30a-5p in HCC cells. g-j qRT-PCR and western blotting analysis were used to detected the relative expression level of RRM2 mRNA and protein in HCC cells transfected with indicated constructs. GAPDH was used as a loading control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; ns, not significant.

Figure 7

miR-30a-5p promotes ferroptosis by regulating RRM2. Huh7 cells transfected with indicated constructs were treated with erastin or RSL3 for 24 h, a, e CCK-8 assay was used to measure the cell viability, b, f flow cytometry was used to measure lipid ROS level, c, g iron detection assay was used to detected intracellular Fe²⁺, and d, h GSH detection assay was used to measure intracellular GSH concentration. Control group represents non-transfected cells. The scale bar is 100µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 8

RRM2 directly regulated GSS to increase GSH synthesis. HepG2 cells transfected with vector or RRM2 were treated with erastin or RSL3 for 24 h, a CCK-8 assay was used to measure the cell viability, b flow cytometry was used to measure lipid ROS level, c iron detection assay was used to detected intracellular Fe²⁺, and d GSH detection assay was used to measure intracellular GSH concentration. Control group represents non-transfected cells. The scale bar is 100µm. e TCGA database was used to analyze the correlation between GSS and RRM2 in HCC. f Western Blotting analysis was used to detected the relative expression level of GSS protein in siNC and siRRM2 HCC cells. GAPDH was used as a loading control. g-h CO-IP and IF analysis were used to valid the interaction between RRM2 and GSS in HCC cells. The scale bar is 10µm. i GSH detection assay was used to measure intracellular GSH concentration in siRRM2 and siRRM2+GSS Huh7 cells treated with erastin or RSL3 for 24 h. The scale bar is 100µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 9

Roles of HCG18 in erastin-induced ferroptosis in vivo. a The change of subcutaneous tumor volume in nude mice after injecting Huh7 cells stably transfected with vector or LV-HCG18 over time. b Comparison of subcutaneous tumor weight in nude mice after injecting Huh7 cells stably transfected with vector or LV-HCG18. c-f The expression of HCG18, miR-30a-5p, RRM2 and Ki-67 in subcutaneous tumor tissues with HCG18 overexpression. The scale bar is 100µm and the magnification scale bar is 20µm. g Iron detection assay was used to detected Fe²⁺, and h Lipid Peroxidation MDA Assay was used to detected MDA content of subcutaneous tumor tissues in nude mice after injecting Huh7 cells stably transfected with vector or LV-HCG18. i The change of subcutaneous tumor volume in nude mice treated with erastin in the presence or absence of siHCG18, siRRM2, or miR-30a-5p agomir over time. j Comparison of subcutaneous tumor weight in nude mice treated with erastin in the presence or absence of siHCG18, siRRM2, or miR-30a-5p agomir. k-l The content of Fe²⁺ and MDA in subcutaneous tumor treated with erastin in the presence or absence of siHCG18, siRRM2, or miR-30a-5p agomir. m Schematic diagram of the regulatory mechanism of HCG18. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.