Identification of SLC7A11-AS1/SLC7A11 pair as a ferroptosis-related therapeutic target for hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC), a prevalent malignancy worldwide, poses significant challenges in terms of prognosis, necessitating innovative therapeutic approaches. Ferroptosis offers notable advantages over apoptosis, holding promise as a novel therapeutic approach for HCC complexities. Moreover, while the interaction between long non-coding RNAs (lncRNAs) and mRNAs is pivotal in various physiological and pathological processes, their involvement in ferroptosis remains relatively unexplored. In this study, we constructed a ferroptosis-related lncRNA-mRNA correlation network in HCC using Pearson correlation analysis. Notably, the SLC7A11-AS1/SLC7A11 pair, exhibiting high correlation, was identified. Bioinformatics analysis revealed a significant correlation between the expression levels of this pair and key clinical characteristics of HCC patients, including gender, pathology, Ishak scores and tumour size. And poor prognosis was associated with high expression of this pair. Functional experiments demonstrated that SLC7A11-AS1, by binding to the 3'UTR region of SLC7A11 mRNA, enhanced its stability, thereby promoting HCC cell growth and resistance to erastin- induced ferroptosis. Additionally, in vivo studies confirmed that SLC7A11-AS1 knockdown potentiated the inhibitory effects of erastin on tumour growth. Overall, our findings suggest that targeting the SLC7A11-AS1/SLC7A11 pair holds promise as a potential therapeutic strategy for HCC patients.

Keywords: SLC7A11; SLC7A11‐AS1; ferroptosis; hepatocellular carcinoma; lncRNA‐mRNA correlation network.

FIGURE 1

Identification of ferroptosis‐related differentially expressed mRNAs and lncRNAs in HCC patients. (A) The Venn diagram illustrates the common DEmRNA between three datasets and ferroptosis‐related genes. (B–D)The heatmap depicts the correlation between lncRNAs and ferroptosis‐related mRNAs in HCCDB25 (B), HCCDB30_HA (C), and HCCDB30_HN (D).

FIGURE 2

Construction of ferroptosis‐related lncRNA‐mRNA correlation network based on person correlation. (A) Constructed lncRNA‐mRNA Correlation Network based on all ferroptosis‐related mRNA and lncRNA from three datasets. The lncRNAs were also organized into three concentric layers based on their node degree, ranging from 1 to 5, 6–10 and 11–35 from the outermost to the innermost layer. (B) Constructed lncRNA‐mRNA Correlation Network based on common ferroptosis‐related mRNA and lncRNA across three datasets.

FIGURE 3

Clinical relevance of SLC7A11‐AS1/SLC7A11 correlation network. (A, B) Heatmap of correlation for the risk model in HCCDB25 (A) and HCCDB30 (B). (C–F) The boxplot demonstrates the relationship between certain clinical features and the expression levels of the SLC7A11‐AS1/SLC7A11 pair in HCCDB25, including gender (C), pathology (D), Ishak score (E), and maximum size (F). (G, H) Kaplan–Meier curve analysis was conducted to examine the overall survival rates of patients with high and low expression of the SLC7A11‐AS1/SLC7A11 pair in HCCDB25 (G) or TCGA‐LIHC (H). SAS, SLC7A11‐AS1/SLC7A11 correlation network.

FIGURE 4

Functional enrichment analysis of the SLC7A11‐AS1/SLC7A11 correlation network. (A, B) Bar graph showing enriched G.O. terms of HCCDB25 (A) and HCCDB30 (B). (C, D) Bar plot showing the enriched KEGG pathways of HCCDB25(C) and HCCDB30 (D). (E, F) Enriched G.O. terms (E) and enriched KEGG pathways (F) from RNA‐Seq results of SLC7A11‐AS1 knockdown.

FIGURE 5

SLC7A11‐AS1 promotes cell proliferation and suppresses ferroptosis in HepG2. (A, B) After knocking down SLC7A11‐AS1 in HepG2 cells, cell proliferation was assessed using the CCK8 assay (A), and clonogenic formation ability was evaluated (B). (C, D) After overexpression SLC7A11‐AS1 in HepG2 cells, cell proliferation was assessed using the CCK8 assay (C), and clonogenic formation ability was evaluated (D). (E) CCK8 assay to determine cell viability after treatment with gradient concentrations of erastin for 24 h in SLC7A11‐AS1 knockdown cells. (F, G) HepG2 cells knocking‐down SLC7A11‐AS1 were treated with erastin for 24 h, GSH levels were measured using a glutathione assay kit (F), lipid peroxidation was assessed by measuring MDA levels (G). (H) CCK8 assay to determine cell viability after treatment with gradient concentrations of erastin for 24 h in SLC7A11‐AS1 overexpressing cells. (I, J) HepG2 cells overexpression SLC7A11‐AS1 were treated with erastin for 24 h, GSH levels were measured using a glutathione assay kit (I), lipid peroxidation was assessed by measuring MDA levels(J). (K, L) the levels of L‐ROS in HepG2 cells knocking‐down (K) or overexpression (L) SLC7A11‐AS1 were detected using flow cytometry with C11‐BODIPY staining. Data are presented as mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001, ns: not significant.

FIGURE 6

SLC7A11‐AS1 binds to SLC7A11 mRNA, enhancing its stability. (A, B) The SLC7A11‐AS1 expression levels in the nuclear and cytosolic fractions derived from HepG2 (A) and HuH7 (B) cells by qRT‐PCR analysis. (C, D) FISH analysis was performed to evaluate the colocalization of endogenous SLC7A11‐AS1 and SLC7A11 RNA in HepG2(C) and HuH7 cells (D). (E) Cell proliferation of each group was measured using CCK8 assay. (F) CCK8 assay was used to measure cell viability of each group after treatment with varying concentrations of erastin for 24 h. (G) Glutathione assay kit measured GSH levels in each group after 24 h of erastin treatment. (H, I) The stability of SLC7A11 mRNA over time was detected by qRT‐PCR relative to time 0 after blocking new RNA synthesis with Actinomycin D (20 μg/mL) in HepG2 cells after knockdown of SLC7A11‐AS1 (H) or overexpression of SLC7A11‐AS1 (I). RNA levels were normalized to 18S rRNA. (J) Structural diagram of SLC7A11‐AS1 and SLC7A11 RNA, as well as a schematic representation of the full length, overlapping region, and non‐overlapping region of SLC7A11‐AS1. The red region indicates the overlapping region between SLC7A11 and SLC7A11‐AS1. 1: Overlapping region 1, 2: Overlapping region 2, 3: non‐overlapping region. (K) RNase protection assay was performed on RNA samples from HepG2 cells, and PCR amplification was used to detect the O.L. region‐1, O.L. region‐2, and the non‐O.L. regions of SLC7A11. (L, M) The RNA–RNA interaction between the F.L. (L) and non‐O.L. region (M) of SLC7A11‐AS1 transcript and SLC7A11 mRNA was detected by RNA pulldown assay. Data are presented as mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001, ns, not significant.

FIGURE 7

SLC7A11‐AS1 promotes tumour growth of cell xenografts in nude mice. (A–C) HepG2 cells from the SLC7A11‐AS1 group or Vector group were injected into nude mice(A), the tumour volumes were measured every 3 days after the cells were injected for 7 days(B), the tumour weight was recorded(C) (n = 5). (D) 5 HepG2 cells from the shSLC7A11‐AS1 group or shNC group were injected into nude mice and treated with erastin after 21 days or not. (E, F) Measure and count the weight of the tumour from nude mice not treated with erastin (E)or treated with erastin (F) (n = 5). (G, H) The tumour volumes were measured every 3 days after the cells were injected for 7 days (n = 5). (I) IHC detected SLC7A11 expression. (J, K) Western blot analysis of SLC7A11 protein levels in tumour tissues and tumour tissues after erastin treatment. Data are presented as mean ± S.D. of three independent experiments. *p < 0.05, **p < 0.01, ***p<0.001, ****p<0.0001, ns: not significant.