Circular RNA circPRKCA represses ferroptosis of hepatocellular carcinoma by promoting SLC7A11

1. Introduction

Globally, hepatocellular carcinoma (HCC) remains a major contributor to cancer mortality, accounting for nearly 90% of primary hepatic carcinoma and consistently ranking among the top four most lethal malignancies worldwide (Forner et al., 2018; Ganesan and Kulik 2023; Bray et al., 2024). Despite the advancements in therapeutic approaches and a decrease in incidence rates, the prognosis for HCC patients continues to be unfavorable, with high rates of recurrence still prevalent (Vyas and Zhang 2020; Llovet et al., 2021). Therefore, elucidating the underlying mechanisms of HCC pathogenesis has become imperative in biomedical research. Recently, ferroptosis has emerged as a new form of iron-dependent administered apoptosis mediated by iron cumulation and lipid oxidation (Mou et al., 2019; Chang et al., 2021). Ferroptosis, a sort of apoptosis, that is distinguished by oxidative stress, which is caused by reactive oxygen species (ROS) and lipid peroxidation (Mou et al., 2019; Jiang et al., 2021; Liang et al., 2022). Research consistently demonstrates that the mode of cell death plays an essential role in preserving normal cellular homeostasis, whereas cancer cells display a significantly enhanced susceptibility to this process (Hassannia et al., 2019; Chang et al., 2021; Lei et al., 2022). Collective accumulating evidence underpins the identification of ferroptosis as a critical therapeutic target within oncology (Liang et al., 2019). From a mechanistic perspective, ferroptosis represents a complex biological process modulated by multifarious factors. Notably, the system Xc⁻—consisted of SLC7A11—which has garnered significant attention in cancer research (Tang et al., 2021). System Xc‐ functions as a cystine‐glutamate antiporter, promotes cysteine uptake for the synthesis of cysteine and the key antioxidant glutathione (GSH) (Li et al., 2020a; Tang et al., 2021). SLC7A11, acting as the core component of system Xc‐, has been characterized as a promising therapeutic target for ferroptosis induction in cancer treatment (Yan et al., 2023). Furthermore, glutathione peroxidase 4 (GPX4), which plays a part in reducing phospholipid hydroperoxides by utilizing GSH, serves as another critical regulator of ferroptosis in malignancies (Koppula et al., 2021).

CircRNAs are primarily formed through the back-splicing of pre-mRNAs transcripts, and resulting in a circular, end-closed structure (Chen et al., 2021). This unique structure provides circRNAs with enhanced stability in physiological environments compared to linear RNAs, making them highly conserved and abundant (Jiang et al., 2021). Functionally, circRNAs have been widely observed to interact with microRNAs to regulate target mRNA expression or directly bind to specific proteins to influence their function (Huang et al., 2020). Numerous studies have shown a strong correlation between circRNAs and various diseases, particularly cancer (Zhang et al., 2020; Li et al., 2020b; Li et al., 2022). However, the effects of circPRKCA on HCC are not fully understood. In this study, we found a significant increase in the expression of circPRKCA in HCC tumors compared to non-cancerous samples. We also discovered that circPRKCA acts as a sponge for miR-384, which in turn modulates SLC7A11 expression, ultimately affecting ferroptosis in HCC.

2. Materials and Methods

3. Results

3.1 circPRKCA is significantly upregulated in HCC

As illustrated in Fig. 1, compared to neighboring healthy tissues, circPRKCA was markedly overexpressed in HCC tumor samples. Consistent with these findings, quantitative real-time PCR (qRT-PCR) demonstrated markedly higher circPRKCA expression levels in multiple HCC cell lines (HLE, MHCC97H, SMMC7721, HCCLM3, BEL7402, Hep3B, HCCLM6, and Huh7) relative to the normal hepatic cell line LO2 (Fig. 1). Among these, HCCLM3 and Huh7 cells exhibited the highest circPRKCA expression and were consequently selected for subsequent functional investigations. Furthermore, FISH analysis revealed predominant cytoplasmic localization of circPRKCA in HCC cells.

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Fig. 1.

circPRKCA expression is upregulated in HCC. (a) Comparative qRT-PCR analysis of 22 paired HCC/non-tumor specimens revealed marked circPRKCA overexpression in malignant tissues. (b) The concentration of circPRKCA in HCC cells and normal hepatic cells was measured by qRT-PCR. (c) The subcellular compartmentalization of circPRKCA in HCC cells was measured by FISH assay. Red: Cy3-labeled circPRKCA, U6 or 18S; Blue: nuclei (scale bar, 50 μm). **p<0.01, ***p<0.001, ns, not significant.

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3.2 Knockdown of circPRKCA induces ferroptosis and suppresses proliferation in HCC cells

To explore the functional significance of circPRKCA in HCC progression, we performed RNA interference-mediated silencing in HCC cells and systematically analyzed subsequent phenotypic alterations. Transfection with two independent siRNAs targeting circPRKCA (si-circPRKCA-1 and si-circPRKCA-2) effectively reduced circPRKCA expression levels in HCC cells, as confirmed by qRT-PCR analysis (Fig. 2a). Functional characterization revealed that circPRKCA knockdown significantly impaired malignant cell behaviors, including reduced proliferative capacity (Fig. 2b) and attenuated migratory potential as demonstrated by transwell assays (Fig. 2c). Notably, circPRKCA depletion induced substantial metabolic alterations characterized by intracellular accumulation of total iron, ferrous iron (Fe²⁺), and lipid reactive oxygen species (ROS) (Fig. 2d), biochemical features consistent with ferroptotic cell death.

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Fig. 2.

circPRKCA knockdown induces ferroptosis and suppresses proliferation in HCC cells. (a) The knockdown effect of circPRKCA in HCC cells was determined by qRT-PCR. (b) Viability of HCCLM3 and Huh7 cells, as detected by CCK-8 assays. (c) Invasive capacity of HCCLM3 and Huh7 cells, as evaluated by performing Transwell assays (scale bar, 50 μm). (d) The concentration of total iron, Fe²⁺, and lipid ROS in HCC cells. **p<0.01, ***p<0.001.

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3.3 Knockdown of circPRKCA suppresses growth of HCC tumor

Subsequently, we further investigated the in vivo effects of circPRKCA on HCC using a xenograft tumor model. Results indicated that knockdown of circPRKCA greatly restrained tumor growth and reduced both tumor volume and weight (Fig. 3a). The concentration of total iron, Fe²⁺, and lipid ROS in xenograft tumors was also suppressed by circPRKCA depletion (Fig. 3b), while western blot results showed analysis demonstrated downregulation of SLC7A11 and GPX4 protein expression (Fig. 3c).

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Fig. 3.

Knockdown of circPRKCA inhibits HCC tumor growth in a xenograft model. (a) Tumor growth curve, size, and weight were estimated (n=5). (b) The concentration of total iron, Fe²⁺, and lipid ROS in tumor tissues. (c) The relative abundance of ferroptosis-related proteins SLC7A11 and GPX4 was determined through western blot. **p<0.01, ***p<0.001.

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3.4 circPRKCA targets miR-384 to regulate SLC7A11 expression in HCC cells

To investigate the molecular mechanisms underlying circPRKCA-mediated ferroptosis regulation, we conducted a systematic analysis of circPRKCA-associated miRNA interactions. Bioinformatic prediction (TargetScan and miRanda algorithms) identified conserved binding complementarity between circPRKCA and miR-384, with parallel alignment revealing potential miR-384 interaction sites in the 3’UTR of SLC7A11 (Fig. 4a). We subsequently engineered luciferase reporter constructs containing either wild-type (WT) or mutant (MUT) miR-384 binding sequences in circPRKCA and the SLC7A11 3’UTR. Transfection efficiency was validated through qRT-PCR quantification of miR-384 levels following mimic transfection (Fig. 4b). Dual-luciferase reporter assays demonstrated miR-384-mediated suppression of WT circPRKCA (56.3% reduction, p<0.01) and WT SLC7A11 3’UTR (62.8% reduction, p<0.001) luciferase activity, while MUT constructs remained unaffected (Figs. 4c-f). Western blot analysis revealed that circPRKCA silencing substantially decreased SLC7A11 protein expression (Fig. 4g). Conversely, circPRKCA overexpression upregulated SLC7A11 protein levels by 2.7-fold (p<0.01), an effect restrained by concurrent miR-384 overexpression (Fig. 4h). These results have shown that circPRKCA functions as a molecular sponge for miR-384, thus post-transcriptionally regulating SLC7A11 expression in HCC cells.

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Fig. 4.

circPRKCA regulates the ferroptosis of HCC cells via miR-384/SLC7A11 axis. (a) Bioinformatic analysis on the binding site of miR-384 on circPRKCA and SLC7A11. (b) Level of miR- 384 in HCCLM3 and Huh7 cells that transfected with miR-384 mimics was measured by qRT-PCR. (c and d) Luciferase activity of circPRKCA reporter gene vectors. (e and f) Luciferase activity of SLC7A11 3’UTR reporter gene vectors. (g and h) The abundance of SLC7A11 protein was detected by immunoblotting. (i) Invasive capacity of HCCLM3 and Huh7 cells, as evaluated by performing Transwell assays (scale bar, 50 μm). (j) The concentration of total iron, Fe²⁺, and lipid ROS in HCCLM3 and Huh7 cells after corresponding processing. **p<0.01, ***p<0.001, ns, not significant.

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4. Discussion

Circular RNAs have attracted significant attention in recent years, and they have played significant roles in various diseases such as cardiovascular diseases, skin disorders, and malignant tumors (Altesha et al., 2019; Wu et al., 2020; Xue et al., 2022; Conn et al., 2024). As a type of non-coding RNAs, circRNAs can interact with miRNAs, thereby influencing the stability and function of miRNAs, and subsequently affecting various biological processes (Kristensen et al., 2019; Gao et al., 2022; Zhang et al., 2022; Chen et al., 2023). miRNAs typically target the 3’UTR region of mRNAs to impede gene translation or induce RNA degradation (Zhou et al., 2021). Therefore, circRNAs act as competing endogenous (ce) RNA to regulate gene expression and participate in various cellular processes, particularly cancer cell phenotypes such as proliferation, apoptosis, metastasis, and nutrient metabolism (Zhou et al., 2020; Zhou et al., 2021). In HCC, there is accumulating evidence highlighting the significant role of circRNAs (Lei et al., 2022). For instance, circRPN2 is reduced in individuals with metastatic HCC, and those with the lower level of circRPN2 exhibit decreased overall survival (Li et al., 2022). Additionally, circRPN2 directly binds to the enolase 1 (ENO1) protein and induces its degradation, thereby stimulating glycolytic reprogramming and inhibiting HCC metastasis by regulating the AKT/mTOR pathway (Li et al., 2022). Simultaneously, circRPN2, acting as a ceRNA for miR-183-5p, can inhibit glucose metabolism in HCC by promoting FOXO1 expression (Li et al., 2022). Moreover, circUHRF1 was substantially overexpressed in HCC tumors compared to matched healthy tissues. Mechanistic investigations have revealed that circUHRF1 suppresses natural killer (NK) cell-mediated immune function through dual regulatory actions: (1) promoting miR-449c-5p degradation via RNA interference mechanisms and (2) enhancing TIM-3 expression at the transcriptional level. This coordinated molecular activity ultimately attenuates the release of pro-inflammatory cytokines TNF-α and IFN-γ from NK cells (Zhang et al., 2020).

In recent years, ferroptosis has emerged as a critical focus in oncology research (Lei et al., 2022). There is growing evidence indicating its pivotal involvement in tumor progression and therapeutic resistance across various malignancies (Mou et al., 2019; Chang et al., 2021; Chen et al., 2021; Liang et al., 2022; Lei et al., 2022). Characterized by intracellular iron accumulation and lethal lipid peroxidation, this oxidative cell death modality presents new opportunities for cancer intervention (Liang et al., 2022). Our investigation of HCC reveals a previously unrecognized regulatory axis where circPRKCA modulates ferroptotic sensitivity through the miR-384/SLC7A11 signaling pathway. Mechanistically, circPRKCA depletion markedly enhanced ferroptotic vulnerability in HCC cells, as evidenced by three hallmark metabolic alterations: elevated total iron content, augmented Fe²⁺ levels, and accelerated lipid reactive oxygen species (ROS) generation. These findings not only establish circPRKCA as a novel ferroptosis regulator in HCC but also elucidate its dual role in maintaining iron homeostasis and suppressing peroxidative damage. The circPRKCA/miR-384/SLC7A11 axis thus represents a potential therapeutic target for overcoming chemoresistance in HCC through ferroptosis induction. In lung cancer, targeting CPT1A, a key rate-limiting enzyme of aliphatic acid oxidation, induces ferroptosis and synergizes with immunotherapy (Ma et al., 2024). Moreover, ferroptosis has also been confirmed to influence the development of stomach cancer. Cancer-associated fibroblasts (CAFs), a crucial component of the tumor microenvironment, secrete miR-552 restrains ferroptosis and facilitates acquired chemo-resistance in gastric carcinoma (Zhang et al., 2020).

An aforetime study reported that protein kinaseCα (PRKCA) functions as an oncogene in several cancers and can be spliced to generate circPRKCAs (Bai et al., 2020). In NSCLC, circPRKCAs/miR-330-5p/PDK1 pathway activates AKT signaling to promote the proliferation and metastasis of NSCLC both in vitro and in vivo. These results support the carcinogenic effect of circPRKCA in cancers and reveal a novel mechanism involving ferroptosis. Several studies have suggested the involvement of circRNAs in ferroptosis during tumorigenesis (Wu and Sun 2022; Zhang et al., 2022; Zuo et al., 2022). For instance, circLRFN5 protein overexpression suppresses cell proliferation, stemness, and growth of GSCs by ferroptosis (Jiang et al., 2022). The mmu_circRNA_0000309 functions as ceRNA for miR-188-3p to increase GPX4 expression, which consequently inactivates the ferroptosis and apoptosis of podocytes in diabetic nephropathy (Jin et al., 2021). In colorectal cancer (CRC), circSTIL promotes cell propagation and impedes ferroptosis by targeting the miR-431-SLC7A11 pathway (Li et al., 2023).

Our findings align with and extend these observations by elucidating how circPRKCA acts as a competing endogenous RNA (ceRNA) for miR-384, hence relieving miR-384-mediated suppression of SLC7A11. Given that SLC7A11 is a key anti-ferroptotic factor that facilitates cystine uptake and GSH biosynthesis (Li et al., 2020a; Tang et al., 2021; Koppula et al., 2021; Yan et al., 2023). Our study provides direct mechanistic insights into how circRNAs modulate ferroptosis in HCC.

And our experimental data demonstrated that genetic silencing of circPRKCA significantly inhibited HCC progression while enhancing susceptibility to ferroptotic cell death in vivo. A comparative analysis of subcutaneous xenograft models revealed that circPRKCA-deficient groups exhibited decreased tumor growth accompanied by increased expression of ferroptosis-related biochemical indicators when compared with control cohorts. Mechanistically, this antitumor effect was attributed to the disruption of cellular defense mechanisms against ferroptosis through the circPRKCA/miR-384 regulatory circuit, which subsequently modulates SLC7A11 expression, a key transporter involved in GSH biosynthesis and redox homeostasis. These findings not only identify circPRKCA as a novel epigenetic regulator promoting hepatocarcinogenesis but also highlight its dual clinical utility: serving as both a promising diagnostic biomarker and a potential therapeutic target. The established molecular axis provides critical insights for developing circRNA-directed treatment strategies against HCC, particularly those leveraging ferroptosis induction approaches.

In conclusion, our study not only identifies circPRKCA as a critical regulator of ferroptosis in HCC but also highlights a novel circPRKCA/miR-384/SLC7A11 axis that governs iron homeostasis and ferroptotic cell death. Targeting this pathway could provide promising therapeutic opportunities for overcoming ferroptosis resistance in HCC.