Circular RNA cIARS regulates ferroptosis in HCC cells through interacting with RNA binding protein ALKBH5

1HCC is a highly refractory and prevalent cancer worldwide, with ~841,000 new cases and 782,000 deaths every year. Rapid advances in diagnosis and treatment had improved patient outcomes. However, the survival gains are stage-specific. Liver resection, transplantation, ablation, or transarterial chemoembolization benefits patients in early or intermediate stages, while treatment for late-stage HCC has remained challenging. The standard first-line systemic drug against advanced HCC is sorafenib (SF, BAY 43-9006, Nexavar), which is currently known as a ferroptosis inducer, exerting only limited effects on overall survival and time to tumor progression,. Therefore, it is imperative to explore novel and effective therapeutic target to improve the cellular sensitivity to SF.

2In recent years, ferroptosis has been confirmed to be an essential mechanism in SF treatment. It is a kind of finely controlled cell death featured with iron-dependent accumulation of lipid hydroperoxides. When HCC cells were exposed to SF, the cystine/glutamate antiporter system (system xc−) was targeted. Subsequently, the cystine uptake was blocked and the biosynthesis of glutathione (GSH) was inhibited, resulting in accumulation of lipid peroxidation products, and eventually inducing ferroptosis. Meanwhile, SF also induces autophagic flux in cells. When autophagy flux is initially induced, MTORC1 dissociates from the ULK1/2/ATG13 complex, leading to dephosphorylation of the complex. Then, phagophore nucleation occurs, recruiting the ATG14/BECN1/PIK3C3/PIK3R4 complex. Next, two ubiquitin-like conjugation systems are involved in phagophore elongation. When de novo autophagosome formation is complete, they are delivered to the lysosome, initiating lysosomal degradation.

3Actually, autophagy is a targetable pathway regulating cellular sensitivity to ferroptosis. The crosstalk between autophagy and ferroptosis has attracted more and more attentions in the recent years. Autophagy inhibition by bafilomycin A1 or chloroquine, or lack of key autophagy associated gene (for example: ATG5, ATG7), will partially suppressed ferroptotic events through ferritinophagy inactivation, reducing the turnover of iron-binding ferritin and the iron release–.

4CircRNAs primarily originate from back-splicing events of pre-mRNAs. The covalently bonded loops are highly stable and abundant. The deregulation of circRNAs is closely related to various human diseases, including cancer–. In HCC, several studies have revealed that circRNAs are involved in multiple malignant behaviors. However, the functions and mechanisms of circRNAs in SF treatment are still not comprehensively understood. In the current research, we found a novel circRNA derived from the IARS gene (ID from circBase: hsa_circ_0008367), named cIARS. cIARS was proven to be a promoter of ferroptosis in HCC cells after SF treatment, which was partially attributed to the activation of autophagy and ferritinophagy. The underlying molecular mechanism of cIARS-mediated ferroptosis was also clarified in this study.

5RNA-seq was performed in three pairs of SF-treated and untreated HCC cell lines (HepG2, SMMC-7721 and Huh7) to screen differentially expressed circRNAs. Cells were treated with 10 μM of SF for 24 h. 11367 unique circRNAs were found. Among these circRNAs, 3963 (35%) had already been reported in circBase, and the other 7404 (65%) circRNAs were newly discovered. Among the 3963 annotated circRNAs, 2506 (63%) were of extremely low abundance with fragments per kilobase million (FPKM) values <1 in cellular samples and were thus excluded from this study. The other 1457 circRNAs comprised two groups: 1320 presented no significant changes in expression levels after SF treatment, and the other 137 were differentially expressed circRNAs (fold change ≥ 2, p-value < 0.05). The volcano plots demonstrated that there were 102 upregulated and 35 downregulated transcripts (Fig. 1a). The results of hierarchical clustering are displayed in a heatmap (Fig. 1b) generated with Heatmap Illustrator and suggest that there were two different clusters of transcripts. The most highly expressed circRNA was cIARS (hsa_circ_0008367) and was marked with black arrow in the volcano plots. According to circBase, cIARS is an exonic circRNA ~226 nt in length originating from the exon 13 and exon 14 of the IARS gene on chr9: 95030455–95032265 (Fig. 1c). qPCR showed that the relative expression levels of cIARS were significantly higher in SF-treated cell lines than in untreated ones (Fig. 1d). Both convergent and divergent primers of cIARS were applied for amplification. The band of cIARS was only observed in cDNA sample not the genomic DNA (Fig. 1e). Sanger sequencing further validated that the sequence around the junction site (about 100 bp around the site) was consistent with the result of RNA-seq and CircInteractome database (Fig. 1f). In addition, cIARS was much more resistant to RNase R (2.5 U/μg) (which degrades linear, but not circular, transcripts) than IARS and GAPDH (Fig. 1g). When actinomycin D (ActD, a transcription inhibitor) was added to HCC cells for the indicated time periods, cIARS was much more stable than its linear counterpart (Fig. 1h). These evidences suggested cIARS to be a highly abundant and stable circular transcript in HCC cells.

6To clarify the biological role of cIARS, we first knockdown cIARS expression with a junction site-specific siRNA vector (si-cIARS). The effects of the si-cIARS was shown in Fig. 2a. CCK-8 assay showed that SF-induced growth inhibition was evidently weakened in si-cIARS transfected cells; Erastin-induced growth inhibition was also attenuated by si-cIARS (Fig. 2b, c). To determine the underlying mechanism, si-cIARS-introduced HCC cells were treated with various cell death inhibitors. Ferrostatin-1, a specific ferroptosis inhibitor, significantly undermined the therapeutic effects of either SF or Erastin in both si-cIARS and NC transfected cells. However, ZVAD-FMK (an apoptosis inhibitor) and Necrosulfonamide (a necroptosis inhibitor) exerted no significant influence on SF or Erastin-induced growth inhibition (Fig. 2c). Simultaneously, malondialdehyde (MDA) and the level of Fe2+ were significantly reduced, while intracellular GSH obviously increased in the cIARS silencing cells following SF or Erastin administration (Fig. 2d). These evidences suggested cIARS to be a positive regulator of ferroptosis in HCC cells.

7cIARS was also found to be an autophagy regulator. Western blot (WB) assay showed that cIARS knockdown significantly decreased LC3 lipidation and increased p62 accumulation (Fig. 3a). Either autophagosomes or autolysosomes were observed via microscopic examination after Ad-mCherry-GFP-LC3 adenovirus transfection. This experiment is applied for concurrent observation of autophagosome and autolysosome. The signal of green fluorescent protein will be quenched during fusion of autophagosome and lysosome. Thus, the red signal of mCherry indicates autolysosome and the merge of green and red signals (yellow puncta) indicates autophagosome. si-cIARS significantly decreased the amount of red (autolysosome) and yellow (autophagosome) puncta per cell, demonstrating an inhibition of autophagy flux (Fig. 3b). TEM visually suggested the autophagic compartments. si-cIARS decreased the number of double-membraned vacuoles to a relatively low level (Fig. 3c). These results showed that cIARS is a positive autophagy regulator in SF-treated HCC cells. Furthermore, the protein levels of FTH1 and NCOA4, the substrate and cargo receptor of ferritinophagy, were determined by WB assay. si-cIARS resulted in remarkable accumulation of both FTH1 and NCOA4 (Fig. 3d). This finding indicated that the ferroptotic events in SF-treated HCC cells, positively regulated by cIARS, may be partially associated with autophagy and ferritinophagy.

8Recent studies have revealed that circRNA–RBP interaction has important roles in diverse biological processes. According to CircInteractome database, we found six RBPs bearing at least two binding sites matching to cIARS, including FMRP (Fragile X Mental Retardation 1), SFRS1 (Serine And Arginine Rich Splicing Factor 1), ALKBH5, HuR (ELAV like RNA binding protein 1), IGF2BP1 (Insulin Like Growth Factor 2 MRNA Binding Protein 1), and LIN28A (Lin-28 Homolog A) (Fig. 4a). RBP immunoprecipitation (RIP) assay in both HepG2 and Huh7 cell lines demonstrated that the relative levels of cIARS in ALKBH5 enriched samples were much higher than in the other five RBPs (Fig. 4b). RNA pulldown and RNA EMSA were performed to determine the interaction using an anti-sense probe spanning the junction site of cIARS. The presence of specific bands demonstrated the physical binding of cIARS and ALKBH5 (Fig. 4c, d). Interestingly, SF administration had no influence on the protein levels of ALKBH5 in both HepG2 and Huh7 cells (Fig. 4e), but remarkably increased the cIARS–ALKBH5 interaction (Fig. 4f), which may be due to SF-induced expression of cIARS.

9ALKBH5 had been previously proven to be an autophagy inhibitor in cancer,. However, its role in SF-treated HCC cells remains unclear. WB assay showed that siRNA against ALKBH5 (si-ALKBH5) significantly promoted the transformation of LC3B I to II and degraded p62 (Fig. 5a). This result suggested ALKBH5 to be a negative regulator of autophagy in HCC cells. qPCR and WB showed that si-cIARS failed to influence ALKBH5 mRNA and protein levels (Fig. 5b, c); similarly, si-ALKBH5 also had no impact on the relative expression of cIARS (Fig. 5d). A previous study revealed that si-ALKBH5 promoted the dissociation of BECN1 and BCL-2, a key step during phagophore nucleation. In SF-treated HCC cells, we gained similar results via IP assay. More importantly, this process can be effectively blocked by cIARS silencing (Fig. 5e). These results demonstrated that cIARS repressed the biological role of ALKBH5 in autophagy.

10To explore whether cIARS-regulated ferroptosis via ALKBH5, we performed several phenotype rescue experiments. First, si-ALKBH5 successfully rescued the effects of si-cIARS in autophagic flux and ferritinophagy (Fig. 6a, b). Second, ALKBH5 knockdown effectively re-intensified the SF cytotoxicity, which was remarkably impaired by si-cIARS (Fig. 6c). Third, si-cIARS-mediated decrease of MDA, Fe2+ and increase of GSH, can be rescued by si-ALKBH5 (Fig. 6d–f).

11Taken together, a novel circRNA in HCC was revealed in our research. We partially clarified its role and mechanism in ferroptosis.

12As a novel class of noncoding RNA, circular transcripts have attracted widespread attention. However, circRNA-regulated ferroptosis in human diseases has not been widely investigated. A small portion of studies focused on the circRNA-mediated autophagy. For instance, Chen et al. reported that circHIPK3 depletion significantly induced autophagy via miR-124-3p-STAT3-PRKAA/AMPKa axis and there was an antagonistic regulation on autophagy between circHIPK3 and linear HIPK3. Du WW et al. showed that the oncogenic circDnmt1-stimulated autophagy flux in breast carcinoma via interaction with both p53 and AUF1. These findings demonstrated the potential of circRNAs in autophagy regulation and prompted us to explore the role of circRNA in ferroptosis, which had been identified as an autophagic cell death.

13In our research, we delineated a mechanism of circRNA-mediated ferroptosis during SF treatment in HCC cells. circRNA cIARS (hsa_circ_0008367) was screened from RNA-seq analysis. Phenotypically, cIARS positively regulated ferroptosis, which may be partially dependent on autophagy and ferritinophagy. Mechanistically, cIARS physically interacted with RBP ALKBH5 and negatively regulated its role in autophagy.

14To comprehensively investigate the complicated circRNA network, it is essential to deeply evaluate its binding partners. In this study, cIARS is found to be an interactor of RBP ALKBH5, which had previously been reported to be a N6-methyladenosine (m6A) eraser. Its role in autophagy regulation is completely different in different tissues and diseases,,–. In lung cancer, ALKBH5 upregulation stabilized UBE2C, an autophagy inhibitor, with maintenance of lower m6A level. In ovarian cancer, ALKBH5 inhibited autophagy through activating PI3K-Akt-mTOR signaling pathway, stabilized BCL-2 mRNA and promoted the interaction between BCL-2 and BECN1. Herein, cIARS was proven to be a pivotal regulator of autophagy, ferroptosis, and ferritinophagy, depending on negatively regulating the biological role of ALKBH5, an autophagy inhibitor in HCC cells.

15In this study, the cIARS–ALKBH5 axis was demonstrated to be a key mechanism regulating ferroptosis during SF treatment. Further studies are still needed at the clinical and mechanistic levels.

16Under humidified conditions with 5% CO2 at 37 °C, the HCC cell lines HepG2, SMMC-7721, and Huh7 (bought from the National Infrastructure of Cell Line Resource) were cultured in Dulbecco’s Modified Eagle’s medium (HyClone, Logan, UT, USA) with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and penicillin (100 U/ml)-streptomycin (100 µg/ml) solution in the medical research center of Shandong Provincial Qianfoshan Hospital, Shandong First Medical University. All of the cell lines mentioned in this research were cultured within ten passages. The transfection experiments were performed, aided by Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA). The RNA oligonucleotides in this work were designed and constructed by GenePharma (Shanghai, China), including siRNAs against cIARS or ALKBH5, and the corresponding negative controls (NC and NC’). Sequences were shown as follows (5′–3′): si-cIARS: GAC UUU GAG GAG AUC AGA CAC; si-ALKBH5: GGA UAU GCU GCU GAU GAA ATT; NC/NC′: UUC UCC GAA CGU GUC ACG U.

17The growth inhibition rate of HepG2 and Huh7 cells was assessed by the CCK-8 (Dojindo Laboratories, Japan) assay. The blank or transfected cells (5 × 103 cells per well) were seeded into 96-well plates with three replicate wells. After the treatment with SF or Erastin or various cell death inhibitors (Ferrostatin-1, ZVAD-FMK or Necrosulfonamide) for 24 h, a 10 µl volume CCK-8 reagent was added to each well. Then, measuring the absorbance at 450 nm after incubation with the CCK-8 solution at 37 °C for 2 h.

18Expression profiles of genome-wide circRNAs in three pairs of HCC cell lines (before and after SF treatment) were explored on an Illumina HiSeq 4000 platform (by Novogene, Beijing, China). The cDNA from divergent primers was subjected to Sanger sequencing (by Zhonghong Boyuan Biological Technology, Jiangxi, China). Agarose gel electrophoresis was used to detect the qPCR amplification of cDNA and genomic DNA after applying with the divergent and convergent primers of cIARS.The relative fold changes in expression were calculated with the formula 2−ΔΔCt. The sequences of all the primers were listed as follows (5′–3′): cIARS F: AGC GAT GAC TTT GAG GAG ATC A, R: CCC AGT AGC ACA GGT CAT TG; IARS F: CAT ATC CAG TTT CTC CAT CGG A, R: TGG ATT TTC CAG GAG CAA TAC T; ALKBH5 F: GCA AGG TGA AGA GCG GCA TCC, R: GTC CAC CGT GTG CTC GTT GTA C; U6 F: CTC GCT TCG GCA GCA CAT A, R: ATT TGC GTG TCA TCC TTG CG; GAPDH F: CAG AAC ATC ATC CCT GCC TCT AC, R: ATG AAG TCA GAG GAG ACC ACC TG.

19RNase R assay (R0301, 20 U/µl, Geneseed, Guangzhou, China) was used for the identification of circRNA. According to the manufacturer’s guidance: 2 µl 10× Reaction Buffer and 2.5 U RNase R/µg RNA were mixed to the total RNA, and then added RNase-Free Water to form a 20 µl reaction solution system. After digestion with RNase R for 15 min at 37 °C, the enzyme then was inactivated at 70 °C for 10 min and then directly perform reverse transcription reaction. The qPCR assay was used to determine the relative expression of cIARS, IARS and GAPDH compared to the mock group.

20ActD assay (HY-17559, MedChem Express, New Jersey, USA) was used to detect the stability of RNA. 1 μg/ml ActD reagent was used to treat HepG2 and Huh7 cells. After 0, 4, 8, 12, or 24 h of administration, the total RNA was extracted respectively to determine the relative expression of cIARS or IARS by qPCR assay.

21Radioimmunoprecipitation assay buffer (RIPA) was applied to lyse cells. Total proteins were then harvested and quantified with bicinchoninic acid assays (Beyotime, Shanghai, China). The target proteins were separated through 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). The membranes were blocked with nonfat milk, incubated with primary antibodies, and then incubated with secondary antibody diluted at a ratio of 1:10,000 (Jackson ImmunoResearch, West Grove, PA, USA). The primary antibodies were anti-LC3B (2775, Cell Signaling Technology, Beverly, MA, USA), anti-p62 (88588, Cell Signaling Technology), anti-HuR (ab28660, Abcam, Cambridge, MA, USA), anti-SFRS1 (ab133689), anti-FMRP (ab17722), anti-ALKBH5 (ab195377), anti-IGF2BP1 (ab82968), anti-LIN28A (ab46020), anti-FTH1 (ab65080), anti-NCOA4 (ab86707), anti-BCL-2 (ab32124), anti-BECN1 (ab62557), and anti-GAPDH (ab9485). The protein signals were visualized with enhanced chemiluminescence detection reagents (ECL, Millipore, Burlington, MA, USA) and quantified with Image Lab software (Bio-Rad, Hercules, CA, USA).

22Ad-mCherry-GFP-LC3 adenovirus (Servicebio Technology, Wuhan, China) was transfected into HCC cells. After 24 h incubation, cells were observed and photographed using a fluorescence microscope (Olympus FSX100, Tokyo, Japan). Autophagic compartments were finely observed through transmission electron microscopy (TEM, HT7700, HITACHI, Tokyo, Japan). The identification of autophagic vacuoles in TEM images was mainly based on the 3rd edition of the Guidelines for the Interpretation of Assays for Monitoring Autophagy.

23RIP and IP were performed to confirm the RNA–protein and protein–protein interactions using RIP Kit (17-700, Millipore) and Immunoprecipitation Kit (ab206996) according to the manufacturer’s guidance. RIP related details had been described in our previous studies,. The IP assay consists of four steps, including antibody binding, beads preparation, bead capture, and elution. The volume of the antibody binding system was made up to 500 µl with lysis buffer containing the protease inhibitor cocktail and gently mixed for 4 h; the protein A/G sepharose (30 µl/reaction) was washed twice with wash buffer, centrifuged at 2000 × g for 2 min and aspirated the supernatant between washes.

24Pierce™ Magnetic RNA-Protein Pull-Down Kit (Thermo) was applied to evaluate RNA–protein interaction using biotin-labeled junction-specific probe and its negative control (designed and synthesized by Viagene Biotech, Jiangsu, China). Detailed procedure strictly followed the manufacturer’s guide. cIARS-probe: 5′-TGT AAA TTA GAG GAG TGT CTG ATC TCC TCA AAG TC-3′; Negative control: 5′-GCA GCC TGA TCA CGA CTG ACT TTA GTG TTT GCA TT-3′.

25The LightShift Chemiluminescent RNA EMSA Kit (ThermoFisher Scientific) was applied to evaluate the interaction between cIARS and ALKBH5, according to the manufacturer’s guidance. The details were described in our as previous research.

26The relative level of MDA was evaluated through Lipid Peroxidation Kit (ab118970) according to the manufacturer’s guidance.

27The relative level of intracellular iron was determined by an Iron Assay Kit (ab83366) according to the manufacturer’s instructions.

28The relative level of intracellular GSH was assessed through a GSH Colorimetric Detection Kit (CS0260, Sigma-Aldrich, St. Louis, USA) according to the manufacturer’s instructions.

29The statistical analyses in this work were carried out using Prism 7. The results from at least three independent tests are shown as the mean value ± standard deviation (SD). The mean values of two groups were compared via unpaired Student t-tests. “p-value < 0.05” was defined as significant. *p < 0.05, **p < 0.01.