Abstract
Circular RNAs (circRNAs) are a novel and unique class of noncoding RNAs that are back-spliced from pre-mRNAs. It has been confirmed that circRNAs are involved in various malignant behaviors of hepatocellular carcinoma (HCC). However, the role of circRNA in the regulation of ferroptosis and the underlying mechanism remain unknown. Here, cIARS (hsa_circ_0008367) was found to be the most highly expressed circRNA after sorafenib (SF) treatment in HCC cells. Small interfering RNA against cIARS (si-cIARS) significantly suppressed the cellular sensitivity to SF or Erastin through inactivating ferroptosis, which may be partially attributed to the inhibition of autophagy and ferritinophagy. Prediction analysis and mechanistic identification revealed that cIARS physically interacted with RNA binding protein (RBP) ALKBH5, which was a negative regulator of autophagic flux in HCC. The dissociation of BCL-2/BECN1 complex, mediated by ALKBH5 silencing was effectively blocked by si-cIARS. Furthermore, the inhibition of ferroptotic events, autophagic flux and ferritinophagy resulted from si-cIARS, were significantly rescued by ALKBH5 downregulation. Overall, cIARS may be an important circRNA, positively regulating SF-induced ferroptosis through suppressing the ALKBH5-mediated autophagy inhibition.
Similar content being viewed by others
Introduction
HCC is a highly refractory and prevalent cancer worldwide, with ~841,000 new cases and 782,000 deaths every year1. Rapid advances in diagnosis and treatment had improved patient outcomes. However, the survival gains are stage-specific2. Liver resection, transplantation, ablation, or transarterial chemoembolization benefits patients in early or intermediate stages3, 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 inducer4, exerting only limited effects on overall survival and time to tumor progression5,6. Therefore, it is imperative to explore novel and effective therapeutic target to improve the cellular sensitivity to SF.
In recent years, ferroptosis has been confirmed to be an essential mechanism in SF treatment7. It is a kind of finely controlled cell death featured with iron-dependent accumulation of lipid hydroperoxides8. 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 complex9. Then, phagophore nucleation occurs, recruiting the ATG14/BECN1/PIK3C3/PIK3R4 complex10. Next, two ubiquitin-like conjugation systems are involved in phagophore elongation11. When de novo autophagosome formation is complete, they are delivered to the lysosome, initiating lysosomal degradation.
Actually, autophagy is a targetable pathway regulating cellular sensitivity to ferroptosis12. 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 release13,14,15.
CircRNAs primarily originate from back-splicing events of pre-mRNAs. The covalently bonded loops are highly stable and abundant16. The deregulation of circRNAs is closely related to various human diseases, including cancer17,18,19. In HCC, several studies have revealed that circRNAs are involved in multiple malignant behaviors20. 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.
Results
Circular transcript cIARS (hsa_circ_0008367) is significantly upregulated in SF-treated HCC cells
RNA-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 circBase21, 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 Illustrator22 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 database23 (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.
cIARS is found to be a significant regulator of SF-induced ferroptosis
To 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-124, 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.
cIARS positively regulates SF-induced autophagy and ferritinophagy
cIARS 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.
cIARS specifically interacted with RBP ALKBH5 (AlkB Homolog 5, RNA demethylase)
Recent 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.
cIARS repressed the role of ALKBH5 in the regulation of autophagy
ALKBH5 had been previously proven to be an autophagy inhibitor in cancer25,26. 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-226, 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.
cIARS regulates ferroptosis through ALKBH5-mediated autophagy
To 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).
Taken together, a novel circRNA in HCC was revealed in our research. We partially clarified its role and mechanism in ferroptosis.
Discussion
As 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.27 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.28 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 death12.
In 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.
To 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 diseases25,26,29,30,31. In lung cancer25, ALKBH5 upregulation stabilized UBE2C, an autophagy inhibitor, with maintenance of lower m6A level. In ovarian cancer26, 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.
In 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.
Materials and methods
Cell culture and transfection assay
Under 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.
Cell Counting Kit-8 (CCK-8) assay
The 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.
RNA extraction and analysis
Expression 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.
Ribonuclease R (RNase R) assay
RNase 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.
Actinomycin D (ActD) assay
ActD 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.
Western blot analysis
Radioimmunoprecipitation 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).
Observation of autophagy flux
Ad-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 Autophagy32.
Immunoprecipitation
RIP 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 studies33,34. 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.
RNA pulldown assay
Pierce™ 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′.
RNA EMSA
The 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 research34.
Lipid peroxidation assay
The relative level of MDA was evaluated through Lipid Peroxidation Kit (ab118970) according to the manufacturer’s guidance.
Iron assay
The relative level of intracellular iron was determined by an Iron Assay Kit (ab83366) according to the manufacturer’s instructions.
Glutathione assay
The 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.
Statistical analysis
The 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.
References
Bray, F. et al. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 68, 394–424 (2018).
Siegel, R. L., Miller, K. D. & Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin. 68, 7–30 (2018).
Forner, A., Reig, M. & Bruix, J. Hepatocellular carcinoma. Lancet 391, 1301–1314 (2018).
Li, J. et al. Ferroptosis: past, present and future. Cell Death Dis. 11, 88 (2020).
Llovet, J. M. et al. Sorafenib in advanced hepatocellular carcinoma. N. Engl. J. Med. 359, 378–390 (2008).
Cheng, A. L. et al. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol. 10, 25–34 (2009).
Dixon, S. J. et al. Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis. Elife 3, e02523 (2014).
Stockwell, B. R. et al. Ferroptosis: a regulated cell death nexus linking metabolism, redox biology, and disease. Cell 171, 273–285 (2017).
Jung, C. H. et al. ULK-Atg13-FIP200 complexes mediate mTOR signaling to the autophagy machinery. Mol. Biol. Cell 20, 1992–2003 (2009).
Itakura, E. & Mizushima, N. Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy 6, 764–776 (2010).
Geng, J. & Klionsky, D. J. The Atg8 and Atg12 ubiquitin-like conjugation systems in macroautophagy. ‘Protein modifications: beyond the usual suspects’ review series. EMBO Rep. 9, 859–864 (2008).
Zhou, B. et al. Ferroptosis is a type of autophagy-dependent cell death. Semin. Cancer Biol. https://doi.org/10.1016/j.semcancer.2019.03.002 (2019).
Torii, S. et al. An essential role for functional lysosomes in ferroptosis of cancer cells. Biochem. J. 473, 769–777 (2016).
Gao, M. et al. Ferroptosis is an autophagic cell death process. Cell Res. 26, 1021–1032 (2016).
Hou, W. et al. Autophagy promotes ferroptosis by degradation of ferritin. Autophagy 12, 1425–1428 (2016).
Memczak, S. et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333–338 (2013).
Bi, W. et al. CircRNA circRNA_102171 promotes papillary thyroid cancer progression through modulating CTNNBIP1-dependent activation of beta-catenin pathway. J. Exp. Clin. Cancer Res. 37, 275 (2018).
Su, Y. et al. circRIP2 accelerates bladder cancer progression via miR-1305/Tgf-beta2/smad3 pathway. Mol. Cancer 19, 23 (2020).
Chen, J. et al. Circular RNA profile identifies circPVT1 as a proliferative factor and prognostic marker in gastric cancer. Cancer Lett. 388, 208–219 (2017).
Wang, M., Yu, F. & Li, P. Circular RNAs: characteristics, function and clinical significance in hepatocellular carcinoma. Cancers 10, https://doi.org/10.3390/cancers10080258 (2018).
Glazar, P., Papavasileiou, P. & Rajewsky, N. circBase: a database for circular RNAs. RNA 20, 1666–1670 (2014).
Deng, W., Wang, Y., Liu, Z., Cheng, H. & Xue, Y. HemI: a toolkit for illustrating heatmaps. PLoS ONE 9, e111988 (2014).
Dudekula, D. B. et al. CircInteractome: A web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA Biol. 13, 34–42 (2016).
Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).
Guo, J. et al. Deregulation of UBE2C-mediated autophagy repression aggravates NSCLC progression. Oncogenesis 7, 49 (2018).
Zhu, H. et al. ALKBH5 inhibited autophagy of epithelial ovarian cancer through miR-7 and BCL-2. J. Exp. Clin. Cancer Res. 38, 163 (2019).
Chen, X. et al. Circular RNA circHIPK3 modulates autophagy via MIR124-3p-STAT3-PRKAA/AMPKalpha signaling in STK11 mutant lung cancer. Autophagy 16, 659–671 (2020).
Du, W. W. et al. A circular RNA circ-DNMT1 enhances breast cancer progression by activating autophagy. Oncogene 37, 5829–5842 (2018).
Song, H. et al. METTL3 and ALKBH5 oppositely regulate m(6)A modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes. Autophagy 15, 1419–1437 (2019).
Chen, Y. et al. m(6)A mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells. Autophagy 1–19, https://doi.org/10.1080/15548627.2020.1720431 (2020).
Li, G. et al. Bone-derived mesenchymal stem cells alleviate compression-induced apoptosis of nucleus pulposus cells by N6 methyladenosine of autophagy. Cell Death Dis. 11, 103 (2020).
Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1–222 (2016).
Yang, G. et al. circ-BIRC6, a circular RNA, promotes hepatocellular carcinoma progression by targeting the miR-3918/Bcl2 axis. Cell Cycle 18, 976–989 (2019).
Liu, B. et al. CircBACH1 (hsa_circ_0061395) promotes hepatocellular carcinoma growth by regulating p27 repression via HuR. J. Cell Physiol. https://doi.org/10.1002/jcp.29589 (2020).
Acknowledgements
This work was supported by the National Natural Science Foundation of China [Grant No. 81772601], Shandong Natural Science Foundation [Grant No. ZR201808200282], and Medical and Pharmacological Technology Development plan, Shandong [Grant Nos. 2017WS188 and 2017WS284].
Author information
Authors and Affiliations
Contributions
Study design: Z.L., X.W., and J.L.; data collection: Q.W., Z.X., and X.W.; data analysis: Z.L., Q.W., and X.W.; cellular experiments: Q.W., Z.L., X.W., and Z.X.; manuscript preparation: Z.L., Q.W., Z.X., and J.L.; figures and manuscript proofing: Z.L., Q.W., J.L., and X.W.; project administration: X.W. and J.L.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Edited by Inna Lavrik
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Liu, Z., Wang, Q., Wang, X. et al. Circular RNA cIARS regulates ferroptosis in HCC cells through interacting with RNA binding protein ALKBH5. Cell Death Discov. 6, 72 (2020). https://doi.org/10.1038/s41420-020-00306-x
Received:
Revised:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41420-020-00306-x
This article is cited by
-
CircRNA as an Achilles heel of cancer: characterization, biomarker and therapeutic modalities
Journal of Translational Medicine (2024)
-
Regulating ferroptosis by non-coding RNAs in hepatocellular carcinoma
Biology Direct (2024)
-
Epigenetic modification of ferroptosis by non-coding RNAs in cancer drug resistance
Molecular Cancer (2024)
-
CircSNX6 promotes proliferation, metastasis, and angiogenesis in hepatocellular carcinoma via miR-383-5p/VEGFA signaling pathway
Scientific Reports (2024)
-
CircNUP54 promotes hepatocellular carcinoma progression via facilitating HuR cytoplasmic export and stabilizing BIRC3 mRNA
Cell Death & Disease (2024)