Oxidative Medicine and Cellular Longevity Hsa_circ_0013731 mediated by E2F1 inhibits ferroptosis in hepatocellular carcinoma cells by sponging miR-877-3p and targeting SLC7A11

Background: The regulatory loop between circular RNAs and microRNAs has a vital role in cell death. Ferroptosis is the form of iron-dependent cell death, which is distinct from necroptosis and apoptosis. Increasing evidences showed that ferroptosis is an important form of cell death in hepatocellular carcinoma. Methods: Real-time PCR were used to examine the expression level of circ_0013731 in hepatocellular carcinoma tissues. Edu and colony formation were performed to detect the cell proliferation. A luciferase reporter assay was used to determine the relationship between circ_0013731, miR-877-3p and SLC7A11. ChIP-qPCR assays were performed to examine the potential binding of E2F1 to the circ_0013731 promoter. Iron Assay Kit (Sigma Aldrich) was used to detect total iron or Fe 2+ . C11 BODIPY 581/591 staining and ow cytometer were used to examine the Lipid ROS. The role of circ_0013731 was examined in xenograft tumors model. Results: We revealed that the expression level of circ_0013731 was elevated in hepatocellular carcinoma. Moreover, E2F1 promote the transcription of circ_0013731. Overexpression of circ_0013731 promoted cell growth and inhibited ferroptosis in SMMC-7721 and QGY-7703 in vitro. miR-877-3p was proved as the direct target of circ_0013731. Then, inhibition of miR-877-3p enhanced cell growth and inhibited ferroptosis. Further mechanism research demonstrated that circ_0013731 upregulated the expression level of SLC7A11 by sponging miR-877-3p. Finally, circ_0013731 promoted HCC growth via miR-877-3p/ SLC7A11 axis in vivo. Conclusions: Our data reveal that circ_0013731 mediated by E2F1 facilitates cell growth and suppressed the ferroptosis via miR-877-3p/ SLC7A11 axis in hepatocellular carcinoma.


Background
Hepatocellular carcinoma (HCC) is a lethal digestive malignancy worldwide and it is considered the second cause of cancer-related death [1,2]. The current problem is that HCC still has high recurrence and mortality rate, even though patients received standardized treatment including operation, chemotherapy and radiotherapy [3]. Moreover, a part of patients are usually diagnosed at late stages, thus missing the time window for radical cure [4]. Thus, nding a novel target for treatment is vital for curing of HCC.
CircRNAs are non-coding RNAs with closed loop and they are lack of 5' and 3' polarity and a polyadenylated tail [5,6]. The different roles of circRNAs have been reported in HCC. circRNA_101237 is acted as a vital prognostic biomarker for HCC [7]. circRNA_104075 promotes YAP-dependent HCC progression via bounding to HNF4a [8].
The reprogramming of cell death is vital for HCC progression. The great progress has been achieved in necrotic cell death and apoptotic [19,20]. Ferroptosis is the form of iron-dependent cell death, which is distinct from necroptosis and apoptosis [21]. In this study, we found circ_0013731 was elevated in HCC and mediated by E2F1. Moreover, circ_0013731 facilitates cell growth and inhibited ferroptosis in HCC. Furthermore, we found that circ_0013731 suppressed ferroptosis through miR-877-3p /SLC7A11 axis.
Tissue samples 45 HCC tissues were enrolled in this study. HCC diagnosis was based on histopathology. The study was carried out under the approval by the Ethics Committee of Lishui Hospital of Zhejiang University. Participants all provided their informed written consent.

EdU assay
EdU assay was performed in HCC cells. Brie y, HCC cells were plated into 96-well plates. HCC cells were cultured for 2 hours after treated with 50 μM EdU. Cells were xed with 4% paraformaldehyde then stained by Hoechst 33342 and Apollp reaction cocktail. Results were counted ve random elds by using a uorescence microscopy.
Cell transfection LV-circ_0013731 and sh-circ_0013731 were purchased from GeneCreate Biological Engineering Co., Ltd.

qRT-PCR
Total RNA was obtained using TRIzol reagent. RT-PCR was conducted using a SYBR Premix Ex Taq (TaKaRa, Dalian, China). Relative expression of genes was calculated by 2 −ΔΔCT .and SLC7A11, GAPDH and Tubulin expression levels were examined using the following speci c primers: Western blot HCC cells and tissues were lysed and protein concentration of total cell lysates was tested by the BCA assay. Protein samples (25-30ug) were loaded to electrophoresis and then transferred to PVDF membrane. After blocked in 5.5% non-fat milk for 2 hours, primary antibodies were used for incubation. Speci c primary antibodies were used in this research included anti-SLC7A11, anti-GAPDH and anti-Tubulin (Abcam, Cambridge, UK, 1:1000) for overnight at 4°C. Finally, the membrane was analyzed by the QuantityOne software (BioRad, Hercules, CA, USA).

Immuno uorescent staining
Histological section was xed by cold acetone for 10 min and incubated in a 1% BSA/PBS solution.

RNA Immunoprecipitation
Magna RIP RNA Binding Protein Immunoprecipitation Kit was used for RNA Immunoprecipitation. HCC cell extract was incubated with RIPA buffer with magnetic beads conjugated with human Ago2 antibody for 9 h. Normal IgG was used for negative control. Finally, immunoprecipitated RNA was collected and subjected to qRT-PCR analysis.
Biotin labeled probe pull down assay HCC cells were lysed in lysis buffer. Next, 3μg biotin labeled probe was added to the buffer lysis and incubated for 4 hours at 25°C. To pull down the circRNA-microRNA complex, strepavidin magnetic beads (Thermo Fisher) were added to the buffer and slowly rotated for another 4 hours. Then, washing step was carried out for 4 times. Finally, the binding RNA was extracted with TRIzol reagent and subjected to qRT-PCR analysis.

Lipid ROS assays
Following instructions [22], HCC cells were treated by C11-BODIPY (10 µM) and incubated for half hour. Afterward, washing step was performed twice times. C11-BODIPY was removed by cold PBS. The uorescence of C11-BODIPY581 was examined by the simultaneous acquisition of green (484/510 nm) and red signals (581/610 nm) using a ow cytometer.

Iron assays
Following instructions [23], Iron Assay Kit (Sigma Aldrich) was used to detect total iron or Fe 2+ in HCC. Following instructions, Iron Assay buffer and Iron Reducer were added in turn to the samples. In dark conditions, samples were mixed well and incubated for half hour after adding Iron Reducer. Samples were incubated for one hour after adding Iron Probe. Lastly, the absorbance was examined at 593 nm (A593

Statistical analysis
All the data were analyzed using SPSS software. Student t test was used to compare the difference of data between groups. P<0.05 represents signi cant difference. The asterisks *, ** and *** stand for p<0.05, p<0.01 and p<0.001, respectively.

circ_0013731 was elevated in hepatocellular carcinoma
To investigate the abundance of circ_0013731, RNase R digestion was used to identify circ_0013731 circular transcripts and MAN1A2 linear transcripts. Results shown that circ_0013731 was more resistant to RNase R digestion than MAN1A2 and GAPDH linear transcripts (Fig. 1a). circ_0013731 in HCC samples (N=45) showed an increased level compared with that in normal subjects as shown in Fig. 1b. Histogram and pie chart of the proportions of HCC samples in which circ_0013731 expression was downregulated (9/45, 20%, blue), no change (2/45, 4.4%, green), or upregulated (34/45, 75.6%, red) as shown in Fig. 1c. Then, the relative expression of circ_0013731 in primary tumor and portal vein tumor thrombus (N=26) was detected by qRT-PCR. Results showed that an increased level in portal vein tumor thrombus than that in primary tumor (Fig. 1d). As shown in Fig. 1e, the expression level of circ_0013731 was elevated in T3-T4 subjects (N=30) than that in T1-2 subjects (N=15). Moreover, the expression level of circ_0013731 was upregulated in N1 samples (N=11) than N0 samples (N=34) (Fig. 1f). Finally, the expression level of circ_0013731 was also elevated in HCC cells (Fig. 1g). Thus, the expression level of circ_0013731 was elevated in HCC.
The expression of circ_0013731 was mediated by transcription factor E2F1 E2F1 is a vital transcription factors and it can upregulate the circRNA expression at transcriptional level by binding to promoter. Based on circBase (http://www.circbase.org/), circ_0013731 was generated from MAN1A2 gene by back-splicing (256 bp). Four putative E2F1-binding sites on MAN1A2 promoter were found by bioinformatics prediction (https://www.gcbi.com.cn/gclib/html/index) and that of wild type or mutant promoter was established (Fig. 2a). Then, SMMC-7721 and QGY-7703 were transfected with pcDNA3.1-E2F1 or si-E2F1. The expression level of MAN1A2 was upregulated in pcDNA3.1-E2F1 group and was downregulated in si-E2F1 group (Fig. 2b-e). Then, correlation analysis revealed positive correlation between the levels of MAN1A2 and E2F1 mRNA in 45 HCC samples (Fig. 2f). Moreover, luciferase reporter assays were performed in SMMC-7721 and QGY-7703. Cells were transfected by pcDNA3.1-E2F1 or si-E2F1. Results showed that luciferase activity was elevated by overexpression of E2F1 and was decreased by knockdown of E2F1 ( Fig. 2g-j). Afterwards, ChIP-qPCR assays were performed in SMMC-7721 and QGY-7703 cells. We proved that putative E2F1 binding site the MAN1A2 promoter was bound to in HCC cells, IgG was used as a negative control (Fig. 2k, l). The expression levels of circ_0013731 in SMMC-7721 and QGY-7703 cells transfected with pcDNA3.1-E2F1 or si-E2F1 were assessed. circ_0013731 was positively regulated by pcDNA3.1-E2F1 and was negatively regulated by sh-circ_0013731 in HCC cells ( Fig. 2m-p). Finally, correlation analysis revealed positive correlation between the levels of circ_0013731 and E2F1 mRNA in HCC samples (N=45) (Fig. 2q). Therefore, E2F1 increased the expression of circ_0013731 through the binding to the MAN1A2 promoter.
Overexpression of circ_0013731 promoted cell growth and inhibited ferroptosis The function of circ_0125310 was furtherly investigated. LV-circ_0013731 was transfected into HCC cells and the e ciency of LV-circ_0013731 was detected by qRT-PCR. Results showed that the expression level of circ_0013731 was successfully upregulated by LV-circ_0013731 in SMMC-7721 and QGY-7703 cells (Fig. 3a, b). EDU assays and colony formation assays indicated that LV-circ_0013731 enhanced the growth of SMMC-7721 and QGY-7703 cells (Fig. 3c-f). Then, erastin or RSL3 was used to induce ferroptosis of HCC cells. Results indicated that circ_0013731 overexpression weakening the effect of erastin or RSL3 on proliferation in SMMC-7721 and QGY-7703 cells (Fig. 3g, h). Moreover, LV-circ_0013731 reduced intracellular concentrations of iron and Fe 2+ in SMMC-7721 and QGY-7703 cells after treating with erastin ( Fig. 3i-l). Moreover, C11 BODIPY 581/591 staining coupled with ow cytometry analysis was performed. To validate the effect of circ_0013731 on ferroptosis, SMMC-7721 and QGY-7703 cells were transfected by sh-circ_0013731. HCC cells were treated by erastin and detected after 0 (red), 4 (green), 8 (orange), 12 (blue) hours. Lipid ROS showed an increased level after transfection with sh-circ_0013731 compared with that in negative control as shown in Fig. 3m, n. Thus, circ_0013731 promoted cell proliferation and inhibited ferroptosis in SMMC-7721 and QGY-7703 cells.
miR-877-3p was the direct target of circ_0013731 As shown in Fig. 4a, we consulted bioinformatics method using http://www.bioinf.com.cn/ and the putative binding sites of predicted miRNAs on circ_0013731 were shown. Luciferase activity of circ_0013731 in HCC cells transfected with ve miRNA mimics was detected. Among the ve candidates, the luciferase activity got the largest reduction compared with the negative control after transfection with mimic-miR-877-3p (Fig. 4b, c). Moreover, the luciferase activity had a largest raise compared with the negative control after transfection with inhibitor-miR-877-3p (Fig. 4d, e). Correlation analysis revealed negative correlation between the levels of circ_0013731 and miR-877-3p in HCC samples (N=45) (Fig. 4f).

circ_0013731 promoted HCC growth in vivo
Then, in vivo assays were carried out to assess the effect of circ_0013731 on HCC progression. The xenograft tumor models of HCC were constructed by using SMMC-7721 and QGY-7703 cells infected with LV-circ_0013731, sh-circ_0013731 and negative control (every group=4). A picture of the xenograft is shown in Fig. 7a. The weight of the xenografts was calculated. The xenograft tumor was signi cantly heavier in the LV-circ_0013731 group than that in the LV-NC group (Fig. 7b). The xenograft tumor was signi cantly lighter in the sh-circ_0013731 group than that in the sh-NC group (Fig. 7c). The sizes of the xenograft in the LV-circ_0013731 and sh-circ_0013731 groups were calculated every 7 days. circ_0013731 overexpression promoted tumor growth and circ_0013731 knockdown suppressed tumor growth ( Fig. 7d-g). Moreover, the miR-877-3p /SLC7A11 axis was determined. The expression level of miR-877-3p was downregulated in the LV-circ_0013731 group, whereas that was upregulated in the sh-circ_0013731 group. The expression level of SLC7A11 was upregulated in the LV-circ_0013731 group, whereas that was downregulated in the sh-circ_0013731 group (Fig. 7h-k). Moreover, the expression level of SLC7A11 was measured in each group tissues by IHC or IF assays. Results showed that SLC7A11 expression was upregulated in the LV-circ_0013731 group, whereas that was downregulated in the sh-circ_0013731 group (Fig. 7l-o). Finally, a schematic diagram of mechanism of circ_0013731/ miR-877-3p /SLC7A11 in HCC progression was provided in Fig. 7p.

Discussion
In this study, we discovered that circ_0013731 was increased in the HCC tissues and HCC cell lines. circ_0013731 greatly promoted proliferation and suppressed ferroptosis in SMMC-7721 and QGY-7703 cells. Moreover, we found that E2F1 enhanced the expression of circ_0013731 through the binding to the circ_0013731 promoter. miR-877-3p was predicted as the target of circ_0013731. We veri ed the direct association between circ_0013731 and miR-877-3p. Inhibition of miR-877-3p enhanced HCC cell growth and inhibited ferroptosis. Then, SLC7A11 was predicted as the downstream target for miR-877-3p. circ_0013731 in HCC could promote growth and suppressed ferroptosis by sponging miR-877-3p and regulating SLC7A11 expression.
The occurrence and development of tumors is closely associated with de ciencies in programmed cell death [24,25]. Because of uncontrolled growth, tumor cells have a higher demand for iron than normal cells. Ferroptosis is the form of iron-dependent cell death, which is distinct from necroptosis and apoptosis [26,27]. Thus, tumor cell is more susceptible to ferroptosis than normal cell because of abnormal iron metabolism [21,28]. Increasing evidences have showed that ferroptosis acts important roles in HCC treatment. Ferroptosis is a vital part of mechanisms for sorafenib, which is rst line therapy for HCC [29]. LDL-DHA nanoparticles, a potential new drug for HCC, cause ferroptosis in HCC tissues [30].
Thus, the role of circ_0013731 in ferroptosis of HCC captured our attention.
It has been elucidated that circ_0013731 expression is increased in HCC tissues and cells. Furtherly, the upstream regulators of circ_0013731 was investigated. Predicted by GCBI (https://www.gcbi.com.cn/gclib/html/index), E2F1, a vital transcription factor for circRNA, was chosen as the upstream regulators of circ_0013731. Our nding elucidated that E2 upregulated the circ_0013731 expression via binding to the circ_0013731 promoter. Then, overexpression of circ_0013731 in HCC inhibited cell growth and ferroptosis. Competing endogenous RNAs (ceRNA) is one of main molecular mechanisms for circRNA [31,32]. Thus, the potential downstream miRNAs of circ_0013731 was investigated by bioinformatics method. miR-877-3p was con rmed as a target of circ_0013731. The role of miR-877-3p in HCC is unknown. We discovered that inhibition miR-877-3p could promote the HCC cell proliferation and repress the ferroptosis.
Solute carrier family 7 membrane 11 (SLC7A11) is a key coding gene of cystine/glutamate antiporter system xc- [33,34]. SLC7A11 promotes the synthesis of glutathione by mediating cystine uptake and glutamate release. Through the above way, SLC7A11 protects cells from oxidative stress, maintains the redox balance of cells, and prevents cell death induced by lipid peroxidation [35,36]. Iron regulates the transcription of SLC7A11 by ROS-Nrf2-ARE axis. It was con rmed that SLC7A11 could inhibit the cell iron death induced by iron overload in murine models [37]. SLC7A11 was con rmed as a target of miR-877-3p. Moreover, we discovered that the expression level of SLC7A11 was induced by circ_0013731, which could be reduced by mimic-miR-877-3p. Therefore, circ_0013731 up-regulated the expression level of SLC7A11 by sponging miR-877-3p in HCC cells. Finally, effect of circ_0013731 on HCC was detected in xenograft tumor models. Our data showed that circ_0013731 promoted HCC growth and regulated miR-877-3p/ SLC7A11 axis in vivo.
In this study, we further veri ed that circ_0013731 has oncogenic roles in HCC progression. Thus, our data discovered that circ_0013731/miR-877-3p/SLC7A11 regulatory loop may be potential therapeutic targets for HCC.

Conclusion
In summary, circ_0013731 is identi ed an important regulator in HCC. circ_0013731 is up-regulated by E2F1 and promoted cell growth in HCC. Furthermore, circ_0013731 suppresses the ferroptosis by regulation of miR-877-3p/ SLC7A11 axis. Thus, circ_0013731 could be acted as potential therapeutical target for HCC.

Data Availability
The data used to support the ndings of this study are included within the article.    c,d Western blot was conducted to determine the expression of SLC7A11 in HCC cells after transfection with inhibitor-miR-877-3p. e The wild or mutant binding sites sequence of SLC7A11 and miR-877-3p were shown. f,g The relative luciferase activities were detected in HCC cells co-transfected with pGL3-SLC7A11-wt, pGL3-SLC7A11-mut or inhibitor-miR-877-3p. h Correlation analysis revealed positive correlation between the levels of miR-877-3p and SLC7A11 HCC tissues. i The protein expression level of SLC7A11 was detected in SMMC-7721 and QGY-7703 transfected by LV-circ_0013731. j,k The relative luciferase activities were detected in SMMC-7721 and QGY-7703 co-transfected with pGL3-SLC7A11-wt,