Hsa_circ_0006988 Promotes Sorafenib Resistance of Hepatocellular Carcinoma by Modulating IGF1 Using miR-15a-5p

Background Hepatocellular carcinoma (HCC) is the most frequently occurring cancer and contributes to the largest number of cancer-associated deaths worldwide. Recent evidence suggests that circular RNAs (circRNAs), which are critical for HCC etiology and metastasis, are distinctly modulated in HCC. Nevertheless, the underlying mechanism of circRNA-mediated sorafenib resistance (SOR) in HCC is yet to be determined. Methods The hsa_circ_0006988, IGF1, and miR-15a-5p contents were quantified via ELISA and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. Cell Counting Kit-8 (CCK-8) was used for the IC50 evaluation. Lastly, associations among hsa_circ_0006988, IGF1, and miR-15a-5p were validated through dual-luciferase reporter (DLR) and RNA immunoprecipitation (RIP) assays. Results Herein, a new circRNA, hsa_circ_0006988, was identified, and its levels were markedly enhanced in SOR-resistant (SOR-R) HCC tissues. Functionally, hsa_circ_0006988 strongly suppressed SOR toxicity in vitro. Our examination of the signaling pathway revealed that hsa_circ_0006988 sequestered miR-15a-5p, a negative modulator of IGF1, thus suggesting that hsa_circ_0006988 deficiency diminished SOR resistance of HCC, and this action utilized the release of excess miR-15a-5p, which suppressed IGF1 levels. Moreover, miR-15a-5p overexpression reversed the hsa_circ_0006988-mediated SOR-R and enhanced IGF1 levels in HCC cells. Conclusion Hsa_circ_0006988 partly promoted the SOR-R of HCC cells through miR-15a-5p sequestering and upregulation of IGF1 levels.


Introduction
With an 18% 5-year overall survival (OS) rate and a 65-80% postsurgical resurgence, hepatocellular carcinoma is the most severe form of primary liver cancer [1]. Recently, new therapeutic strategies such as cancer immunosuppressive therapy have improved patient survival, and the combination of an immune checkpoint inhibitor (ICI) and VEGF inhibitor is targeted as the frst-line treatment for advanced HCC [2][3][4]. In terms of treatment, the multikinase inhibitor sorafenib (SOR) displays superior performance in alleviating HCC. However, HCC patients still sufer poor outcomes due to acquired resistance [5,6]. SOR is an essential factor that limits the long-term OS of HCC patients. Hence, there is an urgent need for new therapeutic candidates to minimize sorafenib resistance (SOR-R) in HCC.
Herein, we examined the diferentially regulated circRNAs in SOR-R HCC tissues using GEO circRNA arrays. Our screening uncovered hsa_circ_0006988 as preserved and markedly enhanced circRNA in SOR-R HCC cell lines and tissues. Loss-and gain-of-function assessments revealed that hsa_circ_0006988 induced SOR-R in HCC tissues. We demonstrated that hsa_circ_0006988 suppression sensitized HCC cells to SOR by modulating miR-15a-5p and its downstream IGF1 levels. Our conclusions will enable a novel understanding of the hsa_circ_0006988-induced mechanisms in tumorigenesis and SOR-R in HCC.

Patients and Tissue Samples.
Matched fresh HCC tissues and corresponding ANTs were obtained from 156 HCC patients and were maintained in liquid nitrogen until further analysis. Te patients were separated into sorafenib-sensitive (SOR-S, n � 82) and SOR-R cohorts (n � 74), following 2 regimens of SOR supplemental therapy. Tumor samples were acquired via surgery before the initiation of therapeutic intervention. Tis work received ethical approval from Xinyu People's Hospital, whereas a written informed consent form was obtained from all participants before the operation.

Cell Culture and Reagents.
Te Chinese Academy of Sciences Cell Bank Type Culture Collection was contacted for HCC, HepG2, and Huh-7 cancer cell lines and the normal human liver cell line LO2. Te cells were maintained in DMEM and RPMI-1640 culture media (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C and in a 5% CO 2 incubator SOR (BAY 43-9006) provided by MedChem Express. It was resuspended in DMSO at <0.1%. To establish SOR-R hepatoma cells, HepG2 and Huh-7 cells were maintained in 1 mmol/L of SOR and gradually elevated by 0.5 mmol/L per month (up to 5 mmol/ L) over a period of 10 months. Subsequently, two SOR-R cell lines were established, which were subsequently termed SOR-R HepG2 (SR-HepG2) and SOR-R Huh7 (SR-Huh7).

RT-qPCR
SYBR green kit (Takara, Dalian, China) was used to evaluate cDNA, and qRT-PCR was conducted via equipment obtained from Bio-Rad Laboratories (Berkeley, USA). Te relative gene expression was computed using the 2 −ΔΔCt method. Te hsa_circ_0006988 and IGF1 expressions were adjusted to GAPDH, and the miR-15a-5p expression was adjusted using U6 levels.
3.1. MTT Assay. After siRNA transfection, 5000 SR-HepG2 and SR-Huh7 cells were seeded in a 96-well plate and treated with SOR for 48 hours. Next, 2 mg/mL of MTT reagent (Sigma-Aldrich) was added to the wells for an additional 4 h. Lastly, all formazan formations were resuspended in 100 μl of dimethylsulfoxide prior to the optical density (OD) assessment at 570 nm on a microplate reader. Te SOR IC50 was determined using GraphPad Prism 7 (GraphPad Software, San Diego, USA).

RNA Pull-Down Assay.
Biotinylated miR-15a-5p (bio-miR-15a-5p) or bio-NC was introduced into SR-HepG2 and SR-Huh7 cells. Following a 48 h incubation and cell lysis, the lysate was incubated with streptavidin-coupled magnetic beads (Invitrogen) for 2 h. Subsequently, following RIP of the biotin-associated RNA, the hsa_circ_0006988interacting RNAs were examined via RT-qPCR following the extraction of associated RNAs.

Elisa
Assay. Te IGF1 expression was predicted in various culture media using an ELISA kit (R&D Systems) following kit directions. Te presented results are the mean of 3 distinct experiments.
3.6. Statistical Analysis. Data are provided as the mean-+ standard deviation of 3 replicates, assessed via Student's ttest and a one-way analysis of variance (ANOVA). Te Spearman rank correlation was employed to assess the associations among hsa_circ_0006988, IGF1, and miR-15a-5p transcript contents in HCC samples. P < 0.05 was considered the signifcance cut-of.

Hsa_circ_0006988 Levels Were Correlated with SOR-Rof
HCC. Our analysis of circRNA expressions in 3 SOR-R and 3 SOR-S samples from the GSE101850 GEO microarray data identifed 250 highly expressed circRNAs in HCC patients, among which the top 5 are presented in Figure 1(a). Te hsa_circ_0006988 levels were markedly enhanced in SOR-R cells and tissues relative to SOR-S cells and tissues (Figures 1(b) and 1(c)). Moreover, the 5-year OS rate of the SOR-R patients with augmented expression was markedly reduced relative to the SOR-S (Figure 1(d)). We also demonstrated that the SOR of IC50 was enhanced in SOR-R cells relative to normal cells (Figure 1(e)).

Hsa_circ_0006988 Silencing Suppressed SOR-R in HCC
Cells. To elucidate the hsa_circ_0006988-mediated regulation of SOR-R in HCC cells, we incorporated SR-Huh7 and SR-HepG2 cells with si-hsa_circ_0006988. Our RT-qPCR data revealed that hsa_circ_0006988 levels exhibited a marked decrease in si-hsa_circ_0006988-incorporated SR-Huh7 and SR-HepG2 cells in comparison with controls ( Figure 2(a)). Based on the MTT assay, the IC50 of SOR was drastically reduced in si-hsa_circ_0006988 incorporated SR-HepG2 and SR-Huh7 cells (Figure 2(b)). Tis evidence suggested a strong infuence of hsa_circ_0006988 silencing in the SOR-R of HCC.

MiR-15a-5p
Targeting IGF1 to Inhibit Chemoresistance in SOR-RHCC Cells. We next estimated candidate miR-15a-5p target genes using an online estimation tool in an attempt to discern the underlying mechanism whilst identifying the docking sites of IGF1 on miR-15a-5p (Figure 4(a)).

Discussion
Herein, we evaluated the signifcance of circRNAhsa_-circ_0006988 on the SOR chemosensitivity of HCC and demonstrated the modulatory signaling behind the miR-15a-5p/IGF1 axis. Our analysis shows that elevated hsa_-circ_0006988 levels enhanced the SOR-R of hepatocellular carcinoma cells. Hsa_circ_0006988 serves as a molecular sequester of miR-15a-5p, which, in turn, disrupts the suppressive efect of miRNA on IGF1. In addition, using DLR and RIP assays, we demonstrated a strong association among hsa_circ_0006988, miR-15a-5p, and IGF1. Collectively, this evidence indicated that hsa_circ_0006988 modulates the SOR-R of HCC, which, in turn, promotes HCC progression.
Over the past decade, drug interventions have markedly enhanced the OS of hepatocellular carcinoma cells in patients with complex diseases. An oral multikinase inhibitor, SOR, is a proliferation and angiogenesis suppressor, and it does so by modulating Raf-1, BRAF Flt3, PDGFR-b, and VEGFR-2/3 [18,19]. SOR, an FDA-approvedanti-HCC therapeutic agent, is highly efcacious against HCC [20]. SOR and NK cells might improve the outcome of applied therapeutic approaches for HCC patients [21]. However, a majority of patients progress to develop drug-resistant diseases, thereby resulting in poor patient outcomes. Te underlying mechanism behind the SOR-R of HCC is rather complicated. To date, the signaling pathways associated with HCC drug resistance remain undiscovered.
Tere has been much focus on circRNA, miRNA, and long noncoding RNA (lncRNA) in recent years [8]. Te circRNA has a CCLS and does not encode proteins [22]. Emerging evidence suggests that circRNA modulates numerous physiological and pathological activities, such as proliferation, cellular diferentiation, angiogenesis, metabolic stress responses, and cell death [23]. Impaired circRNAs behave like tumor suppressor oncogenes in their modulation of cancer development and progression, including HCC [24][25][26]. More reports suggest that numerous miRNAs and lncRNAs regulate SOR-R [27,28]. Similarly, some circRNAs also modulate the SOR-R of HCC. Hence, scientists demonstrated marked alterations in circRNA expressions in a myriad of drugresistant versus drug-sensitive HCC cells. Collectively, these fndings indicate that circRNAs may be used to predict drug efciency and enhance personalized HCC intervention [29].
Recently, high-throughput sequencing technology has massively augmented the investigation of circRNA expression and mechanism. Employing a GEO microarray assay, we assessed the aberrant expression of circRNAs in SOR-R (SR-HepG2) versus parent-HepG2 cells. Based on our analysis, hsa_circ_0006988 was intricately linked to SOR-R within HCC. To further verify the importance of hsa_-circ_0006988 in modulating SOR-R, we conducted a loss-offunction examination by knocking down hsa_circ_0006988 in two SOR-R cell lines (SR-HepG2 and SR-Huh7). Based on our results, hsa_circ_0006988 knockdown dramatically diminished the IC50 of SOR in HCC cells.

Conclusion
In summary, based on our investigation, hsa_circ_0006988 was a novel chief modulator of the miR-15a-5p/IGF1 axis, and it induced SOR-R in HCC cells. Hsa_circ_0006988 competed with the 3′ UTR of IGF1 for interaction with miR-15a-5p, promoting SOR-R in HCC cells. Our demonstration showed that the hsa_circ_0006988/miR-15a-5p/IGF1 axis modulated SOR-R. Tis may facilitate the development of novel therapeutic approaches to overcoming SOR-R in HCC.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.