
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00217-1
10.1016/j.tranon.2024.102090
102090
Original Research
CircCOCH plays a critical role in Hepatocellular carcinoma through modulating miR-450a and activating PI3K/mTOR pathway
Jiang Weiwei a
Wang Yan b
He Wanli a
Wang Peng c
Meng Peng mengpeng0710@163.com
d⁎
Zhang Shanfeng zsf@zzu.edu.cn
e⁎
a School of Medicine, Shangqiu Institute of Technology, Shangqiu, China
b Shangqiu Medical College, Shangqiu, China
c School of nursing and health, Zhengzhou University, Zhengzhou, China
d Department of Obstetrics and Gynecology, Xijing Hospital Affiliated to the Fourth Military Medical University, Xi'an, Shaanxi, China
e School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, China
⁎ Corresponding authors. mengpeng0710@163.comzsf@zzu.edu.cn
23 8 2024
11 2024
23 8 2024
49 10209012 10 2023
29 7 2024
11 8 2024
© 2024 The Authors. Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• hsa_circ_0031431 (circCOCH) is significantly highly expressed in HCC tissues and cell lines.

• IGF2BP3 mediates the biogenesis of circCOCH in HCC.

• The essential role of circCOCH in HCC cell proliferation, migration and invasion.

• circCOCH could act as the “sponge” of miR-450a to regulate the expression of EGFR.

Hepatocellular carcinoma (HCC) is a primary liver cancer with high pathogenicity and extremely poor prognosis. The role of circular RNAs (circRNAs) in HCC carcinogenesis and progression remains to be determined. Based on the analysis of HCC-related databases, as well as the expression analysis and identification of 25 HCC patient tissues and HCC cell lines, we found that the hsa_circ_0031431 (circCOCH) is significantly highly expressed in HCC tissues and cell lines. High circCOCH expression is associated with enhanced tumor proliferation and metastasis, and knocking down circCOCH can inhibit the growth of HCC in vivo and in vitro. Mechanistic studies show that circCOCH upregulates the expression of epidermal growth factor receptor (EGFR) through sponge miR-450a, thereby activating the Phosphoinositide 3-kinases (PI3Ks) cell pathway to promote HCC proliferation and metastasis. Futhermore, we found that IGF2BP3 mediates the biogenesis of circCOCH. The present study provides innovative insights into the role of circRNAs in the etiology of HCC carcinogenesis and might serve as a new promising therapeutic target for HCC.

Keywords

CircRNAs
HCC
CircCOCH
MiR-450a
Therapeutic target
==== Body
pmcIntroduction

Primary liver cancer kills approximately 830,000 people yearly, as shown in the “2020 Global Cancer Report” [19]. Hepatocellular Carcinoma (HCC) accounts for approximately 85 % of all liver cancer cases [9]. As a cancer with high malignancy and poor prognosis, HCC seriously endangers human health. Meanwhile, chemotherapy resistance is becoming more and more common during the treatment of HCC [16]. However, effective therapeutic strategies haven't been found for HCC until now. Therefore, exploring novel drug targets for HCC has become an important research direction in reducing the morbidity and mortality of HCC.

The generation of circRNA is achieved through the back-splicing of precursor mRNA (pre-mRNA), driven by the connection of downstream 5′ to the upstream 3′ splice sites of the introns in reverse order, and forming 3′,5′-phosphodiester bonds between the two sites [1,3,12]. Notably, investigating the biogenesis mechanisms of circRNAs could help us understand how circRNAs generate and accumulate specifically in HCC progression. Most RNA-binding proteins (RBPs) are also engaged in the production of circRNA. For example, RNA processing factor heterogeneous nuclear ribonucleoprotein L (HNRNPL), enhanced the formation of dozens of circRNAs by directly binding to the flanking introns in human prostate cancers [6]. Nevertheless, how RBPs are involved in circRNA formation in HCC is rarely reported yet.

CircRNA exerts its important biological functions in gene expression and translation by functioning as a microRNA/protein “sponge” [8,11]. For example, circTMEM45A can adsorb miR-65 to weaken the inhibitory effect of miR-665 on its target mRNA IGF2, thereby promoting the occurrence of HCC [21]. Circ0097009 regulates the expression of SLC7A11 by adsorbing miR-1261 in liver cancer, thereby inhibiting HCC cell proliferation [14]. Therefore, finding the relationship between circRNA and HCC can help us elucidate the potential mechanisms of HCC pathogenesis and contribute to the current diagnosis and treatment of HCC.

EGFR overexpression occurs in 68 % of human HCC cases and is significantly associated with metastasis, lower patient survival rates, and aggressive tumors [2]. Furthermore, in HCC, approximately 50 % of cases exhibit aberrant activation of the EGFR/PI3K/AKT/mTOR pathway, which involves dysregulated activation of various cellular processes including proliferation, differentiation, autophagy, metabolism, etc. [18]. Due to the strong oncogenic role of the EGFR/PI3K/AKT/mTOR signaling pathway in HCC, inhibition of EGFR can be used for the treatment of HCC [7].

Based on all the above, we determined to study a circCOCH that is highly expressed in HCC. In this study, we found that exons 3–7 of the COCH gene form a circRNA, called circCOCH. In HCC, circCOCH promotes the expression of EGFR and promote the progression of HCC. Most importantly, we found that the Insulin like growth factor 2 mrna binding protein 3 (IGF2BP3) mediates the biogenesis of circCOCH by binding to flanking intronic sequences. Taken together, in this study, we aimed to explore the physiological functions and molecular mechanisms of circCOCH in HCC and explore its potential therapeutic ability for HCC patients.

Materials and methods

Patients and tissue samples

The HCC and corresponding adjacent normal tissue used in this study were provided by HCC patients undergoing surgical resection. The whole study was authorized by the Ethics Committee of Zhengzhou University and all research complied with the principles of the Declaration of Helsinki. The study informed consent was obtained from all patients.

Cell culture

All cell lines were purchased from Yuchi biology Co., Ltd (Shanghai, China) and characterized by DNA fingerprinting and passaged < 6 months. SNU-398 and Hep3B cells were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco, USA). All cell lines were cultured in 5 % CO2 at 37 °C and in a humidified atmosphere.

Reverse transcriptase PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from HCC cell lines and tissue specimens with Trizol reagent (Invitrogen, USA) and a total of 2 μg/sample of RNA was used for reverse transcription with moloney murine leukemia virus (M-MLV) reverse transcriptase (Takara, 639,523). Then, the procedure of reverse transcription was as follows: 42 °C for 85 min and 95 °C for 10 min. RT-PCR and qRT-PCR was performed with 2 × Hieff Canace® Gold PCR Master Mix (YEASEN, 10149ES01) and 2 × Power SYBR® Green PCR Master Mix (Invitrogen, 4,367,659), respectively. The expression levels of genes were calculated and normalized to U6, GAPDH or β-ACTIN following the formula of 2−△△Ct. All primer sequences were listed in Table S1.

Small interfering RNA (siRNA) and plasmid transfection

For circCOCH expression vector, the sequence of circCOCH was amplified from HCC cells using2 × Hieff Canace® Gold PCR Master Mix and cloned into PLO5-ciR plasmid (Geneseed, China) for circular RNA expression. For knock out circCOCH, targeting the junction region of the circCOCH sequence was cloned into PLKO.1-TRC plasmid. sh-circCOCH-1, sh-circCOCH-2, sh-scramble, PLO5-ciR, PLO5-ciR-circCOCH were co-transfected with PMD2.G and psPAX2 into HEK293T cells. The supernatant was harvested at 24 h amd 48 h after transfection and added into HCC cells. Stable HCC cell lines were selected by 1–2 μg/mL puromycin (Sigma-Aldrich) for 72 h. For dual-luciferase reporter analysis, HCC cells were co-transfected with psiCHECK2 (Promega, Madison, WI, USA) containing the circCOCH and 3′ UTR of EGFR sequences of wild-type or mutant binding site of miR-450a and miR-450a mimics/NC mimics. After 48 h transfection, the luciferase activity was assessed using the dual-luciferase reporter kit (YEASEN, 11402ES60, China) based on the manufacturer's instructions. All transfection experiments were conducted with Lipofectamine™ 3000 Transfection Reagent (Invitrogen, USA) following the manufacturer's instructions. Primer sequences were listed in Table S1. All plasmids were confirmed by Sanger sequencing.

ACTD assay

Total RNAs from HCC cells were incubated with 20 U/μL RNase R (Epicentre Biotechnologies, USA) at 37  °C for 15 min. For ACTD assay, HCC cells were treated with 1 μg /mL actinomycin D (Sigma-Aldrich, USA) against new RNA synthesis for 0, 2, 3, 4, 5, and 6 h, respectively.

Nucleocytoplasmic separation

After digesting the cells, add pre-cooled PBS to resuspend the cells. According to the cell concentration, take 107 cells for nucleocytoplasmic separation experiments. Centrifuge at 800 g for 5 min, aspirate all the supernatant, add 100 μL Cell Fractionation Buffer (10 mM HEPES pH=8.0, 10 mM KCL, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 5 % TritonX-100, 1 % Cocktail), mix gently, incubate on ice for 10 min, and then centrifuge at 500 g for 2 min. Wash the supernatant and extract RNA. Wash the precipitated part twice with 1 mL RIPA Buffer, centrifuge and discard the supernatant, and then extract RNA.

Dual-luciferase reporter gene assays

The WT or binding sequences mutant circCOCH, or 3′-UTR of EGFR and miR-450a were co-transfected to SNU-398 and Hep3B cells. After transfection for 24–48 h, Firefly and Renilla luciferase activities were assessed using the dual-luciferase reporter kit (TransGene, China) based on the manufacturer's instructions.

Protein isolation and Western blot

The target cells were lysed with protein lysis buffer containing contain and pmsf, and then the proteins were separated by SDS-PAGE gel. After transferring to polyvinylidene fluoride (PVDF) membrane (Millipore, USA), it was blocked with 5 % skim milk, incubated with specific primary antibody for 1 h at room temperature, and incubated with secondary antibody (Proteintech, USA) for 1 h. Tubulin (Proteintech, USA) were used as loading controls. The antibodies used in this study are listed in Table S2.

In vivo xenograft assay

The 4–5 weeks-old female BALB/c nude (nu/nu) mice were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China) housed under specific pathogen-free (SFP) conditions. Approximately 3–5 × 106 / mL Hep3B cells in Matrigel (Corning, USA) were injected subcutaneously into the BALB/c nude (nu/nu) mice. All animal studies were approved by the Ethics Committee of Zhengzhou University.

Bioinformatic analysis

circRNA expression data in HCC and matched non-tumor tissues were obtained from NCBI GEO database (GEO: GSE164803 and GSE97332) and annotated in circBase by R soft language. HCC mRNA and miRNA data obtained from TCGAbiolinks package, EdgeR or DEseq2 package (version 3.12.1) were used to detect differentially expressed miRNA or mRNAs. Multiple changes > 1.3 and P values < 0.05 were considered as significantly differentially expressed miRNA, circRNAs and mRNAs. The pheatmap packages in R soft language are used to display heat maps.

Statistical analysis

GraphPad Prism 8 (version 8.0, USA) was used for data analysis. Two-tailed Student's t-test and one-way ANOVA was performed to calculate statistical significance. Asterisks denote statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001) and ns indicates no significance in figures. All data were validated at least in three independent experiments and represent as mean ± standard error of the mean (SEM).

Results

circCOCH is up‑regulated in HCC cells and tissues

To identify and characterize circRNAs involved in HCC physiological functions, we obtained HCC and adjacent non-cancerous tissue microarray data sets (GSE164803 and GSE97332) from the GEO database (Table S3). Using bioinformatics analysis, we identified 6 circRNA candidates with significant differential expression in HCC (Fig. 1A). The expression levels of circCOCH in several different HCC cell lines and HCC tissues were detected by qRT-PCR, and we found that circCOCH was most highly expressed in HCC cell lines and tissues (Fig. 1B and C). The circCOCH was formed from exon 3–7 of coagulation factor c homolog (COCH) gene (Fig. 1D). To further confirm the existence of circCOCH, we designed divergent and convergent primers to amplify pre-mRNA of circCOCH. The PCR results showed that circCOCH only existed in cDNA (Fig. 1E). In addition, random primers and oligo dT primers were used to further confirm the circular characteristics of circCOCH. Compared with random primers, the relative expression level of circCOCH was significantly reduced when using oligo dT primers, while the expression of COCH mRNA was not changed (Fig. 1F), indicating that circCOCH does not have a poly (A) tail. Subsequently, the subcellular localization of circCOCH was detected in HCC cells by nucleocytoplasmic fractionation, and we found that circCOCH mainly existed in the cytoplasm of HCC cells (Fig. 1G). To further evaluate the stability of circCOCH, we treated HCC cells with actinomycin D and found that circCOCH was more stable than linear COCH (Fig. 1H).Fig. 1 CircCOCH was up-regulated in HCC. (A) GEO database datasets (GSE97332 and GSE164803) were analyses for differentially expressed circRNAs. The top 6 up or down regulated circRNAs in HCC tissues were at list. (B-C) Relative expression level of 6 circRNAs in HCC tissues and cell lines. (D) Schematic illustration of circCOCH conformation. The exon 3–7 of COCH mRNA formed circCOCH through back splicing. Sanger sequencing of the back-splicing site of circCOCH was shown below. (E) PCR products of the circular nature of circCOCH using divergent and convergent primers in HCC cell lines. (F) qRT-PCR of circCOCH in SNU-398 and Hep3B cell lines by using randon and oligo primers. (G) The location of circCOCH was determined by Nucleo-cytoplasmic separation, U6 and GAPDH were used as nuclear and cytoplasmic markers, respectively. (H) Half-life of relative circCOCH and COCH expression in SNU-398 and Hep3B cells treated with ACTD. P values were calculated using two-tailed Student's t-tests.

Fig. 1

IGF2BP3 regulates circCOCH formation by binding to flanking intron RNA

circRNA is derived from pre-mRNA and is produced by back-splicing. The biogenesis of cricRNA mainly depends on cis-regulatory elements and trans-acting factors [12]. We sought to further identify the molecular mechanisms driving circCOCH upregulation in HCC. Previous studies have shown that 103 RBPs may be involved in the biogenesis of circRNA [11]. To further investigate whether these RBPs may be involved in the formation of circCOCH, by analyzing TCGA data to examine the expression of these RBPs in HCC, we found that 3 RBPs were dysregulated in HCC (Fig. 2A). We further confirmed this result in HCC tissues by qRT-PCR (Fig. 2B). Afterwards, we knocked down these three RBPs in HCC (Fig. 2C). The results showed that knockdown of IGF2BP3 attenuated the expression level of circCOCH, but not linear COCH (Fig. 2D and E). However, it is currently unclear how IGF2BP3 regulates the production of circRNA. In order to further study whether IGF2BP3 may be involved in the formation of circCOCH, we conducted RIP-qPCR experiments to further verify the binding region of IGF2BP3 and circCOCH (Fig. 2F). We found that the upstream and downstream flanking sequences of circCOCH precursor mRNA were significantly enriched in IGF2BP3 (Fig. 2G). This indicates that IGF2BP3 is involved in regulating the production of circCOCH (Fig. 2G). Furthermore, we observed that IGF2BP3 was negatively correlated with circCOCH in HCC tissues. These results indicate that IGF2BP3 regulates the formation of circCOCH by binding to the flanking region of COCH pre-mRNA in HCC (Fig. 2H).Fig. 2 IGF2BP3 regulated the generation of circCOCH. (A) The 3 of 103 RBPs are differentially expressed in HCC of TCGA. (B) The relative expression level of 3 RBPs in HCC tissues (n = 10). (C-E) After knockdown these RBPs in SNU-398 and Hep3B cells, the expression levels of these RBPs, circCOCH and COCH were detected by qRT-PCR. (F) Schematic diagram of the binding region between IGF2BP3 and circCOCH pre-mRNA. (G) RIP-qPCR of flanking intronic sequences of circCOCH binding with IGF2BP3. (H) Correlation analysis was performed after detecting IGF2BP3 and circCOCH through qRT-PCR. P values were calculated using two-tailed Student's t-tests.

Fig. 2

circCOCH is critical for HCC cell proliferation, migration and invasion

To explore the potential biological function of circCOCH in HCC, we designed 2 shRNAs targeting the circCOCH connection site (Fig. 3A). The sh-circCOCH-1 and sh-circCOCH-2 can silence circCOCH in HCC cells without affecting the expression level of COCH and were therefore used for further research (Fig. 3A). Knockdown of circCOCH significantly inhibited the proliferation ability of HCC cells (Fig. 3A and B). Western blotting analysis further confirmed that silencing circCOCH would reduce the expression of cell cycle-related proteins, thereby blocking the cell cycle progression of HCC cells (Fig. 3C). Furthermore, we found that silencing circCOCH inhibited the migration and invasion abilities of HCC cells (Fig. 3D). Next, we established an HCC xenograft model to determine the effect of silencing circCOCH on tumor growth. The results showed that the tumor volume and weight of the circCOCH knockdown group were significantly lower than those of the control group (Fig. 3E and F). These results are consistent with the results of in vitro experiments, indicating that knocking down circCOCH can inhibit the occurrence of HCC.Fig. 3 KD circCOCH inhibits tumor growth. (A) Up: After designing a knockdown vector based on the circCOCH back-splicing site, an SNU-398 and Hep3B cell lines with stable knockdown of circCOCH was constructed, and the relative expression level of circCOCH was detected by qRT-PCR. Down: CCK8 assays for KD circCOCH SNU-398 and Hep3B cell lines. (B) Colony formation assays for KD circCOCH SNU-398 and Hep3B cell lines. (C)The capacity of cell migration and invasion in circCOCH KD SNU-398 and Hep3B cell lines were estimated by transwell assay. (D) Immunoblot (IB) of BCL2 and CDK4 in circCOCH KD SNU-398 and Hep3B cell lines. TUBULIN was used as loading controls. (E) Female BALB/c nude mice (n = 5 per group) were injected with sh-circCOCH or sh-scramble Hep3B cell lines (scale bar, 1 cm). The xenograft tumor weights were counted after euthanizing the animals. P values were calculated using two-tailed Student's t-tests.

Fig. 3

The expression of circCOCH is negatively correlated with the expression of miR-450a

Cytoplasm-localized circRNAs may regulate the expression of genes downstream of miRNAs by enriching miRNAs [8]. As the main component of RNA-induced silencing complex (RISC), Argonaute 2 (Ago2) can target and degrade mRNA by binding to miRNAs [8]. Therefore, if circCOCH can bind to miRNAs, both miRNAs and circCOCH should be enriched by Ago2. We performed RIP experiments of Ago2 in SNU-398 and Hep3B cell lines to verify whether circCOCH can bind to Ago2. The results showed that circCOCH can be significantly enriched in Ago2 (Fig. 4A). This suggests that circCOCH may have the function of enriching miRNAs. In order to further analyze whether circCOCH can bind to miRNAs. By using the bioinformatics website of circular RNA, we found that miR-513a, miR-5010, miR-760, miR-3677 and miR-450a is a potential target of circCOCH (Fig. 4B). Next, we used dual-luciferase reporter assay to detect whether circCOCH can directly bind to these miRNAs.The results of the dual-luciferase reporter gene showed that in HCC cells, only miR-450a could reduce the relative activity of Renilla luciferase by binding to circCOCH in HCC cell lines (Fig. 4C-G). In addition, we measured the expression of miR-450a in HCC tissues, and the results of qRT-PCR showed that miR-450a was significantly decreased in HCC tissues (Fig. 4H). In addition, the expression level of miR-450a was negatively correlated with the expression level of circCOCH (Fig. 4I). Taken together, these results indicate that circCOCH is negatively correlated with the expression of miR-450a in HCC.Fig. 4 CircCOCH “sponge” miR-450a in HCC. (A) RIP assay with an anti-Ago2 antibody in SNU-398 and Hep3B cell lines was used to detect the mRNA levels of circCOCH. GAPDH was used as and negative control. (B) Prediction of potential circCOCH binding with target miRNAs by miRnada and TargetScan. (C-G) The relative luciferase activity of Luc/Rluc was tested in wild-type and mutant circCOCH in SNU-398 and Hep3B cell lines transfected with NC or miR-513a/miR-5010/miR-760/miR-3766/miR-450a mimics. (H) Relative expression level of miR-450a in HCC tissues. (I) Correlation analysis was performed after detecting miR-450a and circCOCH through qRT-PCR. P values were calculated using two-tailed Student's t-tests.

Fig. 4

CircCOCH regulates miR-450a to affect HCC cell proliferation and invasion

To clarify the effects of circCOCH and miR-450a on HCC cell phenotypes, we performed some rescue experiments in circCOCH-overexpressing HCC cell lines by added inhibitors of miR-450a (Fig. 5A). Inhibitors of miR-450a were transfected into cell lines overexpressing circRNA. We found that overexpression of circCOCH greatly promoted the viability of HCC cells, while overexpression of miR-450a impaired this effect through CCK-8 and colony formation assays (Fig. 5B and C). The westering blotting experiments further showed that overexpression of miR-450a basically completely compensated for the malignant phenotype of HCC caused by overexpression of circCOCH (Fig. 5D). Regarding the metastatic ability of HCC cells, circCOCH OE increased the migration and invasion abilities of HCC cells, while overexpression of mir compensated for this phenotype (Fig. 5E).Fig. 5 The oncogenic effect of circCOCH in HCC cells relies on miR-450a. (A) The relative expression level of circCOCH or miR-450a in SNU-398 and Hep3B cell lines with OE circCOCH was detected by qRT-PCR. (B-C) Cell proliferation assay including B) CCK8 analysis, C) colony formation assays for SNU-398 and Hep3B cell lines with co-transfecting with miR-450a mimics and circCOCH. (D) Immunoblot (IB) of BCL2 and CDK4 in SNU-398 and Hep3B cell lines with co-transfecting with miR-450a mimics and circCOCH. TUBULIN was used as loading controls. (E) The capacity of cell migration and invasion in SNU-398 and Hep3B cell lines with co-transfecting with miR-450a mimics and circCOCH were estimated by transwell assay. P values were calculated using one-way ANOVA with Duncan's post hoc test.

Fig. 5

Overall, these data indicate that circCOCH regulates miR-450a to affect HCC cell proliferation, migration, invasion, and apoptosis.

EGFR is a direct target of miR-450a

Previous studies have shown that miR-450a can play a role in various cancers by regulating EGFR, ATP5B, TIMMDC1, among others [13,15]. In our preliminary experiments, we examined the changes in EGFR, ATP5B, TIMMDC1, and other genes in HCC cells transfected with miR-450a mimics. Interestingly, only EGFR showed significant changes in HCC cells transfected with miR-450a mimics (Fig. 6A). Therefore, we decided to further investigate EGFR. To better understand the relationship between miR-450a and EGFR in HCC, we constructed a dual-luciferase vector with a mutated EGFR 3′-UTR region to verify the direct binding of EGFR mRNA to miR-450a (Fig. 6B). The results of the dual-luciferase reporter gene showed that in SNU-398 and Hep3B cells, miR-450a can reduce the relative activity of Renilla luciferase by binding to the 3′-UTR region of EGFR. When the binding site is mutated, the relative activity of Renilla luciferase remains unchanged (Fig. 6B). This indicates that miR-450a can directly bind to the 3′-UTR region of EGFR in HCC cells. We also tested the expression of EGFR in HCC tissues, and the results of western blotting showed that the protein levels of EGFR in HCC tissues were higher than that in normal groups (Fig. 6C). Furthermore, the results of western blotting also showed that knockdown of circCOCH and overexpression of miR-450a could inhibit the expression of EGFR protein levels in two types of HCC cells (Fig. 6D and E).Fig. 6 EGFR is the direct target of miR-450a and circCOCH in HCC. (A) The relative expression levels of EGFR, ATB5B and TIMMDC1 in SNU-398 and Hep3B cell lines transfected with NC or miR-450a mimics. (B) Up: Schematic representation of the binding sites between miR-450a and EGFR 3′UTR; Down: The relative luciferase activity of Luc/Rluc was tested in wild-type and mutant EGFR in SNU-398 and Hep3B cell lines transfected with NC or miR-450a mimics. (C) The protein levels of EGFR were detected by western blotting. TUBULIN was used as loading controls. (D) The relative expression levels of EGFR in SNU-398 and Hep3B cell lines transfected with sh-circCOCH and sh-scramble. (E) The protein levels of EGFR in SNU-398 and Hep3B cell lines transfected with NC, sh-scramble, sh-circCOCH, NC- mimics or miR-450a mimics. P values were calculated using two-tailed Student's t-tests.

Fig. 6

The above results indicate that circCOCH can regulate EGFR expression through miR-450a in HCC cells.

MiR-450a regulates EGFR signaling in HCC cells

Previous studies have shown that miR-450a regulates the PI3K/AKT/mTOR signaling pathway by targeting the 3′UTR of EGFR in glioma cells [13]. Our results showed that miR-450a mimic significantly inhibited the levels of phosphorylated PI3K (p-PI3K), AKT (p-AKT), mTOR (p-mTOR) and ERK1/2 (p- ERK1/2) (Fig. 7A). In addition, the knockdown of circCOCH significantly inhibited the PI3K/AKT/mTOR signaling pathway (Fig. 7A). All these results indicated that circCOCH regulates the expression of EGFR through miR-450a, thereby regulating the EGFR-induced PI3K/AKT/mTOR signaling pathway in HCC cells.Fig. 7 MiR-450a regulates EGFR signaling in HCC cells. (A) The protein levels of mTOR, p-mTOR, AKT, p-AKT, ERK1/2, p-ERK1/2 in SNU-398 and Hep3B cell lines transfected with NC, sh-scramble, sh-circCOCH, NC- mimics or miR-450a mimics. TUBULIN was used as loading controls.

Fig. 7

Discussion

An increasing number of studies have shown that circRNAs are involved in a wide range of biological processes, including the development of cancer [9]. The role of circRNAs in HCC carcinogenesis and progression remains to be determined. In this study, we found that exons 5–9 of the COCH gene form a circRNA, called circCOCH. In HCC, circCOCH expression is upregulated. Mechanistic studies show that in HCC, circCOCH promotes the expression of EGFR by adsorbing miR-450a, thereby activating the mTOR signaling pathway. Most importantly, we found that the RNA-binding protein IGF2BP3 regulates circCOCH production by binding to flanking intronic sequences. Taken together, our results indicate that circCOCH is involved in the progression of HCC by regulating miR-450a.

CircRNA is produced by back-splicing of pre-mRNA [12]. A large number of studies have shown that some RBPs, such as Quaking (QKI), Heterogeneous nuclear ribonucleoprotein L (HNRNPL), Eukaryotic translation initiation factor 4A3 (EIF4A3), etc., bind to specific motifs on the flanking introns of pre-mRNA to regulate the back splicing of circRNA [4,6,22]. Previous studies have shown that 103 RBPs may play a role in the formation of circRNA [11]. To this end, we analyzed the expression levels of these 103 RBPs in HCC inferred from TCGA data, and the results showed that 3 RBPs were dysregulated in HCC. Next, after knocking down these RBPs, we found that only IGF2BP3 could regulate the expression of circCOCH but did not affect the expression level of COCH. This suggests that IGF2BP3 may be involved in circCOCH biogenesis. Further RIP experiments showed that IGF2BP3 regulates the production of circCOCH by binding to the flanking intronic region of the pre-mRNA of circCOCH. However, we have not yet fully resolved whether IGF2BP3 in HCC specifically regulates the formation of circCOCH. This requires further research.

Some circRNAs have been well characterized to act as miRNA sponges in tumorigenesis [5,10,17,20]. To better understand the potential role of circCOCH in HCC, we confirmed that circCOCH is mainly located in the cytoplasm, suggesting that circCOCH may act as a miRNA “sponge” in circCOCH. This prompted us to further explore the interaction between circCOCH and its related miRNAs in HCC. Through a series of experiments, we found that circCOCH can promote the growth of HCC by adsorbing miR-450a. Studies have shown that miR-450a can inhibit the progression of various tumors [13,15]. To our knowledge, the function of miR-450a in HCC has been rarely reported. In this study, we found that circCOCH acts as a regulator of sponge miR-450a, which in turn regulates EGFR degradation through the RISC. Research shows that EGFR plays a role in cancer by activating PI3K signaling [13]. In this study, we found that knocking down circCOCH or overexpressing miR-450a can inhibit PI3K signaling by inhibiting EGFR gene expression, thereby inhibiting the growth of HCC. Although our results demonstrate that knocking down circCOCH has an anti-cancer effect in BALB/c nude mice, there is still a lack of suitable HCC lung metastasis models to further confirm the metastatic outcomes induced by circCOCH under physiological conditions. This requires us to exert more effort in future studies of model construction. Furthermore, some studies suggest that circRNA has additional biological functions, such as RBP sponges and encoding protein functions [3]. In future research, we need to further demonstrate whether circCOCH affects the progression of HCC through other biological functions.

The high stability of circRNA makes it a promising candidate as a biomarker. Furthermore, we found that circCOCH expression in HCC tissues was significantly higher than in normal tissues, suggesting its potential as a biomarker for HCC patients. However, our study lacks more clinical samples for statistical analysis of patient survival. Further research is needed to confirm whether circCOCH can be used as a prognostic factor.

In summary, in this study, we demonstrated that inhibiting the expression of circCOCH can inhibit the malignant progression of HCC. circCOCH promotes the malignant progression of HCC by inhibiting the miR-450a/EGFR/PI3K axis. Importantly, we also demonstrated that IGF2BP3 promotes the generation of circCOCH by binding to the pre-mRNA of circCOCH. These findings have important implications for our understanding of circRNA formation in HCC and highlight the importance of studying the complex circRNA-miRNA regulatory gene network in HCC progression and treatment.

Availability of data and materials

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate

The study was approved by the Ethical Review Committee of the Zhengzhou University. All the patients had signed the informed consent.

Funding

This study was supported by grants from the Henan Province Key Research and Promotion Special Project (No. 202102310057/192102310417 ).

CRediT authorship contribution statement

Weiwei Jiang: Writing – review & editing, Writing – original draft, Methodology, Formal analysis, Data curation. Yan Wang: Software. Wanli He: Validation. Peng Wang: Data curation. Peng Meng: Software, Funding acquisition. Shanfeng Zhang: Writing – review & editing, Supervision, Resources, Funding acquisition.

Declaration of competing interest

All authors declare that they have no competing interests.

Appendix Supplementary materials

Image, application 1

Image, application 2

Acknowledgements

Not applicable.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102090.
==== Refs
References

1 Ashwal-Fluss R. Meyer M. Pamudurti N.R. Ivanov A. Bartok O. Hanan M. Evantal N. Memczak S. Rajewsky N. Kadener S. circRNA biogenesis competes with pre-mRNA splicing Mol. Cell 56 2014 55 66 25242144
2 Bang J. Jun M. Lee S. Moon H. Ro S.W. Targeting EGFR/PI3K/AKT/mTOR signaling in hepatocellular carcinoma Pharmaceutics 15 2023
3 Chen L.L. The expanding regulatory mechanisms and cellular functions of circular RNAs Nat. Rev. Mol. Cell Biol. 21 2020 475 490 32366901
4 Conn S.J. Pillman K.A. Toubia J. Conn V.M. Salmanidis M. Phillips C.A. Roslan S. Schreiber A.W. Gregory P.A. Goodall G.J. The RNA binding protein quaking regulates formation of circRNAs Cell 160 2015 1125 1134 25768908
5 Dou D. Ren X. Han M. Xu X. Ge X. Gu Y. Wang X. Zhao S. CircUBE2D2 (hsa_circ_0005728) promotes cell proliferation, metastasis and chemoresistance in triple-negative breast cancer by regulating miR-512-3p/CDCA3 axis Cancer Cell Int. 20 2020 454 32944002
6 Fei T. Chen Y. Xiao T. Li W. Cato L. Zhang P. Cotter M.B. Bowden M. Lis R.T. Zhao S.G. Genome-wide CRISPR screen identifies HNRNPL as a prostate cancer dependency regulating RNA splicing Proc. Natl. Acad. Sci. USA 114 2017 E5207 E5215 28611215
7 Fuchs B.C. Hoshida Y. Fujii T. Wei L. Yamada S. Lauwers G.Y. McGinn C.M. DePeralta D.K. Chen X. Kuroda T. Epidermal growth factor receptor inhibition attenuates liver fibrosis and development of hepatocellular carcinoma Hepatology 59 2014 1577 1590 24677197
8 Hansen T.B. Wiklund E.D. Bramsen J.B. Villadsen S.B. Statham A.L. Clark S.J. Kjems J. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA EMBO J. 30 2011 4414 4422 21964070
9 Huang A. Yang X.R. Chung W.Y. Dennison A.R. Zhou J. Targeted therapy for hepatocellular carcinoma Signal Transduct Target Ther 5 2020 146 32782275
10 Kong Y. Yang L. Wei W. Lyu N. Zou Y. Gao G. Ou X. Xie X. Tang H. CircPLK1 sponges miR-296-5p to facilitate triple-negative breast cancer progression Epigenomics 11 2019 1163 1176 31337246
11 Li X. Liu C.X. Xue W. Zhang Y. Jiang S. Yin Q.F. Wei J. Yao R.W. Yang L. Chen L.L. Coordinated circRNA Biogenesis and Function with NF90/NF110 in Viral Infection Mol. Cell 67 2017 214 227 e217 28625552
12 Liu C.X. Chen L.L. Circular RNAs: characterization, cellular roles, and applications Cell 185 2022 2016 2034 35584701
13 Liu Y. Yang L. Liao F. Wang W. Wang Z.F. MiR-450a-5p strengthens the drug sensitivity of gefitinib in glioma chemotherapy via regulating autophagy by targeting EGFR Oncogene 39 2020 6190 6202 32820249
14 Lyu N. Zeng Y. Kong Y. Chen Q. Deng H. Ou S. Bai Y. Tang H. Wang X. Zhao M. Ferroptosis is involved in the progression of hepatocellular carcinoma through the circ0097009/miR-1261/SLC7A11 axis Ann. Transl. Med. 9 2021 675 33987373
15 Muys B.R. Sousa J.F. Placa J.R. de Araujo L.F. Sarshad A.A. Anastasakis D.G. Wang X. Li X.L. de Molfetta G.A. Ramao A. miR-450a acts as a tumor suppressor in ovarian cancer by regulating energy metabolism Cancer Res. 79 2019 3294 3305 31101765
16 Ogunwobi O.O. Harricharran T. Huaman J. Galuza A. Odumuwagun O. Tan Y. Ma G.X. Nguyen M.T. Mechanisms of hepatocellular carcinoma progression World J. Gastroenterol. 25 2019 2279 2293 31148900
17 Sang M. Meng L. Liu S. Ding P. Chang S. Ju Y. Liu F. Gu L. Lian Y. Geng C. Circular RNA ciRS-7 maintains metastatic phenotypes as a ceRNA of miR-1299 to Target MMPs Mol. Cancer Res. 16 2018 1665 1675 30072582
18 Sun E.J. Wankell M. Palamuthusingam P. McFarlane C. Hebbard L. Targeting the PI3K/Akt/mTOR Pathway in Hepatocellular Carcinoma Biomedicines 9 2021
19 Sung H. Ferlay J. Siegel R.L. Laversanne M. Soerjomataram I. Jemal A. Bray F. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries CA Cancer J. Clin. 71 2021 209 249 33538338
20 Xu J.Z. Shao C.C. Wang X.J. Zhao X. Chen J.Q. Ouyang Y.X. Feng J. Zhang F. Huang W.H. Ying Q. circTADA2As suppress breast cancer progression and metastasis via targeting miR-203a-3p/SOCS3 axis Cell Death. Dis. 10 2019 175 30787278
21 Zhang T. Jing B. Bai Y. Zhang Y. Yu H Circular RNA circTMEM45A acts as the sponge of MicroRNA-665 to promote hepatocellular carcinoma progression Mol. Ther. Nucleic. Acids. 22 2020 285 297 33230434
22 Zheng X. Huang M. Xing L. Yang R. Wang X. Jiang R. Zhang L. Chen J The circRNA circSEPT9 mediated by E2F1 and EIF4A3 facilitates the carcinogenesis and development of triple-negative breast cancer Mol. Cancer 19 2020 73 32264877
