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. 2023 Apr 26;46(5):1333–1350. doi: 10.1007/s13402-023-00814-9

Aberrant expression of circular RNA DHPR facilitates tumor growth and metastasis by regulating the RASGEF1B/RAS/MAPK axis in hepatocellular carcinoma

Zeyi Guo 1,3, Qingyu Xie 1,3,5, Yanping Wu 1,3, Haiyu Mo 2, Jiajun Zhang 1,3, Guolin He 1,3, Zhongzhe Li 1,3, Luxiang Gan 1,3, Lei Feng 1,3, Ting Li 1,3, Yi Wang 1,3, Yu Fu 1,3, Lei Cai 1,3, Shao Li 1,3, Chao Yu 4, Yi Gao 1,3,✉, Mingxin Pan 1,3,✉, Shunjun Fu 1,3,✉
PMCID: PMC12974678  PMID: 37099250

Abstract

Background

Circular RNAs (circRNAs) are noncoding RNAs. Accumulating evidence suggests that circRNAs play a critical role in human biological processes, especially tumorigenesis, and development. However, the exact mechanisms of action of circRNAs in hepatocellular carcinoma (HCC) remain unclear.

Methods

Bioinformatic tools and RT-qPCR were used to identify the role of circDHPR, a circRNA derived from the dihydropteridine reductase (DHPR) locus, in HCC and para-carcinoma tissues. Kaplan–Meier analysis and the Cox proportional hazard model were used to analyze the correlation between circDHPR expression and patient prognosis. Lentiviral vectors were used to establish stable circDHPR-overexpressing cells. In vitro and in vivo studies have shown that tumor proliferation and metastasis are affected by circDHPR. Mechanistic assays, including Western blotting, immunohistochemistry, dual-luciferase reporter assays, fluorescence in situ hybridization, and RNA immunoprecipitation, have demonstrated the molecular mechanism underlying circDHPR.

Results

CircDHPR was downregulated in HCC, and low circDHPR expression was associated with poor overall survival and disease-free survival rates. CircDHPR overexpression inhibits tumor growth and metastasis in vitro and in vivo. Further systematic studies revealed that circDHPR binds to miR-3194-5p, an upstream regulator of RASGEF1B. This endogenous competition suppresses the silencing effect of miR-3194-5p. We confirmed that circDHPR overexpression inhibited HCC growth and metastasis by sponging miR-3194-5p to upregulate the expression of RASGEF1B, which is regarded as a suppressor of the Ras/MAPK signaling pathway.

Conclusions

Aberrant circDHPR expression leads to uncontrolled cell proliferation, tumorigenesis, and metastasis. CircDHPR may serve as a biomarker and therapeutic target for HCC.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-023-00814-9.

Keywords: Circular RNA, Hepatocellular carcinoma, RASGEF1B, DHPR, Ras/MAPK

Introduction

Hepatocellular carcinoma (HCC) is the sixth most prevalent malignant tumor and the third most common cause of cancer-related deaths worldwide [1]. In China in 2022, there were approximately 431,383 new cases and 412,216 deaths [2]. Hepatocellular carcinoma is one of the most fatal cancers in China because of the high prevalence of hepatitis B virus (HBV) infection, which usually causes end-stage liver cirrhosis [3]. Despite the development of standard systemic therapy, including surgery, chemotherapy, and radiotherapy, patients with HCC still have poor prognoses because of the high frequency of metastasis and tumor recurrence [4]. Therefore, understanding the pathogenic processes and regulatory mechanisms underlying HCC development is crucial.

Circular RNAs (circRNAs), microRNAs (miRNAs), and long noncoding RNAs (lncRNAs) are types of noncoding RNAs, and many researchers have suggested that they play a substantial role in human biological processes [5]. CircRNAs are characterized by back-splicing, which is the connection of a 5’ splice site with the 3’ splice site of a parent pre-mRNA [6]. CircRNAs function as sponges for miRNAs or proteins, and modulate transcription and translation [7]. Increasing evidence has indicated that most circRNAs are involved in cancer progression and anticancer therapy. For example, circNDUFB2 destabilizes IGF2BPs to inhibit the progression of non-small cell lung cancer [8]. Similarly, in HCC, the circular RNA MAT2B acts as a competing endogenous RNA (ceRNA) to sponge miR-338-3p and promotes glycolysis and malignancy in HCC under hypoxic stress by upregulating PKM2 [9]. Circular RNA circβ-catenin encodes a small peptide that promotes liver cancer cells by activating the Wnt pathway [10]. Accumulating evidence suggests that circRNAs are not only byproducts of the transcription process but also have many functions. Nevertheless, only a small group of circRNAs have been investigated in HCC.

In this study, we used a bioinformatics tool to analyze microarray data from the Gene Expression Omnibus (GEO) database and found that circDHPR was downregulated in HCC. We found that circDHPR suppressed HCC progression in vitro and in vivo. Further systematic studies revealed that circDHPR overexpression inhibits the Ras/MAPK signaling pathway by sponging miR-3194-5p. We also found that circDHPR overexpression upregulated Ras-GEF domain-containing family member 1B (RASGEF1B) in HCC. Our findings revealed that circDHPR may be a new therapeutic target for HCC.

Materials and methods

Gene expression omnibus analysis

We downloaded microarray data (GSE94508, GSE97332 and GSE78520) from the GEO database (http://ncbi.nlm.nih.gov/geo/). The “limma” package in R software was used to analyze the differential expression circRNAs in each dataset, and then we took the intersection for narrowing the range of up or down regulated circRNAs between HCC and adjacent tissues. P < 0.05 and fold change > 2 or fold change < 2 were selected as the thresholds for significantly differentially expressed circRNAs.

Clinical HCC specimens

Ten pairs of HCC and adjacent normal tissues were obtained from Southern Medical University Zhujiang Hospital in September 2020. Eighty HCC and 54 normal liver tissue samples were obtained from the Sun Yat-sen University Cancer Center between January 2005 and December 2010. The exclusion criteria were as follows: (1) participants younger than 18 years, older than 75 years, or without full civil capacity; (2) patients who received preoperative anticancer therapies; and (3) the patient was diagnosed with a malignancy in another organ. This study was approved by the ethics committees of Zhujiang Hospital and Sun Yat-sen University Cancer Center. Informed consent was obtained from all the patients. The tumor stages were determined using the tumor-node-metastasis (TNM) system of the 2010 International Union Against Cancer by the American Joint Committee [11]. The histological grade of tumors was determined by the Edmondson Steiner grading system [12].

Cell lines

HCC cell lines (HCCLM3, MHCC97H, Huh7, SMMC7721, and Bel-7402) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). HCC cell lines (Hep3B and HepG2) and the normal human liver cell line HL-7702 were purchased from the American Type Culture Collection (ATCC). Human 293T cells were purchased from ATCC. All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; Basalmedia, China) plus 10% fetal bovine serum (FBS; Gibco, USA) and maintained at 37 ℃ with 5% CO2.

Total RNA extraction, reverse transcription PCR and quantitative real-time PCR

Total RNA from cell lines and tissues was extracted using TRIzol regent (Invitrogen, USA) according to the protocol and then stored at -80 ℃. The quality of the RNA was verified by a Nanodrop2000 (Ther‎mo Fisher, USA). The quality control (QC) criteria were as follows: OD260/280 > 1.9, OD230/260 > 1.8 and concentration > 200 ng/µL. Complementary DNA (cDNA) for circRNA and mRNA was reverse transcribed with the PrimeScript™ RT reagent Kit (Takara, China), and the miRNA 1st strand cDNA synthesis kit (AGBIO, China) for miRNAs. qPCR assays were performed using the SYBR Green Premix Pro Taq HS qPCR Kit II (AGBIO, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize circRNAs and mRNAs. A Small nuclear (U6) was used to normalize the miRNA levels. The expression level of RNAs was determined by the 2−ΔΔCt method. All the primer sequences used in this study are listed in Table S1.

Plasmid construction, lentiviral packaging, and cell transfection

The lentiviral vector overexpressing circDHPR (OE-circDHPR) and the corresponding negative control (vector) were synthesized by Hanheng Biotechnology (Shanghai, China). The structure is shown in Fig S2. The inhibitor and mimic of miR-3194-5p were obtained from Tsingke Biotechnology (Guangzhou, China). The sequences of OE-circDHPR, vector, miR-3194-5p-mimic, and miR-3194-5p-inhibitor are shown in Table S2. Entranster-R4000 (Engreen, Beijing, China) was used to transfect the miRNAs, following the manufacturer’s instructions. Lentivirus cells was transfected at a multiplicity of infection of 20. Seventy-two hours later, cells were cultured in DMEM with 3 µg/mL puromycin (Beyotime, Shanghai, China) for Hep3B cells and 1.5 µg/mL puromycin for SMMC7721 cells for 2 weeks. Transfection efficacy was verified by qPCR.

Western blotting

Sodium dodecyl sulfate lysis buffer (Beyotime, China) containing proteinase and phosphatase inhibitors (Sigma-Aldrich, NY, USA) was used to extract the proteins. Proteins were separated on 8%, 10%, or 12% polyacrylamide gels based on their molecular masses and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). Subsequently, 5% bovine serum albumin (BSA) or skim milk was used to block nonspecific binding for 2 h. The membranes were incubated with antibodies overnight at 4 ℃. The next day, the membranes were washed three times with 0.05% TBS plus 0.1% Tween-20 for 5 min each, incubated with secondary antibodies (Horseradish peroxidase-labeled goat anti-rabbit IgG and goat anti-mouse IgG) for 1.5 h at room temperature and again washed three times with 0.05% TBS plus 0.1% Tween-20 for 5 min each. ECL chemiluminescence kits (Invitrogen) were used to detect the protein signals. The antibodies used in this study are listed in Supplementary Table S3.

Sanger sequencing

Sanger sequencing was used to verify the full-length circDHPR. Total RNA was extracted from Hep3B cells and reverse-transcribed into cDNA. Divergent primers (Tsingke, Guangzhou, China) were designed to amplify the back-splicing sequence of circDHPR. PCR products was subjected to Sanger sequencing (Tsingke) to confirm the head-to-tail junction.

RNase R and actinomycin D treatment

Transcription was inhibited by Actinomycin D (2 mg/ml). Dimethyl sulfoxide (DMSO) (Sigma-Aldrich) was used as the negative control. Total RNA (3 µg) from Hep3B and SMMC7721 cells were incubated with 2 U/µg RNase R (Geneseed, Guangzhou, China) for 15 min at 37 °C. After treatment, qPCR was performed to detect the expression levels of circDHPR, using GAPDH as the reference gene.

Fluorescence in situ hybridization and immunohistochemistry

Probes targeting circDHPR and miR-3194-5p for fluorescence in situ hybridization (FISH) were purchased from RiboBio (Guangzhou, China). The sequences of the RNA FISH probes are listed in Supplementary Table S2. Hep3B and SMMC7721 cells were incubated with the indicated probes at 4 ℃ overnight, and images were taken using a fluorescence microscope (Carl Zeiss Microscopy GmbH, Jena, Germany).

Immunohistochemistry (IHC) was performed according to a standard protocol. Briefly, specimens were deparaffinized using dimethylbenzene and alcohol. After washing with phosphate-buffered saline (PBS), the specimens were subjected to antigen retrieval using 2% sodium citrate, and endogenous enzymes were blocked using bovine serum albumin for 2 h. The specimens were incubated with antibodies at 4 ℃ overnight. The specimens were stained and screened using Pannoramic MIDI (3D HISTECH, Budapest, Magyar Orszag). The intensity of immunohistochemistry staining was scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong (Fig. S1). Each sample was scored in a blinded manner by two investigators who did not have any clinical or pathological information regarding the origin of the samples. Five fields were randomly selected for further statistical analysis.

Cell proliferation and colony formation assays

Hep3B and SMMC7721 cells were transfected with circDHPR overexpression (OE-circDHPR) or control lentiviral vectors and seeded in 96-well plates at a density of 3 × 103 cells/well for SMMC7721 cells and 1 × 103 cells/well for Hep3B cells. The Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was added to the cells and incubated for 2.5 h in the dark. Cell proliferation ability at the indicated times was determined by measuring the absorbance at 450 nm using Multiskan™ FC (Thermo Scientific, MA, USA). Colony forming ability was determined by seeding Hep3B and SMMC7721 cells (1 × 103 cells/well) in 6-well plates and incubating them for 10 days. The colonies formed were fixed using 4% paraformaldehyde and stained with a 0.1% crystal violet solution. The number of cells in each 6-wells was counted for statistical analysis. CCK-8 and colony formation assays are presented as the mean ± SD of at least three independent experiments.

Transwell and wound healing assay

For the migration assay, 3 × 104 targeted cells were seeded into the upper chamber with 200 µL serum-free DMEM, and 800 µl of DMEM with 15% fetal bovine serum was added to the lower chamber. For the invasion assay, Matrigel (Corning, NY, USA) was used to precoat the polycarbonate inserts for 30 min before the experiment to simulate the extracellular matrix (ECM). Target cells (5 × 104) were seeded in the upper chamber as described previously [13]. After 24 h, cells remaining in the upper polycarbonate layer were removed using a cotton swab. The cells in the lower polycarbonate layer were washed three times with PBS and fixed with methanol for 20 min. Cells were stained with 0.1% crystal violet solution. The number of crystal violet-stained cells was counted in three random fields at 100× magnification. For the wound healing assay, 1 × 106 cells were seeded in 6-well plates. The next day, a 1 mL pipette tip was used to create a linear wound. Cells migration across the wound was observed at 0 and 48 h after scraping. Three random fields were selected and photographed at 40× magnification for statistical analysis. Transwell and wound healing assays are presented as the mean ± SD of at least three independent experiments.

Prediction of miRNAs that bind to circRNAs and the downstream genes of miRNAs

StarbaseV3.0 [14], CSCD [15], and CircInteratome [16] databases were used to identify miRNAs that bind to circDHPR. TargetScan Human 8.0 [17], miRwalk [18], and StarbaseV3.0 were used to identify the downstream target genes of miR-3194-5p. The results show the intersection of the three aforementioned databases. GEPIA database [19] was used to perform pan-cancer analysis.

RNA immunoprecipitation assay

The RNA immunoprecipitation (RIP) assay was performed using an Imprint RNA Immunoprecipitation Kit (Sigma-Aldrich) according to the manufacturer’s protocol. Briefly, 20 µL of protein A magnetic beads was premixed with 5 µg of anti-Argomaute 2 antibodies (anti-AGO2, Sigma-Aldrich) or anti-IgG (Sigma-Aldrich) for 1 h at room temperature. Next, the mix was added to cell lysates (plus a protease inhibitor cocktail and a ribonuclease inhibitor) and incubated at 4 °C overnight to perform immunoprecipitation. The enrichment levels of circDHPR and miR-3194-5p were detected using RT-qPCR, as previously described.

Dual-luciferase reporter assay

Luc-circDHPR-WT and Luc-circDHPR-mut luciferase reporter plasmids were synthesized by Hanheng Biotechnology (Shanghai, China). 293T cells (3 × 105 cells/well) were seeded in 24-well plates and incubated overnight. Subsequently, 0.5 µg Luc-circDHPR-WT or Luc-circDHPR-mut plasmids were co-transfected into cells with 100 nM miR-3194-5p-mimic/miR-3194-5p-mimic-NC using Lipofectamine 3000 (Invitrogen). Forty-eight hours later, the luciferase fluorescence intensity of Firefly and Renilla cells was detected using the Dual-Luciferase Reporter Assay kit (YEASEN, Shanghai, China) with a microplate reader (Ynergy H1, BioTek, VT, USA) according to the manufacturer’s instructions. The relative luciferase intensity was quantified using the Firefly/Renilla luciferase assay.

In vivo tumorigenesis and metastasis

Male BALB/c nude mice were purchased from the Guangdong Medical Laboratory Animal Center (Guangzhou, China) and maintained under specific pathogen-free conditions. The experimental protocols were approved by the Animal Ethics Committee of Zhujiang Hospital. Stably overexpressed (OE-circDHPR) or negative control Hep3B cells (vector) (1 × 107 cells) resuspended in 150 µL PBS were subcutaneously injected into mice, followed by growth for 5 weeks. The width (W) and length (L) of the tumors were measured every 7 days to assess tumor growth. After 5 weeks, the mice were euthanized, and the tumors were excised and weighed. IHC and Western blotting were performed to detect the expression of tumor-associated proteins.

For the metastasis experiments, 3 × 106 cells were injected into the tail vein to establish a lung metastasis model. After 4 weeks, an in vivo optical imaging system (IVIS) was used to detect SMMC7721 cells overexpressing circDHPR or vectors with a GFP tag. The mice were then euthanized. The lungs were excised and fixed in formaldehyde solution. Hematoxylin-eosin staining was used to verify metastasis, and the metastatic numbers were counted.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8.0 (La Jolla, CA, USA) or SPSS 21.0 (IBM Corp, NY, USA). Differences between groups were analyzed using Student’s t-test or Analysis of Variance. Categorical variables were compared using the chi-squared test. Kaplan–Meier analysis and log-rank tests were used to analyze the overall survival (OS) and disease-free survival (DFS). The Cox proportional hazards model was used to identify the independent risk factors. Statistical significance was set at P < 0.05.

Results

CircDHPR is downregulated in HCC and associated with poor prognosis

Microarray data (GSE97332, GSE94508, and GSE78520) were obtained from the GEO database to identify the role of circRNAs in HCC. R software was used to identify differentially expressed circRNAs using the limma package (Fig. 1a and S3a-b). Subsequently, we considered the intersections of the aforementioned datasets. Eight differentially expressed circRNAs (five upregulated and three downregulated) were identified (Fig. 1b and S3c). Next, we used RT-qPCR to identify the expression of differentially expressed circRNAs in ten pairs of HCC and corresponding paracancerous tissues. Previously, we selected circRNAs that were upregulated in microarray data from the GEO database for further analysis. Therefore, we investigated the expression level of hsa_circ_0072088 [20] and hsa_circ_0001955 [21, 22], which have been reported to be overexpressed in HCC. However, the expression level of hsa_circ_0001955 and hsa_circ_0072088 were not significantly different between HCC and that corresponding paracancerous tissues (Fig. S4a-4b). Therefore, we verified that the downregulated circRNAs predicted in 10 pairs of tissues. We chose the most significantly differentially expressed circRNAs based on predictions. The RT-qPCR results showed that a novel circRNA (circBase ID: hsa_circ_0069249) derived from the dihydropteridine reductase (DHPR) locus (Fig. S3d) was downregulated (P = 0.0031) in HCC tissues compared to normal tissues (Fig. 1c). Therefore, we named it circDHPR. Moreover, the expression level of circDHPR was lower in HCC cell lines than in the normal liver cell line HL-7702 (Fig. 1d). To confirm circDHPR expression, RT-qPCR was used to investigate additional clinical tissues (54 normal and 80 HCC) from the Sun Yat-sen University Cancer Center. We found that circDHPR expression was downregulated (P < 0.0001) in the HCC tissues (Fig. 1e). Therefore, we focused on circDHPR in the subsequent experiments.

Fig. 1.

Fig. 1

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CircDHPR is downregulated in HCC. a The differential expression of circRNAs in a representative dataset (GSE97332) is shown using a heatmap. b The intersection of downregulated circRNAs in three datasets by Veen diagram was used. c The expression of circDHPR in 10 matched pairs of HCC tissues and normal tissues. d The expression of circDHPR in the normal liver cell line HL-7702 and HCC cell lines was measured by RT-qPCR. e The expression level of circDHPR in 80 HCC tissues and 54 normal tissues. f Kaplan–Meier curves were used to analyze the correlation of circDHPR expression with OS and DFS. g Sanger sequencing using PCR products amplified using divergent primers. Black arrows indicate the back-splicing junction site. h Convergent primers were applied to amplify GAPDH while divergent primers were used for circDHPR with cDNA or gDNA as the templates. i RT-qPCR showed the expression of circDHPR after RNase R digestion. j RT-qPCR measured the expression level of circDHPR after reverse transcription using random primers or oligo dt primers. k Actinomycin D inhibited the mRNA synthesis but did not have much effect on circRNAs. GAPDH was used as a control. The data are presented as the mean ± SD of at least three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Patients were divided into two groups: those with low circDHPR expression (n = 47) and those with high expression (n = 33). Clinicopathological features were analyzed using the chi-squared test. The results showed that decreased circDHPR expression was associated with the advanced T stage (Table 1). Furthermore, Kaplan–Meier survival curves indicated that HCC patients with low circDHPR expression (n = 47) had poorer overall survival (P = 0.01) and disease-free survival (P = 0.02) than those with high circDHPR expression (n = 33) after surgery (Fig. 1f). Univariate analysis revealed that number of tumors, size of the largest tumor, vascular invasion (Fig. S4c), and expression of circDHPR significantly correlated with overall survival (OS) or disease-free survival (DFS). Therefore, we used these variables to perform multivariate analyses. The results showed that decreased circDHPR expression and vascular invasion were independent risk factors for OS. Similarly, decreased circDHPR expression, the size of the largest tumor (>5 cm), multiple tumors, and vascular invasion were independent risk factors for DFS (Fig. 2). These results indicate that circDHPR expression is downregulated in HCC tissues compared to that in normal tissues and that low circDHPR expression is associated with poorer clinicopathological features than of high circDHPR expression. circDHPR may act as an independent prognostic marker in patients with HCC after surgery.

Table 1.

Correlations between the expression of circDHPR and HCC pathological characteristics

circDHPR expression
Category Subcategory Cases Low (n = 47) High (n = 33) P-value
Gender Male 67 39 (82.98%) 28 (84.85%) 0.82
Female 13 8 (17.02%) 5 (15.15%)
Age (years) ≤ 60 74 42 (89.36%) 32 (96.97%) 0.21
> 60 6 5 (10.64%) 1 (3.03%)
HBsAg Positive 70 41 (87.23%) 29 (87.88%) 0.93
Negative 10 6 (12.77%) 4 (12.12%)
AFP (ng/mL) ≤ 400 44 25 (53.19%) 19 (57.58%) 0.70
> 400 36 22 (46.81%) 14 (42.42%)
Size of largest tumor (cm) ≤ 5 23 10 (21.28%) 13 (39.39%) 0.06
> 5 57 37 (78.72%) 20 (60.61%)
Vascular invasion Yes 20 13 (27.66%) 7 (21.21%) 0.60
No 60 34 (72.34%) 26 (78.78%)
Tumor number Single 68 38 (80.85%) 30 (90.91%) 0.22
Multiple 12 9 (19.15%) 3 (9.09%)
Tumor differentiation Well (I) 11 6 (12.77%) 5 (15.15%) 0.47
Middle (II) 68 41 (87.23%) 27 (81.82%)
Poor (III + IV) 1 0 (0.00%) 1 (3.03%)
T stage T0-T2 58 30 (63.82%) 28 (84.85%) 0.03
T3a-T4 22 17 (36.17%) 5 (15.15%)
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Fig. 2.

Fig. 2

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Multivariate Cox proportional hazards regression model illustrating the prognostic factors for OS and DFS

Structural characteristics of circDHPR

CircDHPR derived from exons 8–10 of DHPR, which is located on chromosome 4p15.32. Experiments were performed to verify its circular structure (Fig. 1g, upper panel). CircDHPR PCR products were subjected to Sanger sequencing. The results showed that the back-splice junction of the PCR product amplified by the divergent primers was consistent with that of circBase (Fig. 1g, bottom panel). Convergent primers were used to amplify GAPDH, and divergent primers were used for circDHPR with cDNA or gDNA as templates (Fig. 1h). RT-qPCR was performed to verify the circular character of circDHPR compared to that of GAPDH after RNase R treatment. The results indicated that circDHPR, which lacked 5′ (m7G) and 3′ poly A, resisted RNase R digestion [23] in contrast to the linear structure of GAPDH mRNA (Fig. 1i), DNA agarose gel electrophoresis confirmed the stability of circDHPR after RNase R treatment (Fig. S5a). The reverse transcription was performed using random primers and oligo dt primers. The qPCR results revealed that circDHPR can be reverse-transcribed by random primers or Oligo dt primers (Fig. 1j). Furthermore, the mRNA synthesis can be inhibited by actinomycin D according to indicated time points, and actinomycin D did not have much effect on inhibiting the circDHPR synthesis (Fig. 1k). These results confirm the circular structure of circDHPR.

CircDHPR overexpression suppressed HCC proliferation and metastasis in vitro

To investigate the biological function of circDHPR, we established stable circDHPR overexpressing Hep3B and SMMC7721 cell lines using a lentiviral vector with a green fluorescent protein-tag (OE-circDHPR or vector). RT-qPCR was used to identify the successfully constructed cell lines (Fig. 3a). CCK-8 and colony formation assays were used to test the proliferation and colony formation abilities of the transfected Hep3B and SMMC7721 cell lines. The results indicated that circDHPR overexpression significantly suppressed cell proliferation and colony formation (Fig. 3b-c). The flow cytometry results indicated that circDHPR inhibited cell proliferation by inducing G0/G1 phase cell cycle arrest (Fig. 3d). To further explore the effect of circDHPR on HCC invasion and migration, we used Transwell chambers with or without pre-coated Matrigel. The results showed that circDHPR overexpression inhibited HCC cells invasion and migration (Fig. 3e). Similar results were observed in the wound healing assay (Fig. 3f). We hypothesized that circDHPR inhibits the malignant cancer phenotype by affecting normal cell epithelial-mesenchymal transformation (EMT). To test this hypothesis, we used RT-qPCR and Western blotting to evaluate the expression of EMT markers (E-cadherin, N-cadherin, snail1, vimentin, and ZO-1) [24]. The results showed that circDHPR overexpression upregulated the expression of E-cadherin and ZO-1 and decreased the expression of N-cadherin, snail1, and vimentin at both the mRNA and protein levels, which is consistent with the literature (Fig. 3g and S5b). In summary, circDHPR may act as a tumor suppressor and inhibit HCC proliferation and metastasis in vitro.

Fig. 3.

Fig. 3

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CircDHPR suppresses HCC progression in vitro. a Cells were transfected with vector or OE-circDHPR, and RT-qPCR demonstrated that the transfections were successful. b CCK-8 assays were performed to assess the proliferation ability of Hep3B and SMMC7721 after transfection. c Colony formation assays detected the proliferation ability of Hep3B and SMMC7721 cells after transfection with circDHPR. d FACS analysis was used to measure the number of cells in each phase of the cell cycle (2N = G0/G1 phase, 4N = G2/M phase). e-f Transwell (e) and wound healing (f) assays were performed to assess the changes in invasion and migration abilities induced by circDHPR overexpression. g Western blotting was used to measure the protein expression of EMT markers. β-actin was used as the reference gene. The data are presented as the mean ± SD of at least three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

CircDHPR acts as a ceRNA to sponge miR-3194-5p in HCC

CircRNAs have been widely reported to function as ceRNA to sponge microRNAs (miRNAs) and regulate biological functions and tumorigenesis. Notably, FISH analysis of circDHPR in Hep3B and SMMC7721 cells showed that circDHPR was mostly localized in the cytoplasm (Fig. 4a). We hypothesized that circDHPR regulates downstream genes by sponging miRNAs. To verify this, we performed bioinformatics analysis using online databases (StarbaseV3.0, CSCD, and CircInteratome), and the intersection of the aforementioned three results revealed that circDHPR might absorb miR-3194-5p and miR-1270 to regulate tumor development (Fig. 4b). To further confirm ceRNA function, we performed RIP assays using an antibody against the Argonaute 2 protein (AGO2), which reportedly participates in the formation of RNA-induced silencing complexes [25]. These results indicate that circDHPR could bind AGO2 to attain ceRNA function (Fig. 4c). RT-qPCR was performed to measure the enrichment of miR-3194-5p and miR-1270. The results showed that both miR-3194-5p and miR-1270 were enriched by AGO2 in the Hep3B and SMMC7721 cell lines. However, the enrichment of miR-3194-5p was significantly higher than that of miR-1270 (Fig. 4d). Western blotting was used to evaluate the enrichment induced by the anti-AGO2 antibody (Fig. 4e). Therefore, miR-3194-5p was selected for further analysis. To further confirm the interaction of miR-3194-5p with circDHPR, two pairs of pmirGLO plasmids with circDHPR mut sites (circDHPRmut1 and circDHPRmut2) and wild-type (circDHPRwt) were constructed according to a prediction in StarbaseV3.0 (Fig. 4f, left panel), and a dual-luciferase assay was performed using 293T cells, which showed that overexpressed miR-3194-5p reduced the activity of circDHPRwt and circDHPRmut2 reporter genes. In contrast, the overexpression of miR-3194-5p did not affect the circDHPRmut1 reporter gene expression (Fig. 4f, right panel). The FISH results indicated that circDHPR colocalized with miR-3194-5p in the cytoplasm (Fig. 4g). To further identify the ceRNA mechanism, qPCR was conducted, and the results showed that neither overexpressed circDHPR and miR-3194-5p nor downregulated miR-3194-5p affected the expression of miR-3194-5p and circDHPR, respectively (Fig. 4h). This revealed that circDHPR regulates miR-3194-5p through a sponging effect rather than affecting its expression. Therefore, circDHPR acts as a ceRNA by absorbing miR-3194-5p.

Fig. 4.

Fig. 4

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CircDHPR acts as a ceRNA by sponging miR-3194-5p. a FISH of circDHPR in Hep3B and SMMC7721 cells. b Targeted miRNAs that bind to circDHPR were predicted using StarBase V3.0, CSCD, and CircInteratome. The Venn diagram illustrates the intersection of the three databases. c-d RIP assays with an anti-AGO2 antibody were performed to verify the enrichment of circDHPR and targeted miRNAs predicted by the databases. e Western blotting was used to check for enrichment induced by the anti-AGO2 antibody. f Dual-luciferase assays were performed to explore the circDHPR and miR-3194-5p binding positions in 293T cells. g FISH was used to detect the intracellular localization of circDHPR (green) and miR-3194-5p (red). h RT-qPCR was performed to measure the expression of miR-3194-5p and circDHPR after altering the expression of circDHPR or miR-3194-5p. GAPDH was used as a reference gene The data are presented as the mean ± SD of at least three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01.

miR-3194-5p is also downregulated in HCC

We investigated the biological functions of miR-3194-5p in HCC. RT-qPCR was performed using 10 pairs of HCC tissues and the corresponding paracancerous tissues (Fig. 5a). The results indicated that the expression of miR-3194-5p was downregulated in HCC tissues compared to normal tissues. Similar results were observed in the comparison between the HCC cell lines and the normal liver cell line HL-7702 (Fig. 5b). The results of the CCK-8 and colony formation assays using the miR-3194-5p inhibitor after circDHPR overexpression showed that decreased miR-3194-5p expression promoted HCC cell proliferation and colony formation (Fig. 5c-e). Similar results were obtained in the wound healing and Transwell assays. These results revealed that the inhibition of metastasis induced by ectopically expressed circDHPR was rescued by the miR-3194-5p-inhibitor (Fig. 5f-g). In summary, miR-3194-5p is downregulated in HCC and alleviates the tumor suppressive effect of circDHPR overexpression.

Fig. 5.

Fig. 5

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miR-3194-5p inhibits HCC growth and metastasis. a RT-qPCR was used to detect the expression of miR-3194-5p in 10 matched pairs of HCC and normal tissues. b Expression of miR-3194-5p in normal liver cell line HL-7702 and HCC cell lines. c-d CCK-8 assays were performed to explore the proliferative ability of Hep3B and SMMC7721 cells after transfection with the miR-3194-5p-inhibitor. e Colony formation assays were used to detect the colony formation ability of Hep3B and SMMC7721 cells after transfected with the miR-3194-5p-inhibitor. f-g wound healing (f) and Transwell (g) assays were performed to assess the migration and invasion of Hep3B and SMMC7721 cells after transfection with the miR-3194-5p-inhibitor. The data are presented as the mean ± SD of at least three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

CircDHPR regulates the Ras/MAPK signaling pathway by sponging miR-3194-5p

Target genes that might bind to miR-3194-5p were predicted using TargetScan Human 8.0. Next, we performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) [26] enrichment analysis to identify the signaling pathways that might be affected by miR-3194-5p. This revealed that the Ras/MAPK signaling pathway is most likely regulated by circDHPR (Fig. 6a). To confirm this hypothesis, we performed Western blotting to analyze the critical proteins [27] (Raf1, Erk1/2, and Mek1) involved in the Ras/MAPK signaling pathway. The results indicated that circDHPR upregulation decreased the levels of crucial proteins involved in the Ras/MAPK signaling pathway. However, the inhibition of miR-3194-5p rescued the tumor suppressive effect of circDHPR overexpression (Fig. 6b). These results indicated that circDHPR regulates the Ras/MAPK signaling pathway by sponging miR-3194-5p.

Fig. 6.

Fig. 6

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CircDHPR regulates the Ras/MAPK signaling pathway by sponging miR-3194-5p. a KEGG enrichment analysis was used to explore the pathways affected by miR-3194-5p downstream target genes. b The expression of key proteins in the Ras/MAPK signaling pathway were measured by Western blotting after circDHPR overexpression or transfection with miR-3194-5p-inhibitors. c Targeted genes binding to miR-3194-5p were predicted by TargetScan (green), miRWalk (blue) and StarBase (red), The Venn diagram shows the intersection of the results. d The expression profile in normal tissues (grey) and HCC tissues (red) were obtained using TCGA database. e Dual-luciferase assays were performed to verify the binding position of RASGEF1B and miR-3194-5p in 293T cells. The data are presented as the mean ± SD of at least three independent experiments. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

RASGEF1B is the direct target of miR-3194-5p

To move forward in a single step, potential target genes of miR-3194-5p were selected using databases (TargetScan Human 8.0, miRwalk, and StarbaseV3.0). Surprisingly, 635 genes were identified after this intersection (Fig. 6c). A literature search showed that Ras-GEF domain-containing family member 1B (RASGEF1B) inhibits the Ras/MAPK signaling pathway [28, 29]. Furthermore, The Cancer Genome Atlas (TCGA) database was used to analyze the expression of RASGEF1B in HCC and normal liver tissues. The results showed that RASGEF1B was downregulated in HCC, which was consistent with the ceRNA hypothesis (Fig. 6d). Next, we constructed the RASGEF1B mutant site (RASGEF1Bmut) and RASGEF1B wild type (RASGEF1Bwt) dual-luciferase plasmid according to StarBaseV3.0 and performed dual-luciferase assays. The results revealed that miR-3194-5p binds to the RASGEF1B 3′untranslated region (Fig. 6e).

Next, Pan-Cancer analysis was performed to investigate the expression of RASGEF1B in different cancers using GEPIA database. We found that the expression of RASGEF1B was upregulated in five different types of cancer (Fig. 7a, red) and downregulated in four types of cancer (Fig. 7a, green). Importantly, HCC patients with higher RASGEF1B expression levels (n = 100) had better OS and DFS than patients with lower RASGEF1B expression levels (n = 100) in TCGA database (Fig. 7b). Moreover, higher RASGEF1B expression was associated with better BCLC stage (Fig. 7c). RT-qPCR results from randomly selected clinical tissues (24 normal and 56 HCC tissues) showed that the expression of RASGEF1B was downregulated in HCC tissues (Fig. 7d). Pearson’s correlation analysis showed that the expression levels of RASGEF1B and circDHPR were positively correlated in HCC tissues (r = 0.4919, P = 0.0031; Fig. 7e). Western blotting and IHC assays were performed to determine the expression of RASGEF1B in HCC and normal liver tissues. The results indicated that the protein level was downregulated in HCC, which was consistent with the mRNA expression of RASGEF1B (Fig. 7f-h). Next, we examined the expression levels of RASGEF1B in stably transfected cell lines. Overexpression of circDHPR upregulated the expression of RASGEF1B (Fig. 7i). Western blotting showed that the upregulation of RASGEF1B caused by circDHPR overexpression was suppressed by the miR-3194-5p-inhibitor (Fig. 7j). These results indicated that RASGEF1B is a direct target of miR-3194-5p and is regulated by circDHPR.

Fig. 7.

Fig. 7

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RASGEF1B is downregulated in HCC. a TCGA data were used to perform RASGEF1B Pan-Cancer analyses. b Kaplan–Meier curves were used to analyze the correlation between RASGEF1B expression and OS or DFS. c Violin plot showing the correlation between RASGEF1B expression and the BCLC stage. d RT-qPCR was used to detect the expression level of RASGEF1B in normal (n = 24) and HCC tissues (n = 56). e The correlation between RASGEF1B and circDHPR expression in clinical HCC samples was assessed using Pearson’s correlation analysis. f-g Western blotting was used to measure the protein levels of RASGEF1B in three matched pairs of HCC and normal tissues. h Immunohistochemistry was performed to verify the expression of RASGEF1B in HCC and normal liver tissues. i The mRNA expression of RASGEF1B after circDHPR overexpression was detected using RT-qPCR. j Protein levels of RASGEF1B after transfection with miR-3194-5p-inhibitor at the base of overexpressed circDHPR. The data are presented as the mean ± SD of at least three independent experiments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Overexpression of circDHPR could inhibit HCC tumorigenesis and metastasis in vivo

Next, we investigated the in vivo biological function of circDHPR in HCC tumorigenesis. Hep3B cell lines with successfully transfected OE-circDHPR or vector were subcutaneously injected into the right flanks of BALB/c nude mice. Tumor volumes were measured weekly, and the results revealed that circDHPR overexpression suppressed tumor growth. Nude mice were euthanized at the end of the 5 weeks after inoculation, and the tumors were excised and weighed. These results suggest that circDHPR suppresses tumorigenesis (Fig. 8a-b). Furthermore, IHC and Western blotting results showed that circDHPR overexpression upregulated the expression of RASGEF1B (Fig. 8c) and suppressed the Ras/MAPK signaling pathway (Fig. 8d).

Fig. 8.

Fig. 8

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CircDHPR suppresses HCC progression in vivo. a Representative images of xenograft tumors in five nude mice. b Changes in tumor volume (left panel) and weight (right panel) after circDHPR overexpression. c-d Immunohistochemistry (c) and Western blotting (d) were used to detect the expression of RASGEF1B and proteins involved in the Ras/MAPK signaling pathway using Hep3B cells with overexpressing circDHPR or vector. e-g Representative IVIS images (e) of the mouse lung metastasis model established by SMMC7721 cell lines overexpressing circDHPR or vector. Lungs were excised and subjected to IVIS (f) Statistical analysis was performed to measure the total radiant efficiency (g). h-i Hematoxylin-eosin staining was used to assess the lung metastasis nudes in cells overexpressing circDHPR or vector. ns, not significant; *, P < 0.05; **, P < 0.01.

Next, a lung metastasis model was established by injecting SMMC7721 cells transfected with OE-circDHPR or a lentiviral vector into the tail vein of BALB/c nude mice. IVIS was used to measure the fluorescence intensity of GFP labeled in the lungs at the end of 4 weeks. The fluorescence intensity showed that circDHPR overexpression significantly inhibited HCC metastasis (Fig. 8e and g, left panel). The mice were euthanized, and their lungs were excised, used to verify fluorescence (Fig. 8f and g right panel), and subjected to H&E staining to verify the metastatic nodes. The number of metastatic lung nodes was counted. H&E staining of the dissected lungs revealed that circDHPR overexpression markedly suppressed lung metastasis (Fig. 8h-i). In summary, the ectopic overexpression of circDHPR inhibited HCC tumorigenesis and metastasis in vivo.

Discussion

circRNAs are regarded as noise during gene transcription [30]. With the development of high-throughput sequencing and bioinformatics tools, numerous circRNAs have been identified [31]. circRNAs, which may play a substantial role in disease development, have attracted the attention of researchers. Although several circRNAs have been identified and investigated, their biological functions in various diseases remain unknown.

Many studies have reported that circRNAs function as proto-oncogenes and exert critical effects on the tumorigenesis of the lung [32], gastric [33], pancreatic [34], renal [35], and other carcinomas. However, only a few circRNAs have been identified as tumor suppressor genes in HCC [36–38]. In this study, we used a bioinformatics analysis tool to screen for differentially expressed circRNAs in HCC and normal liver tissues using microarray data from the GEO database. We found that circDHPR expression was downregulated in HCC tissues. Furthermore, qPCR was used to confirm that circDHPR was downregulated in HCC at both the tissues and cell levels. In the prognostic analysis, high circDHPR expression was associated with better OS and DFS in patients with HCC. We also identified a circular structure of circDHPR. Sanger sequencing was used to verify the back-splice junction. CCK-8, colony formation, flow cytometry, Transwell, and wound healing assays were used to confirm that ectopic overexpression of circDHPR inhibited HCC proliferation and metastasis in vitro.

circRNAs have been reported to contain miRNA response elements [39]. Thus, many circRNAs may serve as miRNA sponges to form the circRNA-miRNA-mRNA regulatory axis. Therefore, in this study, we investigated whether circDHPR could absorb miRNAs. RIP and dual-luciferase assays were used to determine whether circDHPR served as a sponge to absorb miR-3194-5p and alleviate the gene-silencing effect caused by miR-3194-5p. Our experiments further demonstrated that miR-3194-5p suppresses HCC proliferation and metastasis. This is the first study to determine the biological functions of miR-3194-5p in HCC.

miRNAs play substantial roles in many biological processes, particularly in disease development and tumorigenesis [40]. These functions mostly depend on downstream target genes regulated by miRNAs. In the present study, the KEGG enrichment analysis was used to identify the target genes of miR-3194-5p, which were mainly enriched in the Ras/MAPK signaling pathway. Studies have reported that the Ras/MAPK signaling pathway exerts critical effects on tumorigenesis and metastasis in different types of cancer [41, 42]. We further found that circDHPR overexpression inhibited the Ras/MAPK signaling pathway. However, this effect was rescued by the miR-3194-5p-inhibitor, suggesting that circDHPR suppresses HCC progression by sponging miR-3194-5p to regulate the Ras/MAPK signaling pathway. However, the underlying mechanism remains unknown.

Thus, we further explored the target gene of miR-3194-5p, specifically RASGEF1B, a suppressor of the Ras/MAPK signaling pathway. Next, we confirmed that miR-3194-5p could bind to the 3′untranslated region of RASGEF1B by using a dual-luciferase assay. Our results also indicated that RASGEF1B is downregulated in HCC. The mRNA expression of RASGEF1B was positively correlated with that of circDHPR, which is consistent with the result of many published studies [43, 44]. Moreover, the protein level of RASGEF1B increased after circDHPR overexpression and was downregulated by inhibiting the expression of miR-3194-5p. Subsequent in vivo experiments confirmed that circDHPR acts as a tumor suppressor by upregulating RASGEF1B and inhibiting the Ras/MAPK signaling pathway.

Our results revealed that circDHPR inhibited HCC proliferation and metastasis by sponging miR-3194-5p to upregulate RASGFE1B expression, which is a suppressor of the Ras/MAPK signaling pathway. Notably, our study has limitations. Firstly, the upstream regulatory mechanisms of circDHPR remain unknown and require further investigation. Secondly, although we found that RASGEF1B was downregulated in HCC, its biological function was not explored. Finally, we performed only animal experiments to verify the function of circDHPR, the tumor-inhibiting effect of miR-3194-5p and RASGEF1B require further investigation.

Conclusion

To the best of our knowledge, this study demonstrates that circDHPR is downregulated in HCC and that low circDHPR expression was associated with poor prognosis. Our results confirm the circular structure of circDHPR and its ability to suppress tumorigenesis and metastasis both in vitro and in vivo. To the best of our knowledge, this is the first study to verify that miR-3194-5p is downregulated in HCC and suppresses HCC progression by affecting the Ras/MAPK signaling pathway. We also found that RASGEF1B was downregulated in HCC. Our study illustrates that circDHPR can inhibit HCC progression by sponging miR-3194-5p, which upregulates RASGEF1B to suppress the Ras/MAPK signaling pathway. Our results suggested that the circDHPR/miR-3194-5p/RASGEF1B axis may have prognostic and therapeutic applications in patients with HCC (Fig. 9).

Fig. 9.

Fig. 9

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Proposed model of the molecular mechanism of circDHPR in hepatocellular carcinoma

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (1.5MB, docx)

Abbreviations

HCC

hepatocellular carcinoma

DHPR

dihydropteridine reductase

ceRNA

competing endogenous RNA

GEO

Gene Expression Omnibus

KEGG

Kyoto Encyclopedia of Genes and Genomes

TNM

tumor-node-metastasis

DFS

disease-free survival

OS

overall survival

DMEM

Dulbecco’s modified Eagle media

FBS

fetal bovine serum

PBS

phosphate buffered saline

ECL

electrochemical luminescence

HR

hazard risk

CI

confidence internal

EMT

epithelial-mesenchymal transformation

RASGEF1B

Ras-GEF domain-containing family member 1B

RAS

rat sarcoma

MAPK

mitogen-activated protein kinase

Author Contribution

Z-YG, S-JF, M-XP, and Y-G conceived the experiment and wrote the manuscript. Z-YG, Q-YX, Y-PW, and H-YM performed the experiments and analyzed the data. J-JZ and Z-YG performed the animal experiments. G-LH, L-XG, F-L, T-L, Y-W, Y-F, Z-ZL, L-C, C-Y, and S-L helped with the experiments and analysis. All authors approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (no.31972926 and no.82072627), and the Guangdong Basic and Applied Basic Research Foundation (no.2021B1515230011 and no.2023A1515010141). The funders had no role in the study design, data collection and analysis, decision to publish, or manuscript preparation.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. In addition, the datasets here are available from the Gene Expression Omnibus (GSE97332, GSE94508, and GSE78520) and The Cancer Genome Atlas databases.

Declarations

Ethical approval

This study was approved by the ethics committees of Zhujiang Hospital and Sun Yat-sen University Cancer Center. Informed consent was obtained from all the patients. All animal experimental protocols were approved by the Animal Ethics Committee of Zhujiang Hospital.

Competing interests

The authors declare no competing interests.

Footnotes

Zeyi Guo, Qingyu Xie, Yanping Wu and Haiyu Mo contributed equally to this work.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Yi Gao, Email: gaoyi@smu.edu.cn.

Mingxin Pan, Email: pmxwxy@sohu.com.

Shunjun Fu, Email: fsj103@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (1.5MB, docx)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. In addition, the datasets here are available from the Gene Expression Omnibus (GSE97332, GSE94508, and GSE78520) and The Cancer Genome Atlas databases.

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