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. 2020 Oct 9;44(2):279–295. doi: 10.1007/s13402-020-00564-y

Circular RNA circ-102,166 acts as a sponge of miR-182 and miR-184 to suppress hepatocellular carcinoma proliferation and invasion

Rong Li 1,2,#, Yinan Deng 3,#, Jinliang Liang 1, Zhongying Hu 1, Xuejiao Li 1, Huanyi Liu 1, Guoying Wang 3, Binsheng Fu 3, Tong Zhang 3, Qi Zhang 4, Yang Yang 3,2, Guihua Chen 1,3,✉, Wei Liu 1,2,✉
PMCID: PMC12980780  PMID: 33034848

Abstract

Purpose

Multiple circular RNAs (circRNAs) have been reported to be dysregulated in hepatocellular carcinoma (HCC). However, their functions and modes of action are still largely unclear. Identifying key circRNAs and revealing their potential functions and molecular mechanisms is considered important for improving the diagnosis and treatment of HCC.

Methods

Dysregulated circRNAs in HCC were identified through integration of three human HCC circRNAs microarray datasets (GSE94508, GSE97332 and GSE 78520), followed by qRT-PCR validation in primary HCC tissues and cell lines. circRNA characteristics were verified through Sanger sequencing, RNase R treatment, northern blotting and intracellular localization analyses. In addition, circRNA functions in HCC development were assessed using CCK8, colony formation, EDU incorporation, flow cytometry, transwell and scratch wound healing assays in vitro and tumor xenograft assays in vivo. Next, underlying molecular mechanisms in HCC were assessed using dual-luciferase reporter, RNA pull-down, RNA immunoprecipitation and western blotting assays.

Results

We found that a novel circular RNA, circ-102,166, was down-regulated in HCC and that its expression level was significantly associated with multiple clinicopathologic characteristics, as well as the clinical prognosis of HCC patients. In vitro and in vivo experiments revealed that circ-102,166 overexpression significantly inhibited the proliferation, invasion, migration and tumorigenicity of HCC cells. Furthermore, we found that circ-102,166 can bind to miR-182 and miR-184 to regulate the expression of several of their downstream targets (FOXO3a, MTSS1, SOX7, p-RB and c-MYC).

Conclusion

Our data revealed a tumor-suppressing role of circ-102,166 in HCC. Down-regulation of circ-102,166 enhanced the proliferation and invasion of HCC cells by releasing the oncomiRs miR-182 and miR-184.

Electronic supplementary material

The online version of this article (10.1007/s13402-020-00564-y) contains supplementary material, which is available to authorized users.

Keywords: Hepatocellular carcinoma; Circ-102,166; miR-182; miR-184; Proliferation; Invasion

Introduction

Primary liver cancer is one of the leading causes of cancer-associated death worldwide, with an incidence ranking sixth and a mortality ranking third among all cancers [1]. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer (comprising 75%–85% of cases) with approximately 750.000 new cases diagnosed and 500.000 deaths reported annually worldwide [2]. Due to a lack of obvious symptoms at early stages and effective early diagnostic methods, only 30–40% of HCC patients are eligible for curative resection. Nevertheless, the 5-year recurrence or metastases rates are as high as 70% [3, 4]. This situation urges for more effective treatment options, as well as more efficient early diagnostic and prognostic biomarkers.

Circular RNAs (circRNAs) comprise a class of RNAs characterized by covalently closed loop structures with neither 5′-3′ polarity nor a poly(A) tail [5, 6]. Most circRNAs originate from protein-coding genes [7, 8]. They are conserved, stable and usually expressed in particular tissues or during specific developmental stages [9, 10] . Although some circRNAs are translatable and produce previously unknown protein isoforms [11–15], most circRNAs do not have protein-coding capacity. Instead, they exert their functions as microRNA sponges or as gene transcription modulators [16–18]. Previous studies have shown that circRNAs may be deregulated in different cancers, such as gastric cancer (circLARP4, circPVT1) [19, 20], esophageal squamous cell carcinoma (circ-ITCH, ciRS-7) [10, 21], colorectal cancer (circCCDC66, circITGA7) [22, 23] and HCC (cSMARCA5, circMTO1) [24, 25], and be involved in their initiation and/or progression, implicating their potential as treatment targets. Moreover, circRNAs have been identified as suitable diagnostic targets due to their stability, conservation and tissue specificity. While the list of cancer-associated circRNAs continues to grow, their clinicopathological relevance and functional roles still remain largely unknown. Therefore, we aimed to identify novel circRNAs involved in the development of HCC, and to reveal their functions and underlying molecular mechanisms.

In this study, circ-102,166 was found to be significantly down-regulated in HCC, and to be associated with multiple clinicopathologic characteristics, as well as a poor clinical prognosis. We also found that circ-102,166 can inhibit HCC cell proliferation and invasion by acting as a sponge for miR-182 and miR-184, both in vitro and in vivo. Overall, our findings suggest that circ102,166 may serve as a novel biomarker and a therapeutic target for HCC.

Materials and methods

Tissue specimens

A total of 136 patients, who were histopathologically and clinically diagnosed as HCC at the third affiliated hospital, Sun Yat-Sen University (Guangzhou, Guangdong, China), was recruited for this study. All procedures with human subjects in this study were conducted in accordance with the Institutional Research Ethics Committee. Informed written consent was obtained from all participants as well. Among them, 77 cases with detailed clinical follow-up data were used to analyze correlations between circ-102,166 expression and clinicopathological and patient survival parameters. The other 59 HCC cases with paired non-tumorous tissues were used to analyze circ-102,166 expression profiles. None of the patients had received pre-operative chemotherapy, radiotherapy or targeted therapy by the time of sample collection. All tumor tissues were confirmed as HCC using hematoxylin and eosin (H&E) staining. Normal liver tissues were dissected at a standard distance (2 cm) from the HCC resection margin and confirmed by pathological evaluation.

Microarray data analysis

Three HCC circRNAs microarray datasets (GSE94508, GSE97332 and GSE78520) were downloaded from the NCBI GEO DataSets site (https://www.ncbi.nlm.nih.gov/gds/?term=). All the circRNA data were already processed and the logFC (log2 (Tumor/Normal)) values provided by their submitters. CircRNAs that met |logFC| > 1 and p < 0.05 were selected for further investigation. The selected circRNAs were filtered by Venny 2.1 (http://bioinfogp.cnb.csic.es/tools/venny/index.html) to extract circRNAs with the same trend in all the three datasets. The circRNA sequences can be obtained in CircBase (http://www.circbase.org/).

Cell lines and culture

HCC cell lines Huh7, HepG2, 97H, SK-Hep1, Hep3B and LM3, immortalized normal liver cell line LO2 and human embryonic kidney cell line 293 T were purchased from the Cell Bank of Type Culture Collection (Shanghai, China). These cell lines were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco, Carlsbad, CA, USA) and 1% penicillin/streptomycin (Invitrogen). The cells were incubated at 37 °C in an environment of 5% CO2.

Genomic DNA and RNA extraction

Genomic DNA (gDNA) was isolated from cultured cells using a Blood & Cell Culture DNA Mini Kit (Qiagen, Chatsworth, CA, USA) according to the manufacturer’s instructions. Total RNA was extracted from tissue specimens and cultured cells using Trizol reagent (Invitrogen, Life Technologies Inc., Germany) as described in the manufacturer’s instructions. The concentration and purity of RNA were measured using a NanoDrop 2000 apparatus (Thermo Fisher Scientific, Waltham, MA, USA). The RNA integrity was evaluated by the ratio of 28S:18S after denaturing formaldehyde agarose gel electrophoresis.

qRT-PCR and RNase R treatment

cDNA was synthesized by reverse transcription using a first strand cDNA synthesis kit for RT-PCR (Roche, Switzerland) following the manufacturer’s instructions. A SYBR FAST universal qPCR kit (Roche) was used for expression detection. qRT-PCR was performed using a LC480 real-time PCR detection system (Roche). All primers used for qRT-PCR were designed using Beacon Designer 7.0 and synthesized by TsingKe Biotech (Chengdu, China). GAPDH served as internal control. The sequences of all primers used are listed in Table S2. RNase R digestion was performed using RNase R purchased from Epicentre Biotechnologies (Madison, WI, USA) as per manufacturer’s instructions, after which the RNA was purified by phenol-chloroform extraction and subjected to qRT-PCR.

Northern blotting

Digoxin-labeled circ-102,166 and β-actin probes for northern blotting were obtained from Axl-Bio (Guangzhou, China). β-actin was used as linear RNA control. Total RNA and RNase R-treated RNA were used to perform northern blotting using a NorthernMax® Kit (Invitrogen, Life Technologies Inc., Germany) according to the manufacturer’s instructions.

Intracellular RNA localization analysis

Nuclear and cytoplasmic RNAs were isolated from cultured cells using a PARIS™ Kit (Thermo Fisher Scientific) for qRT-PCR. GAPDH and U6 were used as endogenous references. The following formula was used: Cytoplasm % = 2^CT(nuclear)/(2^CT(cytoplasm) + 2^CT(nuclear)), Nuclear % = 1- Cytoplasm %.

Western blotting

Western blotting was performed according to a previously described standard method using anti-MTSS1(1:500; Cell Signaling, Danvers, MA, USA), anti-FOXO3a (1:500; Cell Signaling), anti-SOX7(1:500; Abcam, Cambridge, MA, USA), anti-p-RB (ser807/811) (1:1000; Cell Signaling), anti-c-MYC (1:2000; Abcam) and anti-GAPDH (1:3000; Cell Signaling) antibodies. The experiments were independently performed in triplicate.

Plasmids and transfections

The complete sequence of circ-102,166 was obtained from circBase. We inserted this sequence into a retroviral transfer plasmid pLCDH-ciR-puro (Geneseed, Guangzhou, China). circ-102,166 siRNAs were synthesized by RiboBio (Guangzhou, China), targeting the back-splicing junction region of circ-102,166. circ-102,166 shRNAs and negative controls were designed and synthesized by IGEbio (Guangzhou, China). miR-182 mimics, miR-184 mimics, anti-miR-182 oligonucleotides, anti-miR-184 oligonucleotides and their corresponding control oligonucleotides were also purchased from RiboBio (Guangzhou, China). Cells were transfected with plasmids or oligonucleotides using Lipofectamine® 3000 (Invitrogen) following the manufacturer’s instructions. The sequences of the primers and siRNAs are listed in Table S2.

Cell proliferation assays

Cell proliferation was assessed using CCK8, colony formation and EDU incorporation assays. For the CCK8 assays, cells were seeded in 96-well plates (600 cells/well), after which cell proliferation was determined every 24 h using a detection kit purchased from KeyGEN Bio TECH (Jiangsu, China). Briefly, 10 μl CCK8 solution mixed with 90 μl fresh cell culture medium were added to each well and incubated at 37 °C for 2 h. Next, absorbance at 450 nm was measured using a spectrophotometer. For the colony formation assays, cells were seeded in 6-well plates (800 cells/well) and cultured at 37 °C in a humidified atmosphere containing 5% CO2 for 14 days. Next, the colonies were washed with PBS, fixed with 4% paraformaldehyde and stained with 0.1% crystal violet at room temperature for 30 min. After staining, the plates were washed with ddH2O and dried in a ventilated place. Colonies were counted using Image-Pro Plus 6.0. For the EDU incorporation assays a keyFluor488-EdU kit (KeyGEN Bio TECH) was used according to the manufacturer’s protocol. Images were obtained using an inverted fluorescence microscope (Carl Zeiss, Jena, Germany). The proportion of EdU-positive cells was determined as: EDU-positive cells/DAPI-positive cells. Each experiment was repeated at least three times.

Flow cytometry

Cell cycle progression and apoptotic rates were determined using a cell cycle detection kit (KeyGEN Bio TECH) and an Annexin V-FITC apoptosis detection kit (KeyGEN Bio TECH), respectively. The proportions of cells in different phases of the cell cycle and apoptotic rates were measured using a FACS Calibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Next, the data were collected and processed using ModFit LT 4.1 (Verity Software House, USA) and FlowJo VX10 (Leonard Herzenberg, USA).

Transwell invasion assay

Transwell inserts were coated with 10% Matrigel (BD Biosciences) (50 μl / insert) and incubated at 37 °C in an atmosphere of 5% CO2 for 0.5 h. Next, 200 μl cell suspension containing 4 × 104 cells without FBS were seeded into the upper chamber. The lower chamber was filled with 500 μl fresh cell culture medium containing 20% FBS. After incubation for 12-24 h, the medium in the upper chamber was removed. The inserts were washed with 1 × PBS, fixed with 500 μl methanol for 20 min and stained with 1 ml 0.1% crystal violet at room temperature for 30 min. After staining, the cells in the upper chamber were wiped off with wet cotton swabs. Next, the inserts were washed again with ddH2O and dried in a ventilated place. Invaded cells were photographed and quantified in five random 200× magnification fields.

Scratch wound healing assay

Cells were seeded in 6-well plates (4 × 105 / well) and cultured at 37 °C in an atmosphere of 5% CO2 for 24 h. Next, a sterile 10 μl pipette tip was used to scratch a wound. The wounded region was subsequently recorded in five random 100 × magnification fields at 0 h and 24 h.

Luciferase reporter assay

Cells were seeded in triplicate in 48-well plates (3–4 × 104 / well) and cultured at 37 °C in an atmosphere of 5% CO2 for 24 h. Next, the cells were transfected with microRNA mimics, 200 ng luciferase reporter and 2 ng pRL-TK Renilla luciferase construct. After 48 h, the transfected cells were lysed and subjected to luciferase activity measurement using a dual luciferase reporter assay kit (Promega, Madison, WI, USA).

RNA pull-down assay

Biotinylated circ-102,166 probe and a negative control oligo probe were purchased from Axl-Bio (Guangzhou, China). The RNA-pull down assay was carried out as previously described [26]. The circ-102,166 probe was incubated together with streptavidin magnetic beads (Thermo Fisher Scientific) to generate probe-coated beads according to the manufacturer’s protocol. Next, the beads were incubated with cell lysates (~1 × 107 cells were harvested, lysed and sonicated), followed by elution in Trizol (Thermo Fisher Scientific). Enrichment of miR-182 and miR-184 by circ-102,166 was determined using qRT-PCR.

RNA immunoprecipitation

Cells were co-transfected with 10 nM miR-182 mimics or miR-184 mimics together with HA-Ago2. After 48 h, the cells were collected as previously reported [27] after which RNA fragments were extracted using a RNAeasy Kit (Qiagen). IP products were analyzed by qRT–PCR to confirm binding of circ-102,166 to miR-182 or miR-184 in the RNA-induced silencing complex (RISC). GAPDH was used as negative control.

In vivo tumor xenograft model

A total of 3 × 106 cells was subcutaneously implanted into the back of 7-week old BALB/c nude mice (n = 5 per group). After 4–6 weeks, the mice were euthanized and tumors were collected, photographed and weighed.

Statistical analysis

All statistical analyses were performed using the SPSS 13.0 statistical software package and the GraphPad Prism 5.0 software package (GraphPad Software, Inc., San Diego, CA, USA). A chi-square test was used to analyze relationships between circ-102,166 expression levels and clinicopathological features. Survival curves were plotted using the Kaplan-Meier method and analyzed using the log-rank test. Comparisons between two groups were made using Student’s t test. Data are presented as mean ± SD derived from three independent experiments. P values < 0.05 were considered statistically significant.

Results

Circ-102,166 candidate identification and characterization in HCC

In an attempt to identify novel circRNAs involved in the progression of HCC, we integrated three human HCC circRNAs microarray datasets (GSE94508, GSE97332 and GSE78520) to screen commonly dysregulated circRNAs. Sixty-three circRNAs were found to be significantly and aberrantly expressed (|logFC| > 1 and p < 0.05) in HCC tissues compared to paired non-tumorous tissues in all three datasets (Fig. 1a). Among them, 41 were consistently upregulated, 5 were consistently downregulated and 17 were inconsistent among the three datasets (Table S1). Circ-102,166, one of the 5 downregulated circRNAs, attracted our attention with the most significant fold-change (Fig. 1a right). Circ-102,166 is located on chromosome 17q23.3 and originates from the TEX2 gene locus (Fig. 1b). Circ-102,166 consists of 495 nucleotides comprising three exons. To verify the circular form of circ-102,166, we designed divergent primers spanning circ-102,166 back-splicing junctions. The expected circularization sequence of circ-102,166 was amplified using divergent primers and confirmed by Sanger sequencing. In theory, divergent primers only amplify circular RNA from cDNA but not genomic DNA (gDNA), while convergent primers of circ-102,166 can amplify fragments from both cDNA and gDNA. β-actin was used as a linear control. Based on the above primers, PCR was carried out using cDNA and gDNA isolated from HCC cells. The PCR products were subsequently identified by agarose gel electrophoresis. As expected, amplification was only observed from cDNA with the divergent primers, which confirmed the back-splice junction of circ-102,166 (Fig. 1c). The looping structure makes circular RNAs more stable than linear RNAs, rendering them resistant to 3′ to 5′ exoribonuclease. The RNase R digestion reaction was performed as previously reported. We found that, in contrast to the linear control β-actin, circ-102,166 was resistant to RNase R treatment, which confirmed the circular nature of circ-102,166 (Fig. 1d). To further substantiate the circular nature of circ-102,166, we performed northern blotting. No significant changes in the circ-102,166 RNA level with or without RNase R treatment were observed, while the β-actin RNA was almost completely degraded after treatment with RNase R (Fig. 1e). Subsequent qRT-PCR analysis of cell fractions showed that circ-102,166 was localized in the cytoplasm (83.6%) rather than in the nucleus (16.4%) (Fig. 1f).

Fig. 1.

Fig. 1

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Characterization of circ-102,166 in HCC. (a) Venn diagram showing all differentially expressed circRNAs in HCC tissues compared with matched non-tumor tissues. The heat map shows that circ-102,166 exhibits the most significant fold-change among the 5 down-regulated circRNAs. (b) Schematic illustration showing that circ-102,166 is located at chromosome 17q23.3 and cyclized from exons 5–7 of TEX2. The circularization sequence of circ-102,166 was confirmed by Sanger sequencing. (c) Presence and circular form of circ-102,166 established by agarose gel electrophoresis. Circular RNA can only be amplified from cDNA but not gDNA by divergent primers, while convergent primers can amplify fragments from both cDNA and gDNA. β-actin acts as the linear control. (d) In contrast to linear control β-actin RNA, circ-102,166 is resistant to RNase R treatment. (e) Presence and circular form of circ-102,166 confirmed by northern blotting. β-actin was used as a linear control. (f) Distribution of circ-102,166 assessed by qRT-PCR in nuclear and cytoplasmic fractions. Results are presented as mean ± SD (n = three independent experiments). *: p < 0.05 by two-tailed Student’s t test

Circ-102,166 is down-regulated in HCC and significantly correlates with a poor prognosis

In order to validate the above expression pattern of circ-102,166 in HCC, qRT-PCR was performed on tumor tissues and paired non-tumor tissues of 59 HCC patients. We found that circ-102,166 was significantly down-regulated in HCC tissues compared with paired non-tumor tissues (p < 0.001) (Fig. 2a). In contrast, no significant difference in the expression of its parental gene TEX2 was observed (p = 0.751), implying that the downregulated of circ-102,166 in HCC was not a concomitant effect. Next, we analyzed correlations between circ-102,166 expression levels and clinicopathologic characteristics of another 77 HCC patients of whom detailed clinical information was available. Based on qRT-PCR circ-102,166 results, we divided this cohort of HCC patients into low and high circ-102,166 expression groups using the median as cutoff. By doing so, we found that the expression of circ-102,166 significantly correlated with tumor size (p = 0.03), TNM stage (p = 0.032), BCLC stage (p = 0.039) and vascular invasion (p = 0.022) (Table 1). Furthermore, through Kaplan-Meier analysis using the log-rank test, we found that HCC patients in the low circ-102,166 expression subgroup exhibited shorter median overall survival (OS; p = 0.0368) and recurrence-free survival (RFS; p = 0.0313) times compared to those in the high circ-102,166 expression subgroup (Fig. 2b-c). In addition, we assessed the expression of circ-102,166 in HCC cell lines and a normal human liver cell line (LO2) and found that circ-102,166 was also down-regulated in the HCC cell lines compared to the LO2 cell line (Fig. 2d). Taken together, these data indicate that circ-102,166 is down-regulated in HCC, and that low circ-102,166 expression significantly correlates with multiple clinicopathologic characteristics and a poor prognosis.

Fig. 2.

Fig. 2

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Circ-102,166 is down-regulated in HCC and correlates significantly with poor prognosis. (a) Analysis of the expression of circ-102,166 and its cognate gene TEX2 in 59 pairs of HCC tissues carried out by qRT-PCR. (b-c) Kaplan‐Meier plots depicting overall survival and recurrence‐free survival in the 77 HCC patients grouped according to low and high circ-102,166 expression. (d) Analysis of circ-102,166 expression in cultured normal human hepatocyte (LO2) and HCC cell lines performed by qRT-PCR. Results are mean ± SD (n = three independent experiments). *: p < 0.05 by two-tailed Student’s t test

Table 1.

Correlations between circ-102,166 expression levels and clinicopathologic characteristics in 77 HCC casess

characteristics circ-102,166 expression Pearson’s chi-square test (p value*)
Low High
Gender Male 35 30 0.192
Female 4 8
Age < 60 32 28 0.376
≧ 60 7 10
Cirrhosis No 13 13 0.935
Yes 26 25
AFP < 20 11 12 0.746
≧ 20 28 26
Tumor size < 5 cm 15 24 0.03
≧ 5 cm 24 14
Differentiation High 6 12 0.24
Moderate 30 24
Low 3 2
TNM stage I 14 25 0.032
II 8 4
III 17 9
BCLC A + B 21 29 0.039
C 18 9
Vascular invasion No 20 29 0.022
Yes 19 9
Tumor number Single 34 32 0.71
Multiple 5 6
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*Results were considered statistically significant at p < 0.05

Circ-102,166 overexpression inhibits the proliferation, invasion and migration of HCC cells

To decipher whether circ-102,166 may play a role in HCC progression, we chose two HCC cell lines (SK-Hep1 and LM3) with relatively low endogenous circ-102,166 expression levels to establish circ-102,166-overexpression cell lines. We found that exogenous circ-102,166 overexpression had no significant effect on the TEX2 mRNA level in SK-Hep1 and LM3 cells (Fig. 3a). Next, we investigated the effect of circ-102,166 overexpression on HCC cell proliferation. Using CCK8 assays, we found that exogenous circ-102,166 overexpression significantly restrained HCC cell growth compared to the respective vector controls (Fig. 3b). Additional colony formation assays confirmed that circ-102,166 overexpression significantly inhibits the proliferation of HCC cells (Fig. 3c). To subsequently clarify whether the effect of circ-102,166 on the proliferation of HCC cells is achieved through regulating cell cycle progression or apoptosis, we performed flow cytometry. We found that circ-102,166 overexpression significantly promoted G0/G1 arrest and decreased the proportion of S-phase cells, but had no effect on apoptosis (Fig. 3d-e). We next investigated whether circ-102,166 affects HCC cell invasion and migration using transwell and scratch wound healing assays, respectively. The invasion and migration abilities of HCC cells were found to be remarkably decreased after circ-102,166 overexpression (Fig. 3f-g). These data indicate that circ-102,166 overexpression significantly inhibits the proliferation, invasion and migration of HCC cells.

Fig. 3.

Fig. 3

Fig. 3

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Circ-102,166 overexpression inhibits the proliferation, invasion and migration of HCC cells. (a) Stable overexpression of circ-102,166 in LM3 and SK-Hep1 cells confirmed by qRT-PCR. Circ-102,166 overexpression did not affect the expression level of TEX2, the cognate gene of circ-102,166. CCK8 (b) and colony formation (c) assays showing that circ-102,166 overexpression significantly inhibits the growth of LM3 and SK-Hep1 cells. (d) Cell cycle assays confirming that circ-102,166 overexpression significantly promotes G0/G1 arrest and decreases the S-phase fraction in LM3 and SK-Hep1 cells. (e) Annexin V-FITC apoptosis assay validating that circ-102,166 overexpression has no effect on HCC cell apoptosis. (f) Transwell assay showing that circ-102,166 overexpression significantly inhibits the invasion of LM3 and SK-Hep1 cells. (g) Scratch wound healing assay showing that circ-102,166 overexpression markedly inhibits the migration of LM3 and SK-Hep1 cells. Results are presented as mean ± SD (n = three independent experiments). *: p < 0.05 by two-tailed Student’s t test

Circ-102,166 silencing promotes the proliferation, invasion and migration of HCC cells

To validate the effects of circ-102,166 on the proliferation, invasion and migration of HCC cells, we silenced circ-102,166 expression in SK-Hep1 cells using siRNA (Fig. 4a). Subsequent CCK8 and colony formation assays revealed that circ-102,166 silencing significantly enhanced the proliferation of these HCC cells (Fig.4b-c). Cell cycle and Annexin V-FITC apoptosis assays revealed that circ-102,166 silencing significantly reduced G0/G1 arrest and increased the proportion of S-phase cells, but had no effect on apoptosis in SK-Hep1 cells (Fig. 4d-e). Additional transwell and scratch wound healing assays confirmed that circ-102,166 silencing significantly enhances the invasion and migration of HCC cells (Fig. 4f-g). Based on the above results, we wondered whether circ-102,166 silencing may also affect the transformed state of normal liver LO2 cells with a relatively high expression of circ-102,166. We found that circ‐102,166 silencing significantly enhanced the proliferation, invasion and migration of LO2 cells (Fig.S1). Collectively, these in vitro data indicate that circ-102,166 plays an important role in regulating the proliferation, invasion and migration of HCC cells.

Fig. 4.

Fig. 4

Fig. 4

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Circ-102,166 silencing promotes the proliferation, invasion and migration of HCC cells. (a) siRNA-mediated knockdown of the expression level of circ-102,166 in SK-Hep1 cells. CCK8 (b) and colony formation (c) assays showing that circ-102,166 knockdown significantly promotes the growth of SK-Hep1 cells. (d) Cell cycle assay showing that circ-102,166 silencing significantly reduces G0/G1 arrest and increases the S-phase fraction in SK-Hep1 cells. (e) Annexin V-FITC apoptosis assay validating that circ-102,166 silencing has no effect on HCC cell apoptosis. (f) Transwell assay confirming that circ-102,166 knockdown strongly promotes the invasion of SK-Hep1 cells. (g) Scratch wound healing assay showing that circ-102,166 knockdown markedly promotes the migration of SK-Hep1 cells. Results are presented as mean ± SD (n = three independent experiments). *: p < 0.05 by two-tailed Student’s t test

Circ-102,166 inhibits HCC tumorigenicity in vivo

We next explored the effects of circ-102,166 overexpression or silencing on HCC tumorigenicity in vivo. To this end, we first established stably silenced circ‐102,166 HCC cells (SK-Hep1-circ-102,166 Ri) and negative control cells (SK-Hep1-scramble). Next, we subcutaneously implanted SK-Hep1-vector, SK-Hep1-circ-102,166, SK-Hep1-scramble and SK-Hep1-circ-102,166 Ri cells on the back of athymic nude mice. After 4–6 weeks, the mice were anesthetized and euthanized, and tumors were collected for further analysis. We found that circ-102,166 overexpression significantly reduced tumor weight and tumor size (Fig. 5a-5b). Conversely, we found that circ‐102,166 silencing significantly increased tumor weight and tumor size (Fig. 5c-5d). Overall, these in vivo data indicate that circ-102,166 can inhibit the tumorigenicity of HCC cells.

Fig. 5.

Fig. 5

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Circ-102,166 overexpression inhibits HCC tumorigenesis in vivo. Circ-102,166 overexpression significantly decreased tumor weight (a) and size (b). Circ-102,166 silencing markedly increased tumor weight (c) and size (d). Results are presented as mean ± SD (n = 5 mice per group). *: p < 0.05 by two-tailed Student’s t test

Circ-102,166 binds to oncomiRs miR-182 and miR-184

Previous studies have shown that exonic circRNAs can function as miRNA sponges to regulate the expression of their downstream target genes. Given that circ-102,166 is an exonic circRNA and mainly localized in the cytoplasm, we set out to predict potential miRNAs associated with circ-102,166 using the TargetScan, MiRanda and Circular RNA Interactome (https://circinteractome.nia.nih.gov) databases. By doing so, potential binding sites of miR-182 and miR-184 were predicted in circ-102,166 (Fig. 6a). Prior studies showed that miR-182 and miR-184 are upregulated in HCC and can promote the progression of HCC. Therefore, we speculated that circ-102,166 may inhibit the proliferation and invasion of HCC cells through binding to miR-182 and miR-184. To test this hypothesis, we first constructed a circ-102,166 luciferase reporter plasmid to confirm that circ-102,166 can directly bind to miR-182 and miR-184. Subsequent dual-luciferase reporter assays showed that HCC cells transfected with miR-182 or miR-184 mimics exhibited significantly decreased luciferase activities compared to the negative controls (Fig. 6b-6c). Then, we mutated the binding site in circ-102,166 of miR-182 and miR-184. Subsequent co-transfection of mutant circ-102,166 and the respective miRNA mimics didn’t affect the luciferase activity compared to its negative controls (Fig. 6d). To further validate the binding of circ-102,166 to the two miRNAs, RNA pull-down assays were carried out in SK-Hep1 and LM3 cells. qRT-PCR revealed that endogenous miR-182 and miR-184 were significantly enriched using a biotinylated circ-102,166 probe (Fig. 6e). Additional RNA-immunoprecipitation (RIP) analysis using an anti-Ago2 antibody after miR-182 or miR-184 overexpression showed that circ-102,166 can specifically be recruited to the miRNP complex (Fig. 6f). Previously, miR-182 has been reported to promote HCC progression by repressing the expression of FOXO3a and metastasis suppressor 1 (MTSS1) and upregulating their downstream targets, such as c-MYC [28–31]. MiR-184 has also been shown to be upregulated in HCC cell lines and tissues and exogenous expression of miR-184 has been found to lead to downregulation of SOX7, resulting in upregulation of c-MYC and phosphorylation of RB, thereby increasing HCC cell proliferation and tumorigenicity [32, 33]. Therefore, we next tested whether circ-102,166 may affect the expression of miR-182 and miR-184 downstream targets by western blotting. We found that circ-102,166 overexpression significantly upregulated the protein levels of MTSS1, FOXO3a and SOX7, and downregulated the protein level of c-MYC and the phosphorylation level of RB (Fig. 6g). In contrast, knockdown of circ-102,166 reversed the effects. Taken together, these results indicate that circ-102,166 can bind miR-182 and miR-184 to regulate the expression of their downstream target genes.

Fig. 6.

Fig. 6

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Circ-102,166 binds to miR-182 and miR-184. (a) Predicted binding sites (highlighted in red) in circ-102,166 for miR-182 or miR-184. (b-c) pGL3-circ-102,166 dual-luciferase reporter assays in SK-Hep1 and LM3 cells transfected with negative control oligonucleotides, miR-182 mimics or miR-184 mimics. (d) pGL3-circ-102,166 Mut1 or pGL3-circ-102,166 Mut2 dual-luciferase reporter assays in SK-Hep1 and LM3 cells transfected with the indicated oligonucleotides. Sequences of pGL3-circ-102,166 mutants are highlighted in green. (e) RNA pull-down assays showing that endogenous miR-182 and miR-184 can be enriched using a biotinylated circ-102,166 probe. An oligo probe served as negative control. (f) RIP assays showing that circ-102,166 is specifically recruited to miRNP complexes following immunoprecipitation with an Ago2 antibody. IgG was used as a negative control. (g) Western blot analyses of the protein levels of MTSS1, FOXO3a, SOX7, p-RB (ser807/811) and c-MYC, downstream targets of miR-182 and miR-184, in the indicated cells. Data are presented as mean ± SD. *: p < 0.05 by two-tailed Student’s t test

Circ-102,166-mediated inhibition of proliferation and invasion of HCC cells is miR-182 /miR-184-dependent

Next, we examined the roles of miR-182 and miR-184 in mediating the effect of circ-102,166 on HCC cell proliferation and invasion. Using CCK8 assays, we found that cells transfected with miR-182 or miR-184 mimics significantly rescued circ-102,166-induced growth inhibition (7a-7b). EDU incorporation assays confirmed that circ-102,166 overexpression markedly decreased the proliferation of HCC cells (Fig. 7c). As expected, cells transfected with miR-182 or miR-184 mimics remarkably reversed circ-102,166-induced proliferation inhibition. Furthermore, cells transfected with miR-182 or miR-184 mimics showed a significant circ-102,166-induced invasion inhibition (Fig. 7d). We also tested whether rescuing miR-182 or miR-184 has influence on the function of circ-102,166 in regulating the expression levels of MTSS1, FOXO3a, SOX7, p-RB and c-MYC. We found that exogenous circ-102,166 overexpression-induced alternations in the expression levels of MTSS1, FOXO3a, SOX7, p-RB and c-MYC were strongly reversed by miR-182 or miR-184 overexpression (Fig. 7e). Moreover, miR-182 or miR-184 knockdown strongly abolished the enhanced proliferation and invasion induced by circ-102,166 silencing in HCC cells (Fig. S2a-d). Conversely, we found that circ-102,166 silencing-induced changes in the expression of MTSS1, FOXO3a, SOX7, p-RB and c-MYC were significantly reversed by miR-182 or miR-184 inhibitors (Fig. S2e). These data indicate that circ-102,166-mediated inhibition of proliferation and invasion is miR-182/miR-184-dependent in HCC cells.

Fig. 7.

Fig. 7

Fig. 7

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Circ-102,166-mediated inhibition of HCC cell proliferation and invasion is miR-182/miR-184-dependent. CCK8 (a-b) and EDU incorporation (c) assays showing that miR-182 or miR-184 overexpression significantly rescues circ-102,166-mediated growth inhibition in LM3 and SK-Hep1 cells. (d) Transwell assays showing that miR-182 or miR-184 overexpression significantly reverse circ-102,166-mediated invasion inhibition in LM3 and SK-Hep1 cells. (e) Western blot analyses confirming that miR-182 or miR-184 overexpression markedly reverse circ-102,166 overexpression-induced alternations in MTSS1, FOXO3a, SOX7, p-RB and c-MYC levels. (f) Schematic diagram of a hypothetical model. Data are presented as mean ± SD (n = 5 mice/group). *: p < 0.05 by two-tailed Student’s t test

Overall, our results show that circRNA circ-102,166, formed by circularization of exons 5, 6 and 7 of TEX2, exerts tumor-suppressing activity by sponging miR-182 and miR-184. Under normal conditions, circ-102,166 binds to miR-182 and miR-184 to upregulate the expression of FOXO3a, MTSS1 and SOX7, which inhibit the proliferation and invasion of cells. Circ-102,166 downregulation in HCC leads to release of miR-182 and miR-184 and, thus, decreased expression of FOXO3a, MTSS1 and SOX7 and increased expression of c-MYC and p-RB, thereby enhancing the proliferation and invasion of HCC cells (Fig. 7f).

Discussion

Although circRNAs were first discovered almost 40 years ago [34], they were initially considered as errors of normal splicing processes [35]. Only after publication of the regulatory role of ciRS-7 (also named CDR1-as) in 2013 [8, 9], circRNAs began to attract attention. Subsequently, ample studies have indicated that circRNAs can play important roles in normal cellular differentiation and tissue homeostasis as well as in disease development, especially in cancer. As yet, elucidation of the expression and functional role of circRNAs in HCC development is still an ongoing process. Here, we integrated three HCC circRNA microarray-based expression datasets (GSE94508, GSE97332 and GSE 78520) to identify commonly dysregulated circular RNAs. Circ-102,166 was found to be significantly down-regulated in all three datasets. We further investigated the function and mode of action of circ-102,166 using a series of functional assays and found that it plays a tumor-suppressing role in HCC.

Several circRNAs have been found to originate from protein-coding genes, and previous studies have shown that circRNAs can modulate the transcription of their cognate mRNAs [36, 37]. Circ-102,166 originates from TEX2, but we found that it did not affect the expression of TEX2 in HCC. This finding suggests that circ-102,166 plays tumor suppressive roles through other mechanisms in HCC. Sponging miRNAs is another important function of circRNAs. We found that circ-102,166 can bind miR-182 and miR-184 to inhibit their functions. Previous studies have shown that miR-182 and miR-184 are up-regulated in HCC and can promote the development and progression of HCC [28–33]. However, the mechanism underlying miR-182 and miR-184 dysregulation in HCC is still not fully elucidated. Du et al. [38] reported that hypoxia can induce miR-182 up-regulation to promote angiogenesis by targeting RASA1 in HCC. Qin et al. [39] showed that miR-182 levels are significantly increased in HCC patients treated with cisplatin-based chemotherapy and that upregulation of miR-182 increased drug resistance in cisplatin-treated HCC cells by regulating TP53INP1. Leung et al. [40] found that Wnt/β-catenin-activated miR-183/96/182 expression promotes cell invasion in HCC and Wang et al. [41] found that LINC01018 confers a tumor-suppressor role through sponging microRNA-182-5p in HCC. Despite these findings, no additional studies have focused on the regulatory role of miR-182 in HCC. In addition, there are no reports on the regulatory role of miR-184 in HCC. Here, we revealed a novel mechanism for miR-182 and miR-184 regulation in HCC in which a circRNA, circ-102,166, can directly bind and sponge these miRNAs. We further validated that dysregulation of circ-102,166 in HCC leads to deregulation of miR-182 and miR-184 targets, including FOX3a, SOX7, MTSS1, p-RB and C-MYC, highlighting the importance of circRNAs in gene regulatory networks.

In this study, we found that circ-102,166 was significantly down-regulated in HCC tissues compared to paired non-tumorous tissues. In addition, the expression level of circ-102,166 was found to be significantly associated with multiple clinicopathologic characteristics, including tumor size (p = 0.03), TNM stage (p = 0.032), BCLC stage (p = 0.039) and vascular invasion (p = 0.022) in our HCC patient cohort. Moreover, circ-102,166 down-regulation was found to significantly correlate with shorter OS and RFS times in HCC patients. These results suggest that circ-102,166 may serve as a novel diagnostic and therapeutic target for HCC. Previous studies have indicated that circRNAs are remarkably stable and may be abundant in tissues and blood [42–45]. Memczak et al. [46] also found that the expression of circular RNAs in human blood may reveal and quantify the activity of hundreds of coding genes not accessible by classical mRNA-specific assays. In addition, Li et al. [47] reported that some circRNAs were enriched in exosomes and might act as promising biomarkers for cancer diagnosis. These findings indicate that circRNAs may be used as biomarkers for the diagnosis of various diseases, including cancer. Thus, it will be worth to investigate whether circ-102,166 can be detected in blood or even in circulating exosomes and, as such, can serve as a biomarker for HCC.

Although circ-102,166 was selected for investigation in the present study, there are still many other circRNAs exhibiting differential expression between HCCs and matched non-tumor liver tissues. We speculate that several of them may also participate in the development of HCC, especially up-regulated circRNAs which account for the major proportion of differentially expressed circRNAs. Here, we not only identified circ-102,166 as a candidate tumor suppressor in HCC, but also validated its mode of action mediated by miR-182 and miR-184. Together, our findings suggest that circ-102,166 may serve as a novel biomarker and potential therapeutic target for HCC.

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Acknowledgements

This project was supported by the National Key R&D Plan 2017YFA0104304, the National 13th Five-Year Science and Technology Plan Major Projects of China, 2017ZX10203205, the National Natural Science Foundation of China, 81802897, 81702393, 81770648, 81670601, 81972286, the Guangdong Basic and Applied Basic Research Foundation, 2015A030312013, 2017A030311034, 2018A030313259, the Science and Technology Program of Guangdong Province, 2017B020209004, 20169013, 2017B030314027, the Science and Technology Program of Guangzhou city, 2014Y2-00200, 201604020001, 201508020262, 201400000001-3 and Sun Yat-sen University Young Teacher Training Project, 19ykpy18.

Author contributions

Conceptualization: Rong Li, Yinan Deng and Wei Liu; Data curation: Rong Li, Jinliang Liang, Zhongying Hu, Xuejiao Li and Huanyi Liu; Formal analysis: Rong Li, Yinan Deng, Jinliang Liang and Zhongying Hu; Funding acquisition: Rong Li, Guihua Chen, Qi Zhang, Yang Yang and Wei Liu; Investigation: Rong Li, Jinliang Liang, Zhongying Hu, Xuejiao Li and Huanyi Liu; Methodology: Rong Li, Yinan Deng, Jinliang Liang, Zhongying Hu, Xuejiao Li, Guoying Wang, Tong Zhang and Wei Liu; Project administration: Rong Li, Yinan Deng, Qi Zhang, Yang Yang and Wei Liu; Resources: Rong Li, Yinan Deng, Jinliang Liang, Huanyi Liu, Guoying Wang, Tong Zhang, Guihua Chen, Qi Zhang, Yang Yang and Wei Liu; Supervision: Rong Li, Yinan Deng, Qi Zhang, Yang Yang and Wei Liu; Writing – original draft: Rong Li and Jinliang Liang; Writing – review & editing: Rong Li, Yinan Deng, Qi Zhang, Yang Yang and Wei Liu.

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest.

Ethical approval

A total of 136 patients who were histopathologically and clinically diagnosed as HCC at the third affiliated hospital, Sun Yat-Sen University (Guangzhou, Guangdong, China) were recruited for this study. All procedures with human subjects in this study were conducted in accordance with the Institutional Research Ethics Committee. Informed written consent was obtained from all participants. Correlations between circ-102,166 expression and clinicopathological parameters and patient survival were analyzed using 77 HCC specimens with detailed clinical follow-up data. The expression profile of circ-102,166 was determined in the other 59 HCC specimens for which paired non-tumorous tissues were available. All experimental procedures involving animals were conducted in accordance with the Chinese legislation regarding experimental animals and were approved by the Animal Ethical and Welfare Committee (AEWC) of Guangzhou Forevergen Medical Laboratory Animal Center (No. GFAC-AEWC-118).

Footnotes

Publisher’s note

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

Rong Li, Yinan Deng and Jinliang Liang contributed equally to this work.

Contributor Information

Guihua Chen, Email: chgh1955@263.net.

Wei Liu, Email: lwei6@mail.sysu.edu.cn.

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