Circular RNA hsa_Circ_101141 as a Competing Endogenous RNA Facilitates Tumorigenesis of Hepatocellular Carcinoma by Regulating miR-1297/ROCK1 Pathway.

As a novel class of noncoding RNAs, circular RNAs (circRNAs) have been recently reported to be involved in cell development and function. However, the functional role of circRNAs in hepatocellular carcinoma (HCC) remains unclear. In the present study, we found that the expression of human circ_101141 was upregulated in HCC tissues and cells. In addition, downregulation of circ_101141 dramatically inhibited cell proliferation, migration, and invasion in HCC cells. In addition, by using the bioinformatics tools, the potential target of circ_101141 was predicted. Mechanistic investigations indicated that circ_101141 acted as a miR-1297 "sponge"; meanwhile, Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) was a direct target of miR-1297. Further experiments demonstrated that circ_101141 contributed to the progression of HCC by acting as competing endogenous RNA (ceRNA) of miR-1297 to regulate ROCK1 expression. Furthermore, knockdown of circ_101141 attenuated HCC tumorigenesis in vivo. Taken together, these findings indicated that circRNA circ_101141 acted as a ceRNA to facilitate tumorigenesis of HCC by regulating miR-1297/ROCK1 pathway.

Introduction

Hepatocellular carcinoma (HCC) is the most commonly diagnosed type of primary liver cancer, which contributes up to 40% of total liver cancer deaths[1-3]. Chronic hepatitis B virus or hepatitis C virus infection, excessive alcohol consumption, immune-related hepatitis, and obesity are generally considered to be major risk factors for HCC[4,5]. The molecular pathogenesis of HCC is very complex, including gene mutations, chromosomal aberrations, as well as molecular-pathway-related factor alterations[6-8]. In recent years, although the survival rate of patients with liver cancer has improved, the outcomes of HCC patients remain unsatisfactory due to the delayed diagnosis, drug resistance, and high adverse side effects. Therefore, it is necessary to explore the new molecular factors and new pathways involved in HCC tumorigenesis, which may be specific targets for the treatment of HCC.

Circular RNAs (circRNAs) are a new class of noncoding RNAs that were previously considered as a product of abnormal RNA splicing without biological function[9,10]. Recently, increasing evidence suggested that circRNAs are able to regulate gene expression at the transcriptional or post-transcriptional level by contacting with microRNAs (miRNAs) or other molecules[11-13]. CircRNAs are mainly present in the cytoplasm and are characterized as a covalent closed-loop structure lack of 3′ and 5′ ends, which may act as regulators for carcinogenesis in variety of cancers, such as bladder cancer, colorectal cancer, gastric cancer, etc.[13,14]. In HCC, several circRNAs have been identified to be involved in HCC progression. For instance, circRNA-100338 directly interacted with miR-141-3p to regulate invasion potential in liver cancer cells[15]. Another study showed that circ_0005986 could inhibit tumorigenesis by regulating miR-129-5p[16]. CircRNA cSMARCA5 was able to inhibit the growth and metastasis of HCC by regulating miR-17-3p and miR-181b-5p[17]. The above studies indicate that circRNAs may be involved in the development of HCC. However, the underlying mechanism of circRNAs in HCC tumorigenesis is not completely clear, and further research is still needed.

In published literatures, we found that a specific circRNA called circ_101141 was upregulated in acute myeloid leukemia and could target the miR-181 family[18]. To our knowledge, most of the target genes of miR-181 family were involved in the tumor pathological process, suggesting that circ_101141 might be an important regulator in the tumor development. However, the role and mechanism of circ_101141 remain unclear in HCC.

Based on this information, our hypothesis is that circ_101141 might be specifically enriched in HCC cells and may interact with other molecular factors that participate in tumorigenesis. The findings might set a novel sight into pathologic mechanism of HCC and provide new therapeutic strategies in future treatment of HCC.

Materials and Methods

Tumor Sample Collection

The protocols of human studies were approved by the Ethical Committee of The First Affiliated Hospital of Xinjiang Medical University (No. 20150402-05). All patients enrolled in this study have given their written informed consents prior to conduct the clinical research–related procedure. None of these patients had received chemotherapy treatment before the surgery. Samples (60 HCC tussles and adjacent paired nontumor tissues) were collected from The First Affiliated Hospital of Xinjiang Medical University. These fresh tissue samples were stored at −80°C for further analysis.

Cell Culture and Transfection

Cell lines used in this study include human HCC cell lines (MHCC97 H, HCCLM3, SK-HEP-1, Hep3B, and Huh7), normal human liver cell line (LO2), as well as HEK293 cell line, and they were all obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 1/10 of fetal bovine serum (FBS) and 1% penicillin/streptomycin was used as culture medium. The culture environment maintained at 37′°C contains 5% CO2.

For cell transfection, short hairpin RNA (shRNA) targeting circ_101141 (sh1-circRNA and sh2-circRNA) or linear ANAPC7 (sh-ANAPC7) and negative control (sh-NC); miR-1297 mimic, inhibitor, and their control plasmids (NC mimic, NC inhibitor); plasmid pcDNA3.1 Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1); and control vector were purchased from Thermo Fisher Scientific (Carlsbad, CA, USA). Transfection of cells was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. All cells were harvested 48 h after transfection for further tests.

Quantitative Real-Time Polymerase Chain Reaction and RNase R Treatment

Total RNAs from human HCC tissue and HCC cells were extracted using TRIzol (Invitrogen). Then, extracted RNAs were reverse transcribed into cDNA. In addition, for miRNAs, MicroRNA Reverse Transcription Kit (Dalian, Takara Biotechnology, Japan) was used to perform reverse transcription. The primers used in the current experiments were designed and purchased from Sangon Biotech (Shanghai, China), which are listed in Table 1. Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out on ABI 7500 fast PCR System (Thermofisher, Carlsbad, CA, USA). For RNase R treatment, 1 unit of RNase R was added to digest 1 µg of RNA for 15 min at 37°C. Glyceraldehyde 3-phosphate dehydrogenase and U6 applied as internal references for mRNAs and miRNAs, respectively. The relative expressions were derived with 2–ΔΔCT method.

Table 1.

Primers Used in the Present Study.

Gene	Primer sequence
miR-1297	Forward: 5′-CACAGGGAAAGCGAGTGGTTGGTAA-3′
	Reverse: 5′-TTACCAACCACTCGCTTTCCCTGTG-3′
circRNA hsa_circ_101141	Forward: 5′-AGATGGGCGAAAGTTCACTTAGAAA-3′Reverse: 5′-TCTTGCCTGCTGAGTTGAGTATATC-3′
	Forward: 5′-CACAGGGAAAGCGAGTGGTTGGTAA-3′
U6	Forward: 5′-CTCGCTTCGGCAGCACA-3′
	Reverse: 5′-AACGCTTCACGAATTTGCGT-3′
ROCK1	Forward: 5′-GTGCTGTTTCACCGTTCACC-3′
	Reverse: 5′-TGGTAGCTAGCCAGCCAAAC -3′
GAPDH	Forward: 5’-CCGGGAAACTGTGGCGTGATGG-3’Reverse: 5’-AGGTGGAGGAGTGGGTGTCGCTGTT-3’

GAPDH: glyceraldehyde 3-phosphate dehydrogenase; ROCK1: Rho-associated, coiled-coil-containing protein kinase 1.

In Situ Hybridization and Fluorescence In Situ Hybridization

For in situ hybridization (ISH) test, tissue microarray (TMA) was first prepared consisted of triplicate 0.6-mm cores using manual tissue microarrayer (Beecher Instruments, Sun Prarie, WI, USA). Then, circ_101141 was labeled with digoxin-labeled RNA probe (Wuhan, BOSTER, China). The probe sequence was as follows: 5′-dig-GGUCCUCUGCUUUUAUGUCAGUUCUCA-3′. After washing with phosphate buffered saline, the samples were treated with 0.5% Triton X-100 at 4°C for 5 min. Next, TMA was hybridized with circ_101141 probe, as well as its positive control and NC, followed by incubation with antibody against digoxigenin for 30 min at 37°C. Afterward, the signal was determined by diaminobenzidine (DAB) solution.

For fluorescence in situ hybridization (FISH) assay, a cy3-labeled probe for detecting circ_101141 (5′-TCATGCCTGTTTTTGTCCTGTACCAGC-3′) and FAM-labeled probes (5′-GTTCCACCAGCATGCCTGCTGA-3′) for detecting miR-1297 were synthesized by GenePharma (Shanghai, China). After fixation, cells were incubated with prehybridization buffer, and then, hybridization was conducted at 55°C for 2 h. Afterwards, the nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Images were captured using Leica SP8 laser scanning confocal microscope.

Cell Counting Kit-8 Assay

Transfected Hep3B and Huh7 cells (3 × 103 per well) were plated onto 96-well plates. Cell Counting Kit-8 (CCK-8) reagents (ab228554, Abcam, Cambridge, UK) and Dulbecco’s modified Eagle medium were added into each well. After incubation with these reagents, optical density of cells was measured at 450′nm at different time points (24, 48, 72, and 96 h).

EdU Assay

For 5-ethynyl-2’-deoxyuridine (EdU) assay, KeyFluor647 Click-iT EdU Kit (Keygen, Jiangsu, China) was used for our experiments. Hep3B and Huh7 cells were treated with culture medium containing 20 μM EdU reagent. Then, cells were incubated in an incubator for 2 h; afterward, they were fixed with paraformaldehyde. The nuclei were stained with DAPI. The percentage of EdU positive cells were quantified and analyzed.

Cell Cycle Detection

Cell cycle was detected using flow cytometry analysis. Different groups of transfected Hep3B and Huh7 cells (1 × 106) were dissociated with 0.25% trypsin and fixed with 70% ice-cold ethyl alcohol for 24 h. Next, Cells were stained with propidium iodide and treated with RNAse A (0.1 mg/ml, Sigma, St. Louis, MO, USA) at 37°C for 30 min, followed by flow cytometry analysis (BD Biosciences, Carlsbad, CA, USA).

Wound-Healing Assay

Different groups of Hep3B and Huh7 cells were seeded into six-well plates. Cell monolayer was scratched using a pipette tip. After 24 h, cell movements were recorded by an Olympus microscope (100). The percentage of wound closure was analyzed using the following formula: (original width − width after migration)/original width.

Transwell Assay

Transwell assay was carried out for detecting cell migration abilities. Briefly, a number of 5 × 103 cells were seeded into the upper compartments of transwell chambers (Corning, New York, NY, USA) and cultured with basal medium without FBS, while in the lower chamber, the culture medium was supplemented with 10% FBS. The following day, the invaded cells were fixed and stained, and then calculated under an Olympus microscope (400).

Western Blot Assay

Total proteins from HCC cells were extracted using radioimmunoprecipitation assay lysis buffer and qualified by a bicinchoninic acid assay detecting kit (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (20 μg) were boiled at 100°C in sodium dodecyl sulfate (SDS) sample buffer for 5 min and separated with SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane. After blocking, membranes were incubated with primary anti-ROCK1 (ab134181, Abcam, Cambridge, UK; 1:1,000), anti-cylinD1 (ab251892, Abcam, 1:500), anti-p21 (ab218311, Abcam, 1:1,000), anti-E-cadherin (ab194982, Abcam, 1:500), and anti-MMP2 (ab97779, 1:1,000) antibodies for overnight. The following day, secondary anti-rabbit or anti-mouse antibodies (ab6940, ab97035, Abcam, 1:2,000) were added and incubated with membranes for 1 h at room temperature. The captured bands were quantified using Image Lab™ Software (Bio-Rad, Hercules, California, USA).

Dual-Luciferase Reporter Assay

The sequence of circ_101141 and the corresponding miR-1297 mutants were cloned and inserted into the 3′-untranslated region (3′UTR) of mirGLO vector (Promega, Madison, WI, USA). In addition, the fragments from ROCK1 3′UTR containing the predicted miR-1297 binding site or the corresponding mutants created by mutating the miR-1297 seed region binding site were also cloned into the pmiRGLO vector. In six-well plates, HEK293 cells were cotransfected with either wild-type (WT) or mutant luciferase reporter vector (2 μg) and either mimic miRNAs or NC. The relative luciferase activities were performed in Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

RNA Immunoprecipitation Assay

RNA Immunoprecipitation (RIP) assay was performed with EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, MA, USA). Briefly, different groups of Hep3B and Huh7 cells were lysed with RIP lysis buffer; then, magnetic beads conjugated with anti-Ago2 antibody or IgG was added. The expressions of Circ_101141 and miR-1297 were analyzed by qRT-PCR.

Tumor Xenograft Experiments

The protocols for animal experiments were approved by the Ethical Committee of The First Affiliated Hospital of Xinjiang Medical University (Approval No. IACUC-20180225-49). Twelve 5-week-old male BALB/c nude mice (16–20 g) were purchased from Charles River (Beijing, China) and kept under sterile-specific pathogen-free facility. Hep3B cells (2 × 106) transfected with shRNA-circ_101141 or sh-NC were inoculated subcutaneously in the right flank of these mice. The tumor size was monitored every 7 days up to 28 days after initial injection. Mice were sacrificed after 4 weeks and tumor weight was then weighted.

Immunohistochemistry

Expression of ROCK1 and proliferative indicator Ki67 were detected by immunohistochemistry (IHC) in tumor tissues from nude mice. Firstly, tissues were embedded in paraffin and cut into 4 µm sections. Next, endogenous peroxidases were blocked with 3% H2O2 solution for avoiding nonspecific binding of primary antibodies. Then, those sections were incubated with primary anti-ROCK1 antibody (ab45171, 1:1,000, Abcam, Cambridge, MA, UK), anti-Ki67 (ab15580, 1:1,000, Abcam) at 4°C. Twenty-four hours later, the secondary anti-rabbit antibody was added and coincubated with sections, and then sections were immunostained using DAB plus kit. The images were then captured and analyzed.

Statistical Analysis

All cell experiments were independently repeated at least in triplicate. Data were presented as mean ± standard deviation and analyzed using SPSS v19.0 (Inc., Chicago, IL). The figures were made by Graph Pad Prism7. Statistical significance was determined by the Student’s t-test or one-way analysis of variance (ANOVA) test. Tukey honestly significant difference (HSD) test was used as post hoc following ANOVA (*P value < 0.05; **P < 0.01; ***P < 0.001 considered statistically significant). Moreover, Pearson’s correlation coefficient and Kaplan–Meier analysis were used for analyzing statistical correlation and survival curves, respectively.

Results

Circ_101141 was Highly Expressed in HCC Tissues and Cells

In our pre-analysis of GEO data (No. GSE94508, GSE97332), we found that hsa_circRNA_101141 was one of the upregulated circRNAs in HCC. Thus, the expression of circ_101141 was supposed to be upregulated in HCC. To confirm this hypothesis, we detected the expression of circ_101141 in HCC tissue and normal tissue using qRT-PCR. The results showed that the expression of circ_101141 was markedly upregulated in HCC tissues (P < 0.01, Fig. 1A) compared with adjacent normal tissue. Moreover, in ISH image with ×40 and ×100 magnification, we found that the expression of circ_101141 was significantly higher in HCC tissues compared with nontumor tissue (P < 0.01, Fig. 1B). In addition, correlation analysis showed that the expression of circ_101141 was related with lower overall survival rate (P = 0.0427, Fig. 1C). Moreover, we conducted the in vitro experiments, and the expression of circ_101141 was determined in different cell lines including normal liver cell line (LO2) and HCC cell lines (MHCC97 H, HCCLM3, SK-HEP-1, Hep3B, and Huh7). The results demonstrated that the expression of circ_101141 was significantly increased in all HCC cell lines (MHCC97 H, HCCLM3, SK-HEP-1, Hep3B, and Huh7) (P < 0.01, Fig. 1D), especially higher in Hep3B and Huh7 cell lines. Therefore, we selected Hep3B and Huh7 for further experiments. Furthermore, we found linear ANAPC7 but not circRNA ANAPC7 (circ_101141) was degraded by Rnase R in Hep3B and Huh7 cell lines (P < 0.01, Fig. 1E). Finally, we examined the expression of circ_101141 in nuclear and cytoplasm in Hep3B and Huh7 cell lines, respectively. The results showed that circ_101141 expressed not only in nuclear but also in cytoplasm. Moreover, in the cytoplasm, the percentage of circular ANAPC7 (circ_101141) was higher than linear RNA ANAPC7 (P < 0.01, Fig. 1F). Taken the above results together, we concluded that circular circ_101141 was highly expressed in HCC tissues and cells, and it was mainly expressed in cell cytoplasm.

Figure 1.

The expression of circ_101141 was detected in HCC tissues and cells. (A) The expression of circ_101141 in 60 pairs of HCC tissues and normal tissues was detected by qRT-PCR. (B) ISH test was conducted to detect the expression of circ_101141 in HCC tissues and normal tissues (with ×40 and ×100 magnification). (C) The overall survival curves of HCC patients with low and high circ_101141 expression based on median circ_101141 value. (D) qRT-PCR was used to assess the expression of circ_101141 in normal human liver cell line (LO2) and HCC cell lines (MHCC97 H, HCCLM3, SK-HEP-1, Hep3B, and Huh7). (E) qRT-PCR analysis of linear ANAPC7 mRNA and circ_101141 in Hep3B and Huh7 cells treated with Rnase R. (F) The expression of linear ANAPC7 mRNA and circ_101141 in Hep3B and Huh7 cells was determined in nuclear and cytoplasm, respectively. Statistical significance was determined by the Student’s t-test or one-way ANOVA. Data were expressed as mean ± SD. **P < 0.01 represent statistically difference. HCC: hepatocellular carcinoma; ISH: in situ hybridization; qRT-PCR: quantitative real-time polymerase chain reaction; SD: standard deviation.

Downregulation of Circ_101141 Suppressed Cell Proliferation, Migration, and Invasion in HCC Cells

To further investigate the biofunctional role of circ_101141 in HCC, we conducted a series of loss-of-function experiments. At the very beginning, two shRNAs for circ_101141 (sh1-circRNA and sh2-circRNA) and one shRNA for linear ANAPC7 (sh-ANAPC7) were checked for their knockdown efficiency in Hep3B and Huh7 cells. As shown in Fig. 2A, both sh1-circRNA and sh2-circRNA were able to specifically knockdown circ_101141, not linear ANAPC7 in HCC cell lines, indicating that the present knockdown system can be used in further experiments. Furthermore, because of better knockdown efficiency, sh1-circRNA was chosen for further experiments (P < 0.01, Fig. 2A). CCK-8 assay demonstrated that downregulation of circ-101141 with sh1-circRNA significantly suppressed proliferation vitality of Hep3B and Huh7 cells in a time-dependent manner (P < 0.05, P < 0.01, Fig. 2B). Colony formation assay implied that knockdown of circ_101141 decreased the clone number of HCC cells (P < 0.05, P < 0.01, Fig. 2C). Furthermore, the percentage of EdU positive cell was lower in sh1-circRNA group, suggesting that knockdown of circ_101141 could inhibit proliferation of HCC cells (P < 0.01, Fig. 2D). Utilizing flow cytometry, we found that interference of circ_101141 partially blocked cell cycle in G1 phase (P < 0.05, P < 0.01, Fig. 2E).

Figure 2.

Knockdown of circ_101141 suppressed cell proliferation in Hep3 and Huh7 cells. (A) The expression level of circ_101141 was measured by qRT-PCR in Hep3 and Huh7 cells transfected with shRNAs (sh1-circRNA, sh2-circRNA, and sh-ANAPC7). (B) The proliferation of Hep3 and Huh7 cells that were transfected with sh1-circRNA or sh-NC was examined in different time points (0, 24, 48, 72, and 96 h). (C) Colony formation assay was conducted in Hep3 and Huh7 cells that were transfected with sh1-circRNA or sh-NC. (D) Proliferation was detected in Hep3 and Huh7 cells transfected with sh1-circRNA or sh-NC by EdU assay. (E) Cell cycle was determined in Hep3 and Huh7 cells transfected with sh1-circRNA or sh-NC by flow cytometry assay. Statistical significance was determined by the Student’s t-test or one-way ANOVA. Data were expressed as mean ± SD. *P < 0.05, **P < 0.01 represent statistically difference. ANOVA: analysis of variance; NC: negative control; qRT-PCR: quantitative real-time polymerase chain reaction.

Wound-healing assay results demonstrated that knockdown of circ_101141 exhibited a slower closing of scratch wound compared with control. The migration rate was reduced nearly 20% in sh1-circRNA group compared with sh-NC group (P < 0.05, Fig. 3A). Transwell assay showed that transfection with sh1-circRNA suppressed the invasion rate of Hep3B and Huh7 cells compared with sh-NC group (P < 0.05, P < 0.01, Fig. 3B). Overall, these data suggested that downregulation of circ_101141 inhibited cell proliferation, migration, and invasion in HCC cell lines.

Figure 3.

Knockdown of circ_101141 inhibited cell migration and invasion in Hep3 and Huh7 cells. (A) The effect of knockdown of circ_101141 on the migration ability of Hep3 and Huh7 cells was determined by wound-healing assay. (B) Transwell assay was conducted to determine the effect of circ_101141 on invasion ability of Hep3 and Huh7 cells. Statistical significance was determined by the Student’s t-test. Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, represent statistically difference. SD: standard deviation.

Circ_101141 Acts as a miR-1297 “Sponge”

In the recent years, accumulating evidence has suggested that miRNAs play as critical modulators to participate in action of other noncoding RNAs. To further explore the underlying mechanism, we used bioinformatics tool to predict the potential target of circ_101141 (starbase: http://starbase.sysu.edu.cn/). Fig. 4A shows the potential target of circ_101141 called miR-1297 and its potential binding site. Then, circ_101141 WT luciferase plasmids and mutated version (MUT) which containing the potential miR-1297 binding sites or miR-NC was generated. As shown in Fig. 4B, the results of dual-luciferase reporter assay demonstrated that miR-1297 was a direct target of circ_101141 (P < 0.01, Fig. 4B). Furthermore, result from RIP assay confirmed that endogenous level of miR-1297 was specifically increased in cells transfected with circ_101141 overexpression plasmid in AGO2 cargo (P < 0.01, Fig. 4C). Subsequently, FISH test was performed to determine the colocalization of circ_101141 and miR-1297. As shown in Fig. 4D, both two factors were expressed in the cytoplasm of HCC cells. Moreover, the expression of miR-1297 was identified in HCC cells, which transfected with sh1-circRNA or control plasmids (sh-NC). The data revealed that circ_101141 is negatively targeted by miR-1297 (P < 0.01, Fig. 4E). Meantime, the expression of miR-1297 was much lower in HCC tumor tissue compared with normal tissue (P < 0.01, Fig. 4F). Pearson’s correlation analysis showed that the expression of circ_101141 and miR-1297 exhibited a dramatically negative correlation (Fig. 4G) (r = −0.6620, P < 0.001). These data suggested that circ_101141 may act as a miR-1297 “sponge.”

Figure 4.

Circ_101141 inversely interacted with miR-1297. (A) Circ_101141 wide-type (WT) and the mutated-type (MUT) in the miR-1297 binding sites were shown. (B) Luciferase activity of HEK293 T cells cotransfected with miR-1297 mimics or NC mimics and luciferase reporters containing circ_101141 WT or MUT were detected. (C) Endogenous miR-1297 precipitated by AGO2 upon overexpression of circ_101141 were determined by RIP assay in HCC cells. (D) The colocalization of circ_101141 and miR-1297 was determined by FISH assay. (E) The expression of miR-1297 was detected in different groups by qRT-PCR. (F) The expression of miR-1297 was detected in tumor tissues and nontumor tissues by qRT-PCR. (G) The correlation between circ_101141 and miR-1297 was analyzed. Statistical significance was determined by the Student’s t-test or one-way ANOVA. Data were expressed as mean ± SD. **P < 0.01, represent statistically difference. ANOVA: analysis of variance; FISH: fluorescence in situ hybridization; HCC: hepatocellular carcinoma; RIP: RNA immunoprecipitation; qRT-PCR: quantitative real-time polymerase chain reaction; SD: standard deviation.

ROCK1 was a Direct Target of miR-1297

To further explore the underlying mechanism of circ_101141 and miR-1297 in HCC, we tried to find out the direct target of miR-1297. Among the putative targets, we focused on ROCK1, which mediates the expression of a variety of genes in response to HCC cell functions. The binding site of ROCK1 and miR-1297 was shown in Fig. 5A. Subsequently, luciferase reporter assay was performed in HEK 293 cells, which were cotransfected with miR-1297 mimics or NC mimics and ROCK1 3′-UTR WT or ROCK1 3′-UTR MUT transcript. The results indicated that ROCK1 was a target of miR-1297 (P < 0.01, Fig. 5B). Then, the expression of mRNA and protein of ROCK1 were detected in Hep3B and Huh7 cells transfected with miR-1297 mimic, miR-NC, miR-1297 inhibitor, or inhibitor-NC. As shown in Fig. 5C, D, the expression of mRNA and protein of ROCK1 was highly suppressed by overexpression of miR-2197, while increased by miR-1297 inhibitor, suggesting that the expression of ROCK1 was negatively regulated by expression of miR-1297 (P < 0.05, P < 0.01). Further, the mRNA and protein expression levels of ROCK1 were also determined in HCC tumor tissue and nontumor tissues. The results showed that both mRNA and protein expression of ROCK1 were higher in HCC tissues compared with normal tissue (P < 0.01, Fig. 5E, F). In addition, correlation analysis showed that the expression level of ROCK1 was positively correlated with expression of circ_101141 (r = 0.6460, P < 0.001), while negatively correlated with expression of miR-1297 (r = −0.6256, P < 0.001, Fig. 5G). Collectively, these data indicate that ROCK1 was a direct target of miR-1297.

Figure 5.

ROCK1 was a direct target of miR-1297. (A) ROCK1 wide-type (WT) and the mutated-type (MUT) in the miR-1297 binding sites were shown. (B) Luciferase activity of HEK293 T cells cotransfected with miR-1297 mimics or NC mimics and luciferase reporters containing ROCK1 WT or MUT were detected. (C and D) The expression of ROCK1 mRNA and protein level were determined by qRT-PCR and western blot in HCC cells that were transfected with miR-1297 mimic, miR-NC, miR-1297 inhibitor, or inhibitor-NC. (E and F) The ROCK1 mRNA and protein expression levels were determined by qRT-PCR and western blot in HCC tissues and nontumor tissues. (G) The correlation between ROCK1 and circ_101141 or miR-1297 was analyzed. Statistical significance was determined by the Student’s t-test or one-way ANOVA. Data were expressed as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 represent statistically difference. ANOVA: analysis of variance; HCC: hepatocellular carcinoma; NC: negative control; qRT-PCR: quantitative real-time polymerase chain reaction; ROCK1: Rho-associated, coiled-coil-containing protein kinase 1; SD: standard deviation.

Circ_101141 Promoted HCC Progression by Acting as Competing Endogenous RNA of miR-1297 to Regulate ROCK1 Expression

Given that circ_101141 plays a role in regulating HCC cell proliferation, migration, and invasion, meanwhile, it was directly interacted with miR-1297, we hypnotized that circ_101141 might exert its role in regulating cell proliferation, migration, and invasion via targeting miR-1297 and maybe further influence the expression of ROCK1. To this end, we first detected the expression of ROCK1 mRNA in three different groups (sh-NC + inh-NC, sh1-circRNA + inh-NC, and sh1-circRNA + miR-1297 inh), and the results showed that the expression of ROCK1 mRNA was downregulated by knockdown of circ_101141, while this effect was reversed by inhibiting miR-1297 (P < 0.01, Fig. 6A). Further, a series of gain-of-function and loss-of-function experiments were conducted in Hep3B and Huh7 cells that were cotransfected with sh1-circRNA or sh-NC and plasmid pcDNA3.1 ROCK1 or control vector. Then, in transfected Hep3B and Huh7 cells, the proliferation ability, cell cycle, cell migration, and invasion abilities were determined by CCK-8 assay, flow cytometry assay, wound-healing assay, and transwell assay, respectively. As shown in Fig. 6B–E, interference of circ_101141 markedly inhibited the proliferation, cell cycle, cell migration, and invasion abilities of Hep3B and Huh7 cells (P < 0.05, P < 0.01). However, these effects were abolished by overexpression of ROCK1 (P < 0.05, P < 0.01). In addition, the protein level of ROCK1 as well as biomarkers for cell cycle (cylinD1, p21), cell migration, and invasion (E-cadherin MMP2) were determined by western blot in different groups of HCC cells. As shown in Fig. 6F, level of cylinD1, MMP2 were down-regulated, p21 and E-cadherin were up-regulated in circ_101141 inhibited group, however, overexpression of ROCK1 reversed these effects (P < 0.05, P < 0.01, Fig. 6F). These results indicated that circ_101141 could promote HCC progression by acting as competing endogenous RNA (ceRNA) of miR-1297 to regulate ROCK1 expression.

Figure 6.

Circ_101141 promoted HCC progression by acting as ceRNA of miR-1297 to regulate ROCK1 expression. (A) The protein level of ROCK1 was determined in HCC cells cotransfected with sh1-circRNA or sh-NC and miR-1297 inhibitor or control NC. (B–E) CCK-8 assay, flow cytometry assay, wound-healing assay, and transwell assay were conducted in Hep3 and Huh7 cells that were cotransfected with sh1-circRNA or sh-NC and plasmid pcDNA3.1 ROCK1 or control vector. (F) The protein level of ROCK1, biomarkers for cell cycle (cylinD1, p21), cell migration, and invasion (E-cadherin MMP2) were determined by western blot in different groups of HCC cells. Statistical significance was determined by the Student’s t-test or one-way ANOVA. Data were expressed as mean ± SD. **P < 0.05, **P < 0.01, ***P < 0.001 represent statistically difference. ANOVA: analysis of variance; ceRNA: competing endogenous RNA; HCC: hepatocellular carcinoma; NC: negative control; qRT-PCR: quantitative real-time polymerase chain reaction; ROCK1: Rho-associated, coiled-coil-containing protein kinase 1; SD: standard deviation.

Knockdown of Circ_101141 Attenuated HCC Tumorigenesis In Vivo

Finally, the effect of circ_101141 on tumorigenesis in vivo was determined using orthotopic xenograft mouse models. Briefly, male BALB/c nude mice (n = 6 each group) were implanted with a total amount of 5 × 106 Hep3B cells that were transfected with shRNA-circ_101141 or sh-NC by subcutaneous inoculation. Then, the expression of circRNA_101141 and miR-1297 was examined in both groups. As shown in Fig. 7A, compared with control group, the expression of circRNA_101141 was significantly downregulated in shRNA-circ_101141 group, while miR-1297 was upregulated (P < 0.01). For the tumor volume (measured every 7 days, up to 28 days) and tumor weight, as shown in Fig. 7B, C, the tumor volume and weight were dramatically reduced in circRNA_101141 shRNA treated group compared with sh-NC group (P < 0.05, P < 0.01). hematoxylin-eosin (HE) staining of tumor tissues in each group demonstrated that circRNA_101141 shRNA group had less tumor feature than control group (Fig. 7D). Moreover, the expression of ROCK1 and proliferative indicator Ki67 were detected in each group by IHC assay. The results demonstrated that the level of ROCK1 and Ki67 exhibited relatively low expression in shRNA circRNA_101141 group (Fig. 7E). These results revealed that knockdown of circ_101141 inhibited HCC tumorigenesis in vivo.

Figure 7.

Knockdown of circ_101141 attenuates HCC tumorigenesis in vivo. (A) The expression of circ_101141 and miR-1297 was tested in mice inoculated with Hep3B cells transfected with shcircRNA or sh-NC plasmid. (B) Tumors were harvested in day 28. (C) Tumor volume was measured every 7 days after the initial injection and up to 28 days. Tumor weight was measured after tumors were harvested. (D) HE staining was conducted in different groups (shcircRNA and sh-NC). (E) The expression of proliferation marker Ki67 and ROCK1 was detected by IHC assay. Statistical significance was determined by the Student’s t-test. Data were expressed as mean ± SD. **P < 0.01 represent statistically difference. HCC: hepatocellular carcinoma; NC: negative control; ROCK1: Rho-associated, coiled-coil-containing protein kinase 1; SD: standard deviation.

Discussion

Due to the current therapeutic limitations, understanding the underlying molecular mechanism of HCC tumorigenesis has attracted widespread attention from clinicians and researchers. As a novel gene regulator, circRNAs have been confirmed to play important roles in occurrence and progression of HCC as previously reported[15-17]. In our study, we focused on a novel circRNA called circ_101141, which was confirmed highly expressed in human HCC tissue and cell lines. Then, in order to investigate the functional role of circ_101141 in HCC pathologic process, loss-of-function experiments were conducted in circ_101141 knockdown systems. The results showed that downregulation of circ_101141 inhibited the proliferation, migration, and invasion of HCC cells, indicating that circ_101141 indeed plays a functional regulatory role in the pathogenesis and development of HCC.

To further explore the molecular mechanism of how circ_101141 exerts its functional role in HCC progression, we investigated the relationship between circ_101141 and its target miRNAs. As another essential member of noncoding RNA family, miRNAs also play crucial roles in pathologic process of HCC along with circRNAs[19]. Unlike circRNAs that lack of 5′ caps and 3′ polyadenylated tails, miRNAs usually consist of 22–25 nucleotides and are responsible for regulation of many genes post-transcriptionally by targeting 3′-UTRs of mRNA[20]. In some cases, circRNAs might act as ceRNAs, or in other word “sponge,” in the regulatory network referring to circRNAs, miRNA, and target genes [21-24]. So far in the HCC-related studies, Xiong et al. collected three gene chips from the GEO database and constructed a circRNA–miRNA–mRNA network, and they found circRNA_100291 and circRNA_104515 serve as ceRNAs in HCC[25]. Moreover, the expression of circFBLIM1 was also confirmed upregulated in HCC tissues and cell lines. It can be served as ceRNA of miR-346 to promote cell proliferation and inhibit cell apoptosis in HCC cells[26]. Another circRNA termed circRNA-101368 can not only function as ceRNA to regulate miR-200a but also target HMGB1/RAGE signaling pathway[27]. In our study, bioinformatics tools were used to predict the target of circ_101141 and key factors for its functional network. By using dual-luciferase reporter assay, RIP, and qRT-PCR, we found that circ_101141 acted as a “sponge” for miR-1297, and ROCK1 was a direct target of miR-1297. These findings revealed that circ_101141 could positively regulate ROCK1 expression by acting as a ceRNA of miR-1297.

As important downstream factors in our study, miR-1297 and ROCK1 both play important roles in HCC pathogenesis. In the published data, miR-1297 was shown to play an inhibitory role to suppress progression of HCC by inhibiting cell proliferation and enhancing cell apoptosis[28,29]. ROCK1 is a major member of ROCK family. ROCK controls the organization of actin cytoskeleton and cell movement by phosphorylating other factors, such as myosin light chain, myosin-binding subunit (MYPT1), and LIM kinase-1 and 2[30]. In HCC, ROCK1 has been confirmed to be involved in tumor invasiveness. For instance, in Ding et al.’s study, they found that ROCK1 was a target of miR-145 and contributed to the progression and metastasis of HCC[31]. In addition, Zhan et al. showed that miR-199a/b-5p suppressed HCC progression by post-transcriptionally inhibiting ROCK1[32]. In the current study, in order to explore how circ_101141 exert its role in regulating cell proliferation, migration, and invasion, the rescue experiments were carried out by using Hep3B and Huh7 cells cotransfected with sh1-circRNA or sh-NC and plasmid pcDNA3.1 ROCK1 or control vector. The results demonstrated that circ_101141 promoted HCC progression via targeting miR-1297/ROCK1 in HCC. Further in in vivo study, we confirmed that knockdown of circ_101141 inhibited HCC tumorigenesis. Therefore, our study provided a novel insight into the circRNAs–miRNA–mRNA regulatory network in the pathogenesis of HCC. However, in the current study, we only confirmed that the high expression of circ_101141 was negatively correlated with survival rate of HCC patients, and the correlation between circ_101141 expression and other clinical features remains unknown. In the future, in order to comprehensively understand the role of circ_101141 in different stages of HCC, further detailed correlation analysis between circ_101141 and other clinical features will be investigated.

In conclusion, this study provides the evidence of circ_101141 as a regulator of HCC cell functions, including cell proliferation, migration, and invasion. Moreover, circ_101141 may have negative impact on the pathological process of HCC and function as a ceRNA for miR-1297 to modulate ROCK1 expression. Our study demonstrated a novel circRNAs–miRNA–mRNA regulatory network, which contributes to a better understanding of the occurrence and development of HCC.