Hepatocellular carcinoma (HCC) is a dreadful threaten to human health worldwide. Many circular RNAs were reported to influence the malignant development of HCC. The aim of this study was to elucidate the role of circ_0091579 in HCC progression and the molecular fundamentation.
Expression of circ_0091579, microRNA-1225-5p (miR-1225-5p), and phospholipase C, β1 (PLCB1) was examined by quantitative reverse transcription-polymerase chain reaction or Western blotting. Cell viability, clonogenicity capacity, and apoptosis were determined via Cell Counting Kit-8 assay, colony formation assay, and flow cytometry, respectively. Transwell assay was employed to detect cell migration and invasion. Target relationship between miR-1225-5p and circ_0091579 or PLCB1 was demonstrated by dual-luciferase reporter assay. Moreover, role of circ_0091579 in vivo was assessed by Xenograft model assay.
Expression of circ_0091579 and PLCB1 was increased, while miR-1225-5p expression was decreased in HCC tissues and cells. Circ_0091579 or PLCB1 depletion had inhibitory effects on HCC cell proliferation and metastasis. Circ_0091579 sponged miR-1225-5p to upregulate PLCB1 expression in HCC cells. Silencing of miR-1225-5p contributed to HCC progression, which was mitigated by PLCB1 depletion. Circ_0091579 deficiency could suppress HCC tumor growth in vivo.
Circ_0091579 knockdown repressed HCC progression and tumorigenesis by regulating miR-1225-5p/PLCB1 axis, affording a novel molecular basis for HCC development.
Hepatocellular carcinoma (HCC) is a leading contributor of cancer-related mortality all over the world, ranking as the sixth most prevailing malignancy . Hepatitis C virus (HCV), hepatitis B virus (HBV) and nonalcoholic fatty liver disease (NAFLD) remain the top three risk factors of hepatocarcinogenesis . Despite the improved treatment tactics of HCC, the prognosis of HCC patients is still inferior . Therefore, detailed acknowledge of molecular mechanisms implicated with HCC progression will be helpful for HCC therapy.
Circular RNAs (circRNAs) are a category of covalently closed RNA molecules, with great abundance and stability in mammalian cells, and are associated with initiation and progression of multiple malignancies, including HCC . CircRNAs had potential values in HCC diagnosis and treatment and could serve as HCC suppressors of promoters [5, 6]. Through circRNA microarray, circRNA hsa_circ_0091579 was found to be upregulated in tumor tissues of HCC patients, which was derived from glypican 3 (GPC3) . Its gene is situated at chrX:132795757–132888203 and its spliced sequence length is 1145 bp. Regrettably, the functional influence of circ_0091579 on HCC progression has not been entirely clarified.
Functionally, circRNAs could act as microRNA (miRNA) sponges and reduce miRNA activity, thereby altering gene expression . MiRNAs are pivotal regulators in a variety of biological processes during tumor progression . In addition, a great many miRNAs are abnormally expressed in HCC, implying their oncogenic or tumor suppressor roles in HCC development . As for miR-1225-5p, it was demonstrated to be an inhibitor of HCC cell invasion, migration, and proliferation . Moreover, miR-1225-5p was estimated to be a target miRNA of circ_0091579 by circular RNA interactome. Whether miR-1225-5p was necessary for role of circ_0091579 in HCC needs to be elucidated.
MiRNAs could bind to the 3′-untranslated region (UTR) of target mRNAs, inducing transcription suppression or degradation . Phospholipase C, β1 (PLCB1), a member of PLC enzymes, expression of this intracellular signaling molecule, was reported to be deficient in schizophrenia patients . And PLCB1 expression in HCC tissues could indicate the inferior prognosis of patients with HCC . Furthermore, miRDB predicted that PLCB1 was a promising target gene of miR-1225-5p. We deduced that circ_0091579/miR-1225-5p/PLCB1 axis was involved in HCC progression.
In this work, relative abundance of circ_0091579, miR-1225-5p, and PLCB1 in HCC tissues and cells was evaluated. In addition, their function and interaction with each other were investigated, which might be helpful for HCC therapy.
Materials and Methods
Clinical Sample Collection
Twenty-six HCC tissues were resected from patients enrolled at The Second Affiliated Hospital of Xi'an Jiaotong University, as well as matched normal tissues. Before resection, ethical permission and written informed consent were procured from the Ethics Committee of The Second Affiliated Hospital of Xi'an Jiaotong University and all participators, respectively. Clinical specimens were preserved at − 80 °C.
Human liver epithelial-2 cells THLE-2 (CRL-2706; American Type Culture Collection, Manassas, VA, USA), HCC cells HCCLM3 (CC-Y1191; EK-Bioscience, Shanghai, China), Huh-7 (CC-Y1280M; EK-Bioscience), and SNU387 (CRL-2237; American Type Culture Collection) were maintained in the mixture of Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Rockford, IL, USA), 10% (V/V) fetal bovine serum (Thermo Fisher Scientific), and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA) in an incubator containing CO2 (5%) at 37 °C.
Small interfering RNAs (siRNAs) targeting circ_0091579 (si-circ_0091579#1, si-circ_0091579#2, and si-circ_0091579#3), siRNAs against PLCB1 (si-PLCB1#1 and si-PLCB1#2) and their control (si-NC), miR-1225-5p mimic (miR-1225-5p) and its control miR-NC, and miR-1225-5p inhibitors (in-miR-1225-5p#1 and in-miR-1225-5p#2) and their control (in-miR-NC) were all designed and constructed via GenePharma (Shanghai, China). These oligonucleotides were introduced into HCCLM3 and Huh7 cells by feat of Lipofectamine 3000 (Thermo Fisher Scientific). At 48 h after transfection, cells were harvested for further assays (Table 1).
Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)
Total RNA of clinical samples or cells was segregated by employing TRIzol LS Reagent (Ambion, Carsland, CA, USA) and then subjected for complementary DNA (cDNA) synthesis utilizing Reverse Transcription Kit (Qiagen, Frankfurt, Germany) or miScript Reverse Transcription Kit (Qiagen). Afterward, qRT-PCR assay was conducted with SYBR Green PCR Kit (Qiagen) or TaqMan miRNA assays (Thermo Fisher Scientific). Relative expression of circ_0091579, miR-1225-5p, and PLCB1 was assessed via 2−ΔΔCt approach , with β-actin (for circ_0091579 and PLCB1) or U6 (for miR-1225-5p) as internal reference. Sequence of specific primers was: circ_0091579, 5′-TGAGCCAGTGGTCAGTCAAA-3′ (sense) and 5′-TGGAGTCAGGCTTGGGTAGT-3′ (anti-sense); PLCB1, 5′-CAACTCACCAAGTCTCCAGTGG-3′ (sense) and 5′-AGTGCCACCATCTGACAACCTG-3′ (anti-sense); β-actin, 5′-CCTCTCCCAAGTCCACACAG-3′ (sense) and 5′-GGGCACGAAGGCTCATCATT-3′ (anti-sense); miR-1225-5p, 5′-GCCGAGGTGGGTACGGCCCA-3′ (sense) and reverse 5′-CTCAACTGGTGTCGTGGA-3′ (anti-sense); U6, 5′-CTCGCTTCGGCAGCACA-3′ (sense) and 5′-AACGCTTCACGAATTTGCGT-3′ (anti-sense).
Clinical samples or cells were treated with Radio-Immunoprecipitation Assay (RIPA) buffer in order to prepare protein samples. After quantification, 30 μg samples were mixed with loading buffer (Solarbio, Beijing, China); then the mixture was loaded on sodium dodecyl sulfate (SDS)–polyacrylamide gels (PAGE), followed by transfer onto polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Subsequently, membranes were subjected for blockage with 5% non-fat milk and incubation with primary rabbit antibody and then probed with goat anti-rabbit secondary antibody (ab205718, Abcam, Shanghai, China). And primary antibodies included anti-PLCB1 (ab182359, Abcam), anti-Cyclin D1 (ab16663, Abcam), anti-BCL2-associated X (anti-Bax, ab32503, Abcam), anti-matrix metalloproteinase 9 (anti-MMP9, ab76003, Abcam), and loading control anti-β-Actin (ab124964, Abcam). Protein signal was excited using a chemiluminescence kit (Millipore) and analyzed by ImageJ software (NIH, Bethesda, MD, USA).
Assessment of Cell Viability
Transfected HCCLM3 and Huh7 cells were placed in of 96-well plates (3.0 × 103 cells each well), following by adding of 10 μL Cell Counting Kit-8 (CCK-8) solution (Sigma-Aldrich) at indicated time points. 4 h later, cell viability was assessed by measuring the absorbance of every well at 450 nm exploiting a microplate reader (Thermo Fisher Scientific).
Evaluation of Clonogenicity Ability
After transfection, 8 × 102 HCCLM3 and Huh7 cells were sowed in 6-well plates and cultured in a conventional way for 10 days. Cell medium was removed, and then generated colonies were incubated with 4% paraformaldehyde (Sigma-Aldrich) for 15-min immobilization, then dyed by crystal violet (Solarbio), and counted exploiting ImageJ software.
Examination of Cell Apoptosis
For cell apoptosis determination, Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BD Biosciences, San Jose, CA, USA) was used complying with manufacturer’s recommendation. Transfected HCCLM3 and Huh7 cells were rinsed with phosphate buffer solution (PBS; Solarbio) and then re-suspended in binding solution. Later, Annexin V-FITC and propidium iodide (PI) were added to stain cells. Finally, a flow cytometer (BD Biosciences) was utilized to identify apoptotic cells. Apoptotic rate was the percentage of apoptotic cells.
Detection of Cell Metastasis
Transwell chambers (8 μm pores; BD Biosciences) pre-enveloped with Matrigel (BD Biosciences) were applied for cell invasion analysis, and chambers without Matrigel adhesion were used for cell migration detection. In brief, transfected HCCLM3 and Huh7 cells were put in top chambers containing non-serum medium; meanwhile, complete medium was added into bottom chambers. After 24 h, cells remaining on the top surface of the chambers were discarded, while those invaded or migrated through pores were fixed and dyed by 4% paraformaldehyde and crystal violet, respectively, followed by photographing. Then, five randomly selected fields were subjected for counting utilizing an inverted microscope (Olympus, Tokyo, Japan).
Dual-Luciferase Reporter Assay (DLRA)
The target miRNAs of circ_0091579 and target genes of miR-1225-5p were forecasted by circular RNA interactome (https://circinteractome.nia.nih.gov/index.html) and miRDB (http://www.mirdb.org/cgi-bin/search.cgi), respectively.
Fragmentary sequence of circ_0091579 and PLCB1 3′-UTR embracing complementary region with miR-1225-5p was severally inserted into pMirGLO reporter vector (Promega, Beijing, China) to construct WT-circ_0091579 and WT-PLCB1 3′-UTR. After mutating corresponding binding sites, mutant-type reporters (MUT-circ_0091579 and MUT-PLCB1 3′-UTR) were generated. Subsequently, these constructs and miR-NC or miR-1225-5p were co-transfected into Huh7 and SNU387 cells for 48 h. Later, luciferase density was determined by dual-luciferase reporter system (Promega) according to the user’s manual.
In Vivo Experiments
This assay was approved by the Ethics Committee of The Second Affiliated Hospital of Xi'an Jiaotong University. Ten male BALB/C nude mice (5 weeks old) were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). HCCLM3 cells (2 × 106) stably transfected with lentiviral small hairpin RNA (shRNA) targeting circ_0091579 (sh-circ_0091579) or the control sh-NC were inoculated into subcutaneous area of right flank of mice to generate tumors, called sh-circ_0091579 group and sh-NC group (n = 5). Afterward, generate tumors were observed once a week, and the volume (mm3) was calculated using following formula: 0.5 × width2 × length. After 4 weeks, all tumors were cut off, weighed and subjected for expression level analysis.
All experiments were implemented in triplicate. After processing by SPSS 21.0, data were exhibited in the modality of mean ± standard deviation. Data difference was determined by Student’s t test or one-way analysis of variance. Correlation between expression of miR-1225-5p and circ_0091579 or PLCB1 in HCC tissues was analyzed by Pearson correlation analysis. P < 0.05 suggested statistically significant.
Circ_0091579 and PLCB1 Were Highly Expressed in HCC Tissues and Cells
Results from qRT-PCR assay showed that circ_0091579 expression in HCC tissues was higher than that in normal tissues (N = 26) (Fig. 1a). Also, in contrast to THLE-2 cells, circ_0091579 was upregulated in HCC cells (HCCLM3, Huh7, and SNU387 cells) (Fig. 1b). In addition, the mRNA and protein levels of PLCB1 were significantly increased in HCC tissues and cells, relative to corresponding control (Fig. 1c–f). Above data indicated the upregulation of circ_0091579 and PLCB1 in HCC tissues and cells.
Knockdown of Circ_0091579 Impeded Proliferation and Metastasis of HCC Cells
In view of the upregulation of circ_0091579 in HCC tissues and cells, we then conducted loss-of-function assays to explore its role in HCC development. At first, siRNAs targeting circ_0091579 (si-circ_0091579#1, si-circ_0091579#2, and si-circ_0091579#3) were introduced into HCCLM3 and Huh7 cells to silence circ_0091579 expression, with si-NC as negative control, and si-circ_0091579#1 and si-circ_0091579#3 exhibited better knockdown effects (Fig. 2a). By performing CCK-8 assay, we found that silencing of circ_0091579 could reduce the cell viability of HCCLM3 and Huh7 cells (Fig. 2b). The following colony formation assay witnessed the circ_0091579 knockdown and induced the declined clonogenicity ability of HCC cells (Fig. 2c). As expected, circ_0091579 depletion strikingly promoted cell apoptosis of HCC cells, which was demonstrated by flow cytometry (Fig. 2d). Transwell assay uncovered that circ_0091579 silencing evidently suppressed migration and invasion of HCC cells (Fig. 2e, f). Moreover, HCCLM3 and Huh7 cells with circ_0091579 knockdown exhibited downregulation of Cyclin D1 and MMP9 proteins, as well as upregulation of Bax protein (Fig. 2g, h). These results revealed that circ_0091579 might exert oncogenic role in HCC development.
Interference of PLCB1 Also Repressed HCC Cell Proliferation and Metastasis
Next, the functional impact of PLCB1 on the malignant behaviors of HCC cells was studied. Transfection with siRNAs of PLCB1 (si-PLCB1#1 and si-PLCB1#2) prominently decreased PLCB1 expression in HCCLM3 and Huh7 cells, with si-NC as negative control (Fig. 3a). Functional assays disclosed that PLCB1 depletion conspicuously reduced cell viability (Fig. 3b) clonogenicity ability (Fig. 3c), migration and invasion (Fig. 3e, f), and elevated apoptotic rate (Fig. 3d), as well as downregulated Cyclin D1 and MMP9 proteins and upregulated Bax protein (Fig. 3g, h) of HCC cells. Collectively, PLCB1 might serve as an oncogene in HCC.
Circ_0091579 Sponged miR-1225-5p and miR-1225-5p Targeted PLCB1
Circular RNA interactome and miRDB predicted that both circ_0091579 (5′-UACCCA-3′) (Fig. 4a) and PLCB1 3′-UTR (5′-GUACCCA-3′) (Fig. 4e) endowed binding sites with miR-1225-5p, respectively. Dual-luciferase reporter assay suggested that relative to miR-NC, gain of miR-1225-5p induced an apparent reduction in the luciferase activity of WT-circ_0091579 (Fig. 4b) and WT-PLCB1 3′-UTR (Fig. 4f) group, while it changed little in the MUT-circ_0091579 (Fig. 4b) and MUT-PLCB1 3′-UTR (Fig. 4f) group. In addition, miR-1225-5p expression was decreased in HCC tissues and cells compared to corresponding control (Fig. 4c, d). Collectively, circ_0091579 sponged miR-1225-5p and miR-1225-5p to target PLCB1.
Circ_0091579 Positively Regulated PLCB1 Expression by Sponging miR-1225-5p
As exhibited in Fig. 5a, circ_0091579 depletion obviously upregulated miR-1225-5p expression in HCCLM3 and Huh7 cells. And miR-1225-5p expression in HCC tissues was negatively correlated circ_0091579 expression (R = − 0.5459, P = 0.0039) (Fig. 5b). Then, HCCLM3 and Huh7 cells with miR-1225-5p inhibition were established by introduction with miR-1225-5p inhibitors (in-miR-1225-5p#1 and in-miR-1225-5p#2) (Fig. 5c). Moreover, miR-1225-5p inhibition could increase PLCB1 expression in HCC cells, at mRNA and protein levels (Fig. 5d, e). There existed an inverse correlation between the expression of miR-1225-5p and PLCB1 mRNA in HCC tissues (R = − 0.5561, P = 0.0032) (Fig. 5f). Furthermore, circ_0091579 knockdown could decrease the mRNA and protein expression levels of PLCB1 in HCCLM3 and Huh7 cells, which was largely remitted by miR-1225-5p inhibitor (Fig. 5g, h). Taken together, circ_0091579 positively regulated PLCB1 expression in HCC cells by segregating miR-1225-5p.
MiR-1225-5p Inhibition Could Facilitate the Aggressive Progression of HCC
After silencing miR-1225-5p expression in HCCLM3 and Huh7 cells, role of miR-1225-5p in HCC development was investigated. We found that miR-1225-5p inhibition resulted in the elevation of cell viability (Fig. 6a) and clonogenicity ability (Fig. 6b), decline of cell apoptosis (Fig. 6c), migration and invasion promotion (Fig. 6d, e), as well as the upregulation of Cyclin D1 and MMP9 and downregulation of Bax (Fig. 6f) in HCC cells. Therefore, miR-1225-5p might be a tumor suppressor in HCC.
Circ_0091579 Depletion Suppressed HCC Cell Proliferation and Metastasis by Increasing miR-1225-5p Expression
In view of the target relationship between circ_0091579 and miR-1225-5p, their interaction in HCC progression was explored. As illustrated in Fig. 7a, miR-1225-5p inhibition greatly weakened circ_0091579 knockdown-induced the decreased cell viability (Fig. 7b) and clonogenicity ability (Fig. 7c), the raised apoptotic rate (Fig. 7d), the repressed migration and invasion (Fig. 7e, f), the downregulation of Cyclin D1 and MMP9 proteins, as well as upregulation of Bax protein (Fig. 7g, h) in HCC cells. To sum up, circ_0091579 depletion might block HCC cell proliferation and metastasis by upregulating miR-1225-5p expression.
MiR-1225-5p Inhibition Aggravated HCC Progression by Upregulating PLCB1 Expression
Having known that miR-1225-5p targeted PLCB1 in HCC cells, their co-effects on HCC development were studied. As exhibited in Fig. 8a, b, miR-1225-5p inhibitor-induced the overexpression of PLCB1 in HCCLM3 and Huh7 cells were markedly undermined by PLCB1 depletion. Furthermore, miR-1225-5p inhibitor-induced the elevation on cell viability (Fig. 8c) and clonogenicity ability (Fig. 8d), and reduction on cell apoptosis (Fig. 8e), migration and invasion promotion (Fig. 8f, g), as well as the upregulation of Cyclin D1 and MMP9, and downregulation of Bax (Fig. 8h, i) in HCC cells were almost reversed by PLCB1 silencing. Thus, the tumor suppressor role of miR-1225-5p in HCC was attributed to targeting PLCB1.
Depletion of Circ_0091579 Curbed Tumor Growth In Vivo
Xenograft model in nude mice was constructed to clarify the role of circ_0091579 in vivo. As shown in Fig. 9a, b, tumors in sh-circ_0091579 group exhibited smaller volume and lighter weight than those in sh-NC group. Additionally, circ_0091579 (Fig. 9c) and PLCB1 (Fig. 9e, f) were downregulated, while miR-1225-5p was upregulated (Fig. 9d) in tumors in sh-circ_0091579 group, when compared to those in sh-NC group. These results indicated that circ_0091579 deficiency reduced tumor growth in vivo.
Dysregulated circRNAs have been uncovered to participate in modulation of the cancer pathophysiological processes and aggressive development of HCC . In this project, circ_0091579 was found to be upregulated in HCC tissues and cells. And we were the first to manifest that circ_0091579 deficiency reduced the tumorigenic properties of HCC cells through miR-1225-5p/PLCB1 pathway.
Zhang et al.  substantiated the potential of circ_0091579 to be a biomarker for HCC diagnosis. In addition, circ_0091579 expression was increased in liver cancer tissues and cells, and circ_0091579 knockdown could reduce the proliferative and metastatic capacities of HCC cells . Liu et al.  alleged that circ_0091579 depletion brought repressed impact on HCC cell growth and metastasis, which was attributed to miR-490-5p/CASC3 axis. And, silencing of circ_0091579 suppressed HCC cell growth in vitro and in vivo through miR-940/TACR1 axis . In keeping with the above reports, we also detected the upregulation of circ_0091579 in HCC tissues and cells. Likewise, we discovered the circ_0091579 deficiency-induced the suppressive effects on HCC cell proliferation and metastasis in vitro, as well as on tumor growth in vivo, suggesting that circ_0091579 was indeed a proto-oncogene in HCC.
It was evidenced that circ_0091579 could play carcinogenic role in HCC by sponging miRNA [17,18,19]. In this study, circular RNA interactome was searched to seek another miRNA interacted with circ_0091579. Then, circ_0091579 was predicted to endow with binding sequence with miR-1225-5p, which was testified by DLRA. Previous studies corroborated that miR-1225-5p could exert cancer-suppressing roles in glioblastoma , gastric carcinoma , laryngeal cancer , thyroid cancer , osteosarcoma , non-small cell lung cancer , etc. In HCC, miR-1225-5p was downregulated in HCC cells and tumor tissues of HCC patients. And miR-1225-5p impaired HCC cell growth, migration, and invasion by targeting NFκB p65 . From our data, miR-1225-5p expression was also decreased in HCC tissues and cells. Loss-of-function assays revealed that interference of miR-1225-5p promoted the aggressive progression of HCC. Furthermore, miR-1225-5p inhibition largely recovered the circ_0091579 knockdown-induced the HCC proliferation and metastasis repression, implying that circ_0091579 play a role in HCC by sponging miR-1225-5p.
Anterior reports suggested that miR-1225-5p served as tumor suppressor by targeting certain genes [11, 23, 24]. Here, PLCB1 was identified as a target gene miR-1225-5p. Upregulated expression of PLCB1 was correlated with unfavorable survival of patients with breast cancer, and PLCB1 could accelerate migration of breast cancer cells . PLCB1 could also serve as prognosis biomarker for patients with non-small cell lung carcinoma  and HCC . In addition, PLCB1 was involved in the malignant progression of ovarian cancer , colorectal cancer , and HCC . In this project, we detected the apparent upregulation of PLCB1 in HCC tissues and cells, in concordance with a previous publication . Silencing of PLCB1 not only hindered HCC proliferation and metastasis, but also ameliorated miR-1225-5p inhibition-induced the aggressive properties of HCC cells. Taken together, circ_0091579 contributed HCC progression by sponging miR-1225-5p and increasing PLCB1 expression.
In conclusion, circ_0091579 and PLCB1 are upregulated, while miR-1225-5p was downregulated in HCC tissues and cells. Knockdown of circ_0091579 repressed the proliferative and metastatic abilities of HCC cells through miR-1225-5p/PLCB1 pathway, highlighting a novel mechanism involving HCC progression.
Availability of data and materials
The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.
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The present study was approved by the ethical review committee of The Second Affiliated Hospital of Xi'an Jiaotong University. Written informed consent was obtained from all enrolled patients.
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Zhang, D., Zhang, Y., Zhang, X. et al. Circ_0091579 Serves as a Tumor-Promoting Factor in Hepatocellular Carcinoma Through miR-1225-5p/PLCB1 Axis. Dig Dis Sci (2021). https://doi.org/10.1007/s10620-021-06861-2
- Hepatocellular carcinoma