MicroRNA-144 inhibits cell proliferation, migration and invasion in human hepatocellular carcinoma by targeting CCNB1
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Hepatocellular carcinoma (HCC) is one of the most common malignancies with a high morbidity and mortality worldwide. MicroRNAs are key regulators of HCC genesis. However, the regulatory role and underlying mechanisms of microRNA in HCC is still limited.
Cyclin B1 (CCNB1) mRNA levels were examined in non-tumor and liver cancer of The Cancer Genome Atlas (TCGA) cohort. CCNB1 was knockdown to evaluate the HCC cell proliferation, migration and invasion. MicroRNA-144 targeting CCNB1 was identified with TargetScan analysis and confirmed with reporter assay. Overexpression of MicroRNA-144 was achieved using microRNA mimics and function of microRNA-144 was tested in vitro HCC cell line proliferation and in vivo tumor formation experiments.
Here, we found that the high level expression of CCNB1 was closely associated with poor prognosis in HCC patients. Knockdown of CCNB1 by RNA interference significantly inhibited cell proliferation, migration and invasion in HCC. Furthermore, we found that miR-144 directly targeted CCNB1 and inhibited CCNB1 expression. Moreover, in vivo experiments of subcutaneous tumor formation further demonstrated that miR-144 delayed tumor formation by negative regulation of CCNB1.
Therefore, we conclude that microRNA-144/CCNB1 axis plays an important role in human HCC. Therapies targeting microRNA-144 could potentially improve HCC treatment.
KeywordsMicroRNA-144 Hepatocellular carcinoma CCNB1 Proliferation Migration Invasion
Hepatocellular carcinoma (HCC) is one of the most common malignancies with a high morbidity and mortality worldwide [1, 2]. However, the precision pathogenesis of HCC is unclear and the effective prevention and treatment are still lacking. Thus, studying the molecular mechanism of HCC proliferation and metastasis, accurately predicting prognosis for patients, preventing HCC metastasis, and taking effectively preventive measures could be a breakthrough to improve the efficacy of prognosis and treatment.
Cyclin B1 (CCNB1), which expression varies periodically throughout the cell cycle is a key initiator and quality control step of mitosis . Recent studies have shown that CCNB1 is highly expressed in several different human cancers including breast cancer, cervical cancer, lung cancer and melanoma [4, 5, 6, 7]. Porter et al. found that the inhibition of CCNB1 nuclear export could induce apoptosis and the accumulation of CCNB1 in the nucleus was sufficient to trigger apoptosis . CCNB1 has also been demonstrated to have significant predictive power in overall survival of ER+ breast cancer patients and HBV-related HCC recurrence [9, 10]. However, the detailed functions of CCNB1 in HCC and how CCNB1 is regulated are still remaining elusive.
MicroRNAs (miRNAs) are an endogenous small non-coding RNAs, which regulates a broad-spectrum of genes involved in developmental and oncogene pathways at the post-transcriptional level. MiRNA can participate in many cellular processes such as cell cycle, proliferation, apoptosis and metastasis . Several studies have demonstrated that miRNAs play important roles in hepatocarcinogenesis and malignant transformation. MicroRNA-144 (miR-144) is a highly conservative miRNA that participates in the process of tumor genesis and development in different cancers [12, 13]. It was reported that miR-144 suppressed cell proliferation, migration, and invasion in hepatocellular carcinoma by targeting SMAD4 .
In this study, we firstly analyzed the Cancer Genome Atlas (TCGA) database and found that the expression of CCNB1 was abnormally elevated and closely related to the malignant phenotype. We confirmed that CCNB1 knockdown significantly increased HCC cell apoptosis, while cell migration and invasion was decreased. Furthermore, we found that miR-144 directly inhibited CCNB1 expression by binding to CCNB1 3′-UTR. Consistently, the expression of miR-144 significantly decreased in liver cancer tissue compared with that in non-tumor tissue. Overexpression of miR-144 in HCC cell lines inhibited the cell proliferation in vitro and delayed the subcutaneous tumor formation in vivo. In conclusion, we demonstrated that miR-144, serving as tumor suppressor, played an inhibit role in the occurrence and development of HCC by negative regulation of CCNB1 expression, thereby contributing to the progress of HCC. MiR-144/CCNB1 pathway plays an important role in the development of HCC, which provides a potential promising target for HCC treatment.
Materials and methods
TCGA database analysis
The liver cancer gene expression profiles in the TCGA database were downloaded using the R package TCGA-assembler, including 424 samples of mRNA expression profiles (374 cases of liver tumor specimens, 50 cases of adjacent tumor specimens). Expression profiles and gene annotation files were imported by BRB-Arraytools (version V4.5.0 Beta 2) to get the gene expression value. Follow-up data were also downloaded for matching and subsequent analysis.
HEK293T cells, Human HCC cell line, HepG2 and 7221 were from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were maintained in DMEM medium (Thermo Fisher, Waltham, MA, USA), supplemented with 10% fetal bovine serum (FBS; Hyclone, USA), 100 U/mL penicillin and 100 μg/mL streptomycin.
Mice and tumor model
All animals were treated in accordance with the Guide for the Care and Use of Laboratory Animals, and all experiments were approved and carried out according to the guidelines of the Ethics Committee of Zhengzhou University. Male BABL/c nude mice (5–6 week old, 18–22 g weight), maintained under a specific pathogen-free condition. SMMC-7721 cells were stably infected with/without the miR-144 overexpression mimic vectors. A total of 5 × 106 viable cells were injected into the right flanks of nude mice. Tumor sizes were measured using a vernier caliper every 5 days, and tumor volume was calculated using the formula: volume = 1/2 × length × width2. At 30 days after implantation, the mice were killed, tumors dissected, and tumor weights were measured.
siRNA targeting CCNB1 (CCNB1-siRNA, GAAUUCUGCACUAGUUCAA), Scramble siRNA control (Scramble-siRNA, GAUUAGUCACCUGACUAUA), miR-144 mimics (GGAUAUCAUCAUAUACUGUAAG), miR-144 inhibitor (CUUACAGUAUAUGAUGAUAUCC) and Scramble controls (GAGUACUACUUAUACAGUAUAG) were designed and synthesized by Shanghai GenePharma Company (Shanghai, China). Cells were seeded into 96-well plates in antibiotic-free growth medium at the density of 4 × 103 cells per well. Transfection with was performed when the cells reached 70–80% confluence using Lipofectamine 2000 (Invitrogen, USA) in accordance to the manufacturer’s protocol.
Real-time quantitative PCR
Total RNA extraction was extracted from the cell lines using Trizol reagent (Invitrogen, USA). Complementary DNA was synthesized using AMV reverse transcriptase (GIBCO, USA) through reverse transcription. PCR reactions were performed using following primers: 5′-GACCTGTGTCAGGCTTTCTCTG-3′ (forward) and 5′-GGTATTTTGGTCTGACTGCTTGC-3′ (reverse) for CCNB1; 5′-CAGGAGGCATTGCTGATGAT-3′ (forward) and 5′-GAAGGCTGGGGCTCATTT-3′ (reverse) for GAPDH. Relative expression levels of CCNB1 were normalized to endogenous control GAPDH using 2−ΔΔCT methods. miRNA were extracted using an miRNA isolation kit miRNeasy kit (Qiagen, Germany) according to the manufacturer’s protocol.
Cell proliferation assay
Cell proliferation was determined using the MTT assay. Cells were seeded at a density of 3 × 103 per well in 96-well plates, and each well was transfected with CCNB1-siRNA, miR-144 mimics, miR-144 inhibitor or the proper negative control. At 48 h after transfection, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (Sigma-Aldrich, USA) solution was used to replace culture medium and incubated for 4 h. A microplate reader (Bio-Rad, Hercules, CA) measured the absorbance at 570 nm for all the samples.
Apoptosis analysis by Annexin-V FITC/PI double staining
The HCC cell line HepG2 and SMMC-7721 were transfected with CCNB1-siRNA or negative control (Scramble-siRNA). To identify apoptotic cells, Annexin V and PI staining was performed using an Annexin V-FITC Apoptosis Detection kit (Becton, Dickinson and Company, San Jose, CA, USA) after 48 h. Apoptosis was detected using a Calibur Flow Cytometer (Becton). Apoptotic cells were defined by positive staining of Annexin V and negative staining of PI.
Cell invasion assay
The invasion assay was performed using a transwell chamber, consisting of 8 mm membrane filter inserts (Corning Incorporated, Corning, NY, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). Briefly, cells were trypsinized and suspended in serum-free medium. Next, 2 × 104 cells were added to the upper chamber, and the lower chamber was filled with medium containing 10% fetal bovine serum. After 36 h of incubation, cells that had invaded the lower chamber were fixed with 4% paraformaldehyde, stained with hematoxylin, and counted using a microscope.
Wound-healing assay was performed using HepG2 and SMMC-7721 cells. Cells were trypsinized and seeded in equal numbers into six-well tissue culture plates, and allowed to grow until confluent (approximately 24 h). Following serum starvation for 24 h, an artificial homogenous wound (scratch) was created onto the cell monolayer with a sterile 100 μL tip. After scratching, the cells were washed with serum-free medium, complete media was added, and microscopic images (20× magnification) of the cells were collected at 0, 36, and 48 h.
Luciferase reporter assay
The 3′-untranslated region (3′-UTR) of CCNB1 mRNA, which contained the predicted miR-144 binding site, was amplified by PCR using the Takara PCR Amplification Kit (Takara, Dalian, China). The following PCR primers were used for cloning 3′-UTR of CCNB1 mRNA (Forward: AAGGCTGTGGCAAAGGTGTAAC; Reverse: TCCCCAGGTAAACCAAAAGGAGT). The Quick Mutagenesis Stratagene kit (Stratagene, La Jolla, CA) was used to create mutant 3′-UTR of CCNB1 mRNA. The PCR products were then enzymatically digested and cloned into the pGL3-CCNB1-3′-UTR vector (Promega, USA). Cells (5 × 104) were seeded in 24-well plates and cultured for 24 h. Reporter plasmids (200 ng the pGL3-CCNB1-3′-UTR or pGL3-CCNB1-3′-UTR-mut, 1 ng pRL-TK renilla plasmid, Promega) and 100 nmol/L miR-144 mimics were co-transfected into HEK293 cells mediated by Lipofectamine 2000 (Invitrogen). After 48 h, the cells were lysed and reporter activity was determined using a Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions.
Western blotting was performed using standard methods. Membranes were probed with polyclonal rabbit antibodies against anti-CCNB1 (Abcam, Cambridge, MA, USA). The proteins on membranes were then stripped and re-probed with an anti-beta-actin rabbit polyclonal antibody (Abcam) as a loading control. The blot was developed using enhanced chemiluminescence solution (Beyotime, Haimen, China) and photographed using the FluorChem imaging system (Alpha Innotech Corp., San Leandro, CA, USA). The intensity of each spot was analyzed with AlphaEaseFC software.
Ki67 immunohistochemistry staining
To evaluate tumor tissue histological changes, tumor sections were processed and immunohistochemical staining of the paraffin sections was performed using a microwave-based antigen retrieval technique, specimen slides were incubated overnight at 4 °C with primary antibodies that were raised against Ki67 (D2H10, Cell Signaling Technology), then Dako LSAB2 System-HRP (K0675), and Dako Liquid DAB + Substrate Chromogen System (K3468).
Unpaired two-tailed Student’s t test was used for calculation of p values between two groups. One-way analysis of variance (ANOVA) with the Tukey post hoc test and two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test were used tfor comparison between multiple groups. All p values < 0.05 were considered significant. Statistical analysis was performed with Graphpad Prism 6.
Bioinformatics analysis of CCNB1 expression in HCC tissues and normal liver tissue
CCNB1 knockdown promotes apoptosis and suppresses proliferation in HCC cell line HepG2 and SMMC-7721
CCNB1 knockdown inhibits HCC cell migration and invasion
MiR-144 inhibits CCNB1 expression by binding to CCNB1 3′-UTR
MiR-144 mimics elicit similar proliferation repressive effect in HCC cell
MiR-144 overexpression suppresses the tumor formation of SMMC-7721 cells in nude mice
Accumulating evidences suggested that miRNAs are important regulators in various cellular processes and are recently extensively studied in the development of HCC [17, 18, 19, 20]. However, the precise role of miRNAs in the development of HCC is still largely unclear as a single miRNA could have multiple targets and a single mRNA could be targeted by various miRNAs . Further understanding the functions of miRNAs in HCC helps to better reveal the underlying mechanism of HCC pathogenesis and progression. In this study, we demonstrated that miR-144 inhibited the proliferation, migration and invasion of HCC cells in vitro by targeting CCNB1. In vivo tumor inoculation nude mice model confirmed that overexpression of miR-144 significantly inhibited growth of HCC tumors. Thus, our results suggest that miR-144/CCNB1 axis could not only be used as a novel biomarker but also a potential gene therapeutic target of HCC.
CCNB1 regulates the G2 to M phase transition in mitosis. Overexpression of CCNB1 is correlated with poor survival in most solid tumors, which suggests that the expression status of CCNB1 is a significant prognostic parameter in solid tumors . Na Chai et al. reported that Foxm1 played an important role in proliferation of HCC via transcriptional activation of CCNB1 expression, which suggested the Foxm1-CCNB1 axis could be a potential target for the treatment of HCC patients . CDCA5 was found to be positively associated with increased CDK1 and CCNB1 expression in HCC tissues . Interestingly, integrated bioinformatics analysis identified CCNB1 as a potential therapeutic target in HCC . In this study, we explored liver cancer expression profiles from TCGA public databases and found the expression level of CCNB1 was abnormally elevated in hepatocarcinoma tissues. Kaplan–Meier analysis showed that the survival time of patients with low expression of CCNB1 was significantly better than that of patients with high expression of CCNB1, suggesting that the expression of CCNB1 is closely related to the clinical prognosis of patients with liver cancer. Meanwhile, further cell function tests confirmed that the proliferation of hepatocellular carcinoma cell lines decreased significantly after CCNB1 knockdown, and the migration and invasion ability and tumorigenic ability decreased significantly. The apoptosis rate of hepatoma cell lines was significantly increased after CCNB1 gene knockdown, suggesting CCNB1 played an important role in the occurrence and development of liver cancer.
As a mechanistic study to find the molecular mechanism of CCNB1 regulation based on its abnormally high expression in liver cancer, we also conducted bioinformatical analysis to identify the miR-144 was targeting CCNB1 by binding to its 3′ UTR. Previous studies demonstrated that miR-144 expression was decreased in various cancers, including gastric cancer, breast cancer and bladder cancer [12, 13, 26]. Our analysis revealed that the expression level of miR-144 was significantly decreased in liver cancer tissues, and the survival time of liver cancer patients with low expression of miR-144 was significantly worse than that of patients with high expression of miR-144, suggesting miR-144 may also be involved in HCC. Overexpression of miR-144 in hepatocarcinoma cells has similar effects on hepatoma cell proliferation, migration and invasion as CCNB1 knockdown, suggesting that miR-144/CCNB1 regulatory axis may play an important role in the occurrence and development of liver cancer. As the occurrence and development of tumors are complicated biological processes which are regulated by multiple factors in tumor cells and tumor microenvironment. The effect of miR-144 overexpression on the metastasis ability of hepatoma cells was also confirmed by the mouse tumor-bearing experiment.
However, miR-144-CCNB1 axis in the occurrence and metastasis of liver cancer, and further substantial follow-up studies still need further investigation. Further, it should also be noted that miR-144 regulate a broad-spectrum of biological processes and the targets of miR-144 is not limited to oncogenes. Moreover, CCNB1 is involved in important processes such as mitogenesis and the side effects of miR-144 gene therapy should also be scrutinized.
In sum, we found that the high level expression of CCNB1 was closely associated with poor prognosis in HCC patients. Knockdown of CCNB1 by RNA interference significantly inhibited cell proliferation, migration and invasion in HCC. Bioinformatics study identified that miR-144 directly targeted CCNB1 and inhibited CCNB1 expression. In vivo experiments of subcutaneous tumor formation further demonstrated that miR-144 suppressed tumor formation by negative regulation of CCNB1. Therefore, these evidences suggest that microRNA-144/CCNB1 axis is an important diagnostic and therapeutic potential target in HCC treatment.
JG and XL designed and carried out the study. JG, XL, JL, and YH participated in experiments and statistical analysis. JG and XL wrote the manuscript. JG revised the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
All the data supporting our findings can be found in the “Results” section of the paper. Please contact authors for data request.
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