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Liver cancer is a globally prevalent cancer with poor prognosis. The present study investigated the link between microRNA-378a (miR-378a) expression and the sensitivity of hepatocellular carcinoma (HCC) and hepatoblastoma (HB) cancers to sorafenib therapy. miR-378a expression was determined in liver tissue samples from healthy candidates and patients with liver cancer using the reverse transcription-quantitative polymerase chain reaction. The antitumor effects of miR-378a alone and in combination with sorafenib were investigated in the HB cell line HepG2 and the HCC cell line SMMC-7721 with methyl thiazoyl tetrazolium, colony formation, flow cytometry and Transwell migration assays. The underlying mechanisms were investigated using western blot analysis. miR-378a expression was decreased in tissue samples from patients with liver cancer. HCC and HB cell line proliferation and invasion ability was inhibited by miR-378a. The combination of miR-378a and sorafenib provided the greatest inhibition. Western blot indicated that mitogen activated protein kinase signaling pathway proteins, vascular endothelial growth factor receptor, platelet derived growth factor receptor β, Raf-1 proto-oncogene, serine/threonine kinase and matrix metallopeptidase 2 were regulated by miR-378a alone and to a greater extent when combined with sorafenib. Results suggest that miR-378a can inhibit liver cancer cell growth and enhance the sensitivity of liver cancer cells to sorafenib-based chemotherapies.
Introduction
Liver cancer is a globally prevalent and aggressive cancer with poor prognosis (1). Examples include hepatocellular carcinoma (HCC) and hepatoblastoma (HB): HCC is estimated to be the second leading cause of cancer-associated mortality in males and the sixth in females worldwide (2), whereas HB is the most common pediatric liver tumor in the US (3,4). Currently approved therapeutic drugs for liver cancer are ineffective and result in poor patient outcomes (5). Sorafenib is a systemic drug used as a first line therapy for advanced HCC and prolongs the overall survival of patients with HCC from 7.9 to 10.7 months (6). Recent studies have indicated that sorafenib can also induce apoptosis in HB cells and inhibit the progression of HB (7,8). Sorafenib is a small molecule multiple tyrosine kinase inhibitor that can induce apoptosis in cancer cells via the downregulation of vascular endothelial growth factor receptor (VEGFR), platelet derived growth factor receptor β (PDGFRβ), KIT proto-oncogene receptor tyrosine kinase and fms like tyrosine kinase 3 expression levels, as well as through the inhibition of the mitogen-activated protein kinase (MAPK) and Wnt/β-catenin signaling pathways (9,10). However, not all patients with liver cancer benefit from sorafenib therapy and drug resistance is often acquired within 6 month (11).
MicroRNAs (miRNAs) are a class of small, non-coding RNA molecules consisting of 20–22 nucleotides (12). Accumulating evidence has demonstrated that miRNAs have an important role in regulating the growth of various tumors, including those in HCC and HB (13–17). Several studies have reported the important physiological role of miRNA-378a (miR-378a). Chen et al (18) reported that miR-378a inhibited prostate cancer via downregulating the MAPK1 signaling pathway. Wei et al (19) demonstrated that miR-378a promotes myoblastic differentiation by regulating histone deacetylase 4 in skeletal muscle development and Zhang et al (20) indicated that miR-378a activated the pyruvate-phosphoenolpyruvate futile cycle and regulated lipolysis. To the best of the authors' knowledge, the effects of miR-378a alone and in combination with sorafenib in HCC and HB treatment has not yet been studied.
The present study analyzed expression levels of miR-378a in liver tissue samples from healthy donors and patients with liver cancer. The effects of miR-378a alone or in combination with sorafenib therapy on the proliferation and invasion capacities of the HepG2 HB cell line and the SMMC-7721 HCC cell line was also investigated. Results indicated that miR-378a alone inhibited the proliferation and invasion capacity of both the HB and HCC cell lines. miR-378a also enhanced the sensitivity of HB and HCC cells to sorafenib by targeting VEGFR, PDGFRβ and Raf-1 proto-oncogene, serine/threonine kinase (c-Raf). These results may provide a new avenue for the development of liver cancer therapies.
Materials and methods
Clinical samples
A total of 32 liver cancer and 32 adjacent normal liver tissue samples were obtained from February to October 2016 (Taihe Hospital, Shiyan, China). The mean ± standard deviation (SD) age of candidates was 43.6±7.9 years (range, 36–58 years). A total of 18 male and 14 female samples were obtained. The Ethics Committee of the Shiyan Taihe Hospital Institutional Review Board approved the present study and patients' permission was obtained prior to surgery. Following the surgery, tissue samples were stored at −80°C. Pathological information was obtained following the operation according to the Union of International Cancer Control's TNM classification of Malignant Tumors 2010 (21). Among the patients, 8 cases of stage 3B, 8 cases of stage 3C and 16 cases of stage 4A cancer were identified.
Cell culture
HepG2 and SMMC-7721 cell lines (American Type Culture Collection, Manassas, VA, US) were cultured in Dulbecco's modified Eagle's medium (DMEM) and supplemented with 10% fetal bovine serum (FBS; 10438026) (both from Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 IU/ml penicillin and streptomycin (Thermo Fisher Scientific, Inc.) in a humidified incubator containing 5% CO2 at 37°C.
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis
Total RNA was extracted from tissue samples using TRIzol (Thermo Fisher Scientific, Inc.). Primer was ordered from Thermo Fisher Scientific, Inc. (assay ID: 478349_mir; Thermo Fisher Scientific, Inc.). Reverse transcription was subsequently performed using the TaqMan MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) and the thermo-cycling conditions were as follows: 16°C for 30 min, 42°C for 30 min and followed with 85°C for 5 min, then staying at 4°C. miRNA was then examined using the TaqMan MiRNA PCR kit (Applied Biosystems; Thermo Fisher Scientific, Inc.), along with the small nuclear RNA U6 (assay ID: 001973; Thermo Fisher Scientific, Inc.) as an endogenous control, with the following thermo-cycling conditions: 95°C for 20 sec (1 cycle), 95°C for 3 sec and 60°C for 30 sec (40 cycles). miRNA expression was measured using the ∆∆Cq method (22). ∆Cq was calculated by subtracting the Cq of U6 RNA from the Cq of each miRNA of interest. The ∆∆Cq was calculated by subtracting the ∆Cq of the control sample from the ∆Cq of each sample. Fold change was generated by using the equation 2−ΔΔCq.
Luciferase reporter assays
Using TargetScan (
Transient transfection
miR-378a mimic (3′-ACUGGACUUGGAGUCAGAAGGC-5′) and its corresponding negative control (3′-UCAGGAGCGUUGCCUGGCUCGG-5′) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). Cells (5×105/well) were cultured to 60–70% confluence prior to incubation with the miR-378a mimic or miR-378a mimic control at a final concentration of 0.1 µM using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in a serum-free medium for 48 h.
Western blot analysis
Cells were lysed for 30 min on ice in radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) supplemented with phosphatase inhibitor cocktail (CST Biological Reagents Co., Ltd., Shanghai, China). Protein concentrations were determined using the bicinchoninic acid kit (cat. no. BCA1-1KT; Sigma-Aldrich; Merck KGaA) Proteins (50 µg) were separated on 12% SDS-PAGE prior to transfer onto polyvinylidene difluoride membranes. Membranes were incubated in blocking buffer (I-block, T2015; Thermo Fisher Scientific, Inc.) at room temperature for 2 h then incubated at 4°C overnight with the following primary antibodies: Phospho-(p)-VEGFR-pTyr1333 (1:1,000, cat. no. SAB4504006; Sigma-Aldrich; Merck KGaA); VEGFR (1:1,000, cat. no. ab46154; Abcam, Cambridge, MA, USA); p-PDGFRβ Tyr751 (1:1,000, cat. no. 3161; CST Biological Reagents Co., Ltd., Danvers, MA, USA); PDGFRβ (1:1,000, cat. no. Sc-374573; Santa Cruz Biotechnology, Inc., Dallas, TX USA); p-c-Raf, Ser259 (1:1,000, cat. no. 9421); c-Raf; (1:1,000, cat. no. 9422); p-extracellular signal-related kinase 1/2 (p-ERK1/2) Thr202/204 (1:1,000, cat. no. 9101); ERK1/2 (1:1,000, cat. no. 9102); and MMP2 (1:1,000; cat. no. 4022) (all from CST Biological Reagents Co., Ltd.) and poly ADP ribose polymerase (PARP, 1:1,000; cat. no. ab32071; Abcam). Membranes were subsequently incubated at room temperature for 2 h with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin (Ig)G (1:2,000, cat. no. 7074) or HRP conjugated anti-mouse IgG (1:2,000, cat. no. 7076) (both from CST Biological Reagents Co., Ltd.) secondary antibodies. β-actin (1:5,000, cat. no. AB8229; Abcam) was used as a loading control. Proteins were visualized using SuperSignal West Pico Plus Chemiluminescent Substrate (cat. no. 34577; Thermo Fisher Scientific, Inc.). ImageJ software version 1.50i (National Institutes of Health, Bethesda, MD, USA) was used for density analysis.
Cell viability assay
Untransfected and miR-378a mimic transfected HepG2 and SMMC-7721 cells (48 h after transfection) were seeded into a 96-well plate (1,500 cells/well). Cells were treated with sorafenib (S7397, Selleck Chemicals, Houston, TX, USA; 0.25, 0.5, 1, 2, 4, 8 and 16 µM) for 72 h prior to incubation with 1 mg/ml of methyl thiazoyl tetrazolium (MTT; Sigma-Aldrich; Merck KGaA) for 4 h at 37°C in a CO2 incubator. Mitochondrial reduction of MTT in DMSO to formazan was tested by culture at 37°C for 15 min. The amount of formazan absorbed was measured at 570 nm absorbance, with 450 nm used as the reference wavelength. IC50 values were calculated and expressed as a mean ± SD of three independent experiments.
Colony formation assay
The cells were seeded at a density of 200 cells/ml into 6-well culture plates for 24 h, then washed with PBS and cultured with DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) in the presence or absence of sorafenib (EC10=1.8±0.2 µM, concentration set as 0.25, 0.5, 1, 2, 4, 8 and 16 µM). Colonies were allowed to grow for 14 days. Cells were subsequently fixed in 70% ethanol for 15 min at room temperature and stained with 0.2% crystal violet at room temperature for 20 min. Positive colony formations (>50 cells/colony) were counted. EC50 was calculated as the concentration of sorafenib eliciting a half-maximal response.
Flow cytometry assay
Following the treatment with sorafenib (0.25, 0.5, 1, 2, 4, 8 and 16 µM) for 72 h, cells were fixed with 70% cold ethanol for 30 min at 4°C, stained with 200 µl propidium iodide (50 µg/ml) at room temperature for 15 min and analyzed by flow cytometry. The data were analyzed using Cellquest Pro version 5.1 software (BD Biosciences, San Jose, CA, US). The experiments were repeated at least three times.
Cell invasion assay
Matrigel (356235; BD Biosciences, San Jose, CA, USA) was pre-coated on the upper compartment of Millicell cell culture inserts containing 8.0-µm pores (PSET010R5; EMD Millipore, Temecula, CA, USA). DMEM (~600 µl) with 10% FBS was added to the lower compartment and 200 µl serum free culture medium with 0.1% BSA containing 2×105 HepG2 cells were seeded to the upper compartment. After culture with sorafenib (0.25, 0.5, 1, 2, 4, 8 and 16 µM) for 24 h at 37°C in a humidified 5% CO2 environment. Cells that had migrated to the lower compartment were fixed with 70% ethanol for 15 min at room temperature and stained with 0.2% crystal violet at room temperature for 20 min and counted under the phase contrast microscope (Olympus CX41; Olympus Corporation, Tokyo, Japan), magnification, ×100.
Statistical analysis
Statistical analysis was completed with SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA) and presented as the mean ± standard deviation for repeated measures. All experiments were repeated at least three times. A one-way analysis of variance test was used for comparison of differences between groups. Tukeys multiple comparison was used as the post hoc test. P≤0.05 was considered to indicate a statistically significant difference.
Results
Sorafenib can suppress tumor growth through the inhibition of multiple tyrosine kinases, including VEGFR, PDGFRβ and c-Raf. These three genes were input into TargetScan to identify seed matched miRNA predictions. miR-378a was predicted to target all three genes. Thus, miR-378a expression levels were investigated in liver tissue samples from healthy donors and patients with liver cancer. RT-qPCR indicated that the expression level of miR-378a in liver cancer samples was significantly reduced compared with normal liver samples (Fig. 1A). Western blot analysis indicated increased expression of VEGFR, PDGFRβ and c-Raf in liver cancer samples (Fig. 1B). These data suggest that miR-378a has effects similar to sorafenib and therefore may have the potential to treat liver cancer.
The ability of miR-378a to target VEGFR, PDGFRβ and c-RAF mRNA was subsequently confirmed through the construction of luciferase expression vectors harboring wild-type and mutant 3′UTR target sequences of the genes: VEGFR-3UTR, VEGFR-3UTR-m; PDGFRβ-3UTR, PDGFRβ-3UTR-m; Raf-3UTR and Raf-3UTR-m (Fig. 1C). The assay revealed that the wild-type target sequences had significantly lower luciferase activity than their respective mutants in 293 cells in the presence of the miR-378a mimic. By contrast, the miR-378a mimic did not reduce luciferase activity when miR-378a seed sequences at 3′UTR of these three gene were mutated. (Fig. 1D). These data indicated that miR-378a may target VEGFR, PDGFRβ and c-Raf mRNA in 293 cells.
The miR-378a mimic or the miR-378a mimic control were transfected into HepG2 and SMMC-7721 cells. The expression of miR-378a was significantly upregulated in miR-378a mimic transfected HepG2 and SMMC-7721 cells, compared with untransfected and miR-378a mimic negative control transfected cells (Fig. 1E). Western blotting also confirmed that VEGFR, PDGFRβ and c-Raf expression levels were significantly lower in miR-378a mimic transfected HepG2 and SMMC-7721 cells compared with cells transfected with the mimic control (Fig. 1F).
The inhibitory capacity of sorafenib on cell proliferation in untransfected or miR-378a mimic transfected liver cancer cells was evaluated by MTT assay (Table I). After 72 h, sorafenib displayed anti-proliferative activity in untransfected HepG2 and SMMC-7721 cells with IC50 values of 5.5±0.5 and 4.7±0.4 µM, respectively. Liver cancer cells transfected with miR-378a mimic had significantly lower IC50 values for sorafenib, at 3.2±0.4 µM in HepG2 and 2.8±0.3 µM in SMMC-7721 cells.
The antitumor activity of sorafenib in liver cancer cells was further confirmed in colony formation experiments. The calculated EC50 values of sorafenib in untransfected HepG2 and SMMC-7721 cell lines were 2.1±0.2 and 1.8±0.2 µM, respectively. EC50 values of sorafenib in miR-378a mimic transfected HepG2 and SMMC-7721 were significantly lower, at 1.6±0.2 and 1.3±0.2 µM, respectively (Fig. 2A). Changes in cell cycle distribution and apoptosis were subsequently analyzed in both untransfected and miR-378a mimic transfected HepG2 and SMMC-7721 cells with a flow cytometry assay. Treatment with sorafenib for 72 h increased the proportion of cells in the G0-G1 phase and decreased the proportion of cells in the S and G2/M phase (data not shown). These results suggest that the inhibition of proliferation by sorafenib in HepG2 and SMMC-7721 cells is in part due to a G1 phase arrest. Furthermore, a larger proportion of cells were dead cells in the miR-378a mimic transfected group compared with untransfected cells. Sorafenib increased the proportion of dead cells in the miR-378a transfected group from 50±5 to 61±6%, demonstrating a possible synergistic inhibitory effect of miR-378a and sorafenib on liver cancer cell growth (Fig. 2B).
Invasion capacity is an important characteristic of malignant cells. Therefore, the ability of miR-378a to reduce HepG2 cell invasion activity was investigated using Transwell assays. Compared with controls, miR-378a mimic transfection reduced the invasive potential of HepG2 cells by 35.4%, and sorafenib treatment in miR-378a mimic transfected HepG2 cells reduced the invasive potential by 86.0% (Fig. 3; Table II). This inhibition rate was significantly higher than that of sorafenib treated untransfected HepG2 cells (75.6% reduction). These data demonstrate that sorafenib or miR-378a alone affect the invasive potential of HepG2 cells in vitro and miR-378a enhances the sensitivity of HepG2 cells to sorafenib.
Raf kinases are key regulators of the MAPK cascade, and upregulated signaling in this pathway has an important role in various types of cancer. The expression levels of MAPK pathway proteins in untransfected and miR-378a mimic transfected HepG2 cells were analyzed using western blotting in order to investigate the mechanism underlying the inhibition of tumor growth by sorafenib (Fig. 4). Results indicated that sorafenib reduced the level of three tyrosine kinase targets: p-PDGFRβ, p-VEGFR and p-c-Raf, in both untransfected and miR-378a mimic transfected HepG2 cells. MAPK signaling pathway proteins activity were significantly downregulated by sorafenib in HepG2 cells (P<0.05). Invasion regulatory protein matrix metallopeptidase 2 (MMP2) was downregulated by sorafenib in HepG2 cells. Concurrent with these results, the pro-apoptotic protein PARP was significantly increased in sorafenib treated HepG2 cells. It was demonstrated that miR-378a also regulated the expression level of PARP and MMP2. Furthermore, the expression level of MAPK signaling proteins was significantly lower in miR-378a mimic transfected cells compared with untransfected HepG2 cells. These data suggest that sorafenib or miR-378a alone can regulate MAPK signaling and the expression of its subsequent downstream proteins and miR-378a can enhance the sensitivity of HepG2 cells to sorafenib.
Discussion
Sorafenib is the mainstay therapy recommended for the treatment of liver cancer, despite its low clinical response rate (6,11). miRNAs have a critical role in the biological behavior of liver cancer (23). Various studies have indicated the effectiveness of miRNAs against HCC and HB is due to their ability to inhibit cell growth, invasion ability, angiogenesis and induce tumor cell apoptosis (24–27). miR-378a has been reported to regulate multiple physiological activities, including myoblast differentiation, adipogenesis, and tumor growth (28), such as those found in colorectal cancer and rhabdomyosarcoma (19,29–31). A recent study reported that miR-378a enhances the sensitivity of MCF-7 breast cancer cells to tamoxifen by targeting golgi transport 1A protein (32). However, to the best of our knowledge, the effect of miR-378a in combination with sorafenib treatment in liver cancer cells has not yet been studied.
The present study revealed that miR-378a levels were decreased in liver cancer tissues. Upregulated miR-378a may inhibit proliferation and induce apoptosis of HepG2 and SMMC-7721 cells. To further examine the mechanisms underlying these effects, VEGFR, PDGFRβ and c-Raf were selected as candidate miR-378a target genes by in silico algorithms. These three genes are also important targets of sorafenib. Experiments revealed that mRNA levels of these three candidate genes were downregulated in 293 cells transfected with miR-378a mimic, which was consistent with the computational analysis. The luciferase reporter assay suggested that miR-378a regulates the expression of VEGFR, PDGFRβ and c-Raf by directly targeting the 3′UTR of these genes. VEGFR, PDGFRβ and c-Raf regulate various cellular processes, including proliferation, apoptosis, the stress response and mitotic checkpoints in cancer cells (33–36). In addition, these target genes mediate the activation of the MAPK signaling pathway. Inhibition of the MAPK/ERK1/2 pathway has been demonstrated to be beneficial in numerous cancer studies (33,37,38). The present study indicated miR-378a or sorafenib alone can inhibit MAPK/ERK1/2 signaling and enhance the expression of the pro-apoptotic protein PARP. A synergistic effect was observed in tumor cells transfected with miR-378a and subsequently treated with sorafenib. miR-378a also enhanced the G1 phase arrest and anti-invasion effects of sorafenib on liver cancer cells (data not shown).
In conclusion, the inhibitory effect of sorafenib on tumor cell proliferation and invasion activities was enhanced by combined treatment with miR-378a. These findings could potentially be applied to develop alternative approaches to liver cancer therapy.
Table I.Anti-proliferative activity of miR1-378a and sorafenib on HepG2 and SMMC-7721 cells.
| IC50 | ||
|---|---|---|
| Treatment | HepG2 | SMMC-7721 |
| Sorafenib | 5.5±0.5 µM | 4.7±0.4 µM |
| miR-378a mimic | – | – |
| miR-378a mimic control | – | – |
| Sorafenib + miR-378a mimic | 3.2±0.4 µMa | 2.8±0.3 µMa |
| Sorafenib + miR-378a mimic control | 4.8±0.6 µM | 4.9±0.5 µM |
P<0.05 vs. sorafenib alone. Untransfected and miR-378a mimic transfected HepG2 and SMMC-7721 cells were treated with sorafenib. After 72 h of treatment, IC50 values were calculated. miRNA-378a, microRNA-378a; mimic-con, mimic control.
Table II.miR-378a alone or combined with sorafenib affected the invasion capacity of HepG2 cells.
| Groups | Cell count | Inhibition rate (%) |
|---|---|---|
| Non-treatment | 8,884±214 | – |
| Sorafenib | 2,168±99b | 75.6 |
| miR-378a mimic | 5,740±203a | 35.4 |
| miR-378a mimic control | 8,657±375 | 2.6 |
| Sorafenib + miR-378a mimic | 1,245±88b | 86.0 |
| Sorafenib + miR-378a mimic control | 2,215±102b | 75.1 |
P<0.05
bP<0.01. Untransfected and miR-378a transfected HepG2 cells were treated with sorafenib for 24 h. Results were reported as mean migrated cells/5 field ± standard deviation of three independent experiments. The inhibition rate was calculated according to the formula: Inhibition rate (%) = [Cell count (non-treatment) - cell count (treatment)]/cell count (non-treatment) × 100. miRNA-378a, microRNA-378a; mimic-con, mimic control.
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