A total of 47 individual primary HCC tissues and matched adjacent non-tumor tissues were collected from patients who underwent surgical resection at the Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine (Shanghai, China) between May 2014 and January 2016. None of these HCC patients had been treated with chemotherapy or radiotherapy before surgery. All tissues specimens were flash-frozen in liquid nitrogen immediately after collection and stored at −80°C. This study was approved by the Ethics Committee of the Seventh People's Hospital of Shanghai University of Traditional Chinese Medicine, and written informed consent was also obtained from all participants prior to their participation in the study.

Three human HCC cell lines (SMMC-7721, Hep3B, and Huh7) and immortalized normal liver epithelial cell line (THLE-3) were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China).

All HCC cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; bth from Gibco, Grand Island, NY, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). THLE-3 cell line was maintained in Bronchial Epithelial Cell Growth Medium (Clonetics Corporation, Walkersville, MD, USA) containing 10% FBS, 5 ng/ml EGF and 70 ng/ml Phosphoethanolamine (both from Sigma-Aldrich, Merck KGaA). All cell lines were grown at 37°C in humidified atmosphere with 5% CO₂.

Mature miR-495 mimics and miRNA mimics negative control (miR-NC) were chemically synthesized by GeneCopoeia (Shanghai, China). Insulin-like growth factor receptor-1 (IGF1R) overexpression plasmid (pcDNA3.1-IGF1R) and empty plasmid (pcDNA3.1) were acquired from Shanghai GenePharma Co., Ltd. (Shanghai, China). For cell transfection, cells were seeded into 6-well plates at a density of 5×10⁵ cells per well. After incuabtion overnight, cell transfection was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA), according to the manufacturer's instructions.

Total RNA was isolated from tissue specimens or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) in accordance with the manufacturer's instructions. For miR-495 quantification, complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the TaqMan microRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Quantification of miR-495 was performed using a TaqMan microRNA Assay kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 small nuclear RNA was used as an endogenous control. To quantify IGF1R mRNA, corresponding cDNA was obtained form total RNA with a PrimeScript RT Reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China), according to the manufacturer's protocols. The quantitative PCR was carried out in an Applied Biosystems^® 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.), with GAPDH as an internal control. All reactions were performed in triplicate and fold changes were calculated based on relative quantification using the 2^−ΔΔCq method (23).

CCK-8 assay was utilized to determine the HCC cell proliferative ability. Transfected cells were collected at 24 h post-transfection, and mechanically dissociated into single cell suspension. Afterwards, transfected cells were seeded into 96-well plates at a density of 3,000 cells/well, and incubated at 37°C with 5% CO₂ for 0, 24, 48, and 72 h. At each time point, CCK-8 assay was performed according to the manufacturer's protocols. Briefly, each well was treated with 10 ul CCK-8 reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). After incubating at 37°C for additional 2 h, the optical density (OD) at a wavelength of 450 nm was determined using a Spectramax M5 microplate reader (Molecular Devices LLC, Sunnyvale, CA, USA). Each assay was performed with 5 replications.

Cell invasion assay was performed using Transwell insert chambers (8-µm pore size; Corning, Inc., Corning, NY, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). After transfection 48 h, cells were harvested and suspended in FBS-free DMEM. Transfected cells (5×10⁴) in 300 µl FBS-free DMEM were seeded into the upper chamber. The lower chambers were then filled with DMEM containing 10% FBS. Following a 24 h incubation at 37°C with 5% CO₂, non-invaded cells were removed using a cotton swab. Invasive cells were fixed with 4% polyoxymethylene, stained with 0.5% crystal violet, washed with PBS and dried in air. Finally, invasive cells were counted in five randomly selected visual fields under an inverted microscope (magnification, ×200; Olympus Corporation, Tokyo, Japan).

The TargetScan (http://www.targetscan.org/) and PicTar (http://pictar.mdc-berlin.de/) were used to predict the potential targets of miR-495.

Bioinformatic analysis indicated a potential miR-495 binding site in the 3′-UTR region of IGF1R. Lciferase reporter plasmids, pMIR-IGF1R-3′-UTR wild-type (Wt) and pMIR-IGF1R-3′-UTR wild mutant (Mut), were chemically synthesized by Shanghai GenePharma Co., Ltd. Cells were seeded into 24-well plates at a density of 1.5×10⁵ cells per well. After incubaiton overnight, cells were co-transfected with the wild-type or mutant 3′-UTR of IGF1R plasmid, and miR-495 mimics or miR-NC using the Lipofectamine 2000 reagent, according to the manufacturer's protocol. After transfection 48 h, the relative luciferase activity was detected using the Dual-Luciferase Reporter assay system (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. Renilla luciferase activity served as an internal control. Each assay was performed in 3 replicates and repeated at least three times.

Total protein was isolated from tissue specimens or cells with Radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate and 1 mg/ml aprotinin. The concentration of total protein was quantified with BCA assay kit (Beyotime Institute of Biotechnology). Equal amount of protein was separated by 10% SDS-PAGE and electronically transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica MA, USA). After blocking with 5% nonfat milk in TBST for 2 h at room temperature, the membranes were incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study include mouse anti-human monoclonal IGF1R antibody (sc-81464; 1:1,000 dilution), mouse anti-human monoclonal p-AKT (sc-271966; 1:1,000 dilution), mouse anti-human monoclonal AKT (sc-81434; 1:1,000 dilution), mouse anti-human monoclonal p-ERK (sc-81492; 1:1,000 dilution), mouse anti-human monoclonal ERK (sc-514302; 1:1,000 dilution), and mouse anti-human monoclonal GAPDH antibody (sc-47724; 1:1,000 dilution; all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequent washing three times with TBST, the membranes were probed with goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology) at room temperature for 2 h. Protein bands were visualized using an enhanced chemiluminescence kit (EMD Millipore), and analyzed using ImageJ 1.49 (National Institutes of Health, Bethesda, MD, USA). GAPDH served as a loading control.

Data are shown as the mean ± standard error of at least three independent experiments, and analyzed with Student's t-test or one-way analysis of variance. SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA) was used to perform statistical analysis. Spearman's correlation analysis was adopted to investigate the association between miR-495 and IGF1R mRNA expression level in HCC tissues. P<0.05 was considered statistically significant.

To determine the potential roles of miR-495 in HCC, RT-qPCR was performed to detect miR-495 expression levels in 47 primary HCC tissues and matched adjacent non-tumour tissues. Our results demonstrated that miR-495 was significantly downregulated in HCC tissues compared with that in matched adjacent non-tumour tissues (P<0.05; Fig. 1A). The miR-495 expression was also examined in three HCC cell lines, namely, SMMC-7721, Hep3B and Huh7, and in immortalized normal liver epithelial cell line, namely, THLE-3. In Fig. 1B, the miR-495 expression levels in the four HCC cell lines were lower than that of THLE-3 (P<0.05). Among these HCC cell lines, Hep3B yielded the lowest miR-495 expression levels and thus were selected for further functional studies. These results suggested that miR-495 may be involved in the formation and progression of HCC.

The patients with HCC were subsequently divided into either miR-495 low-expression group (n=24) or miR-495 high-expression group (n=23) to elucidate the clinical significance of miR-495 in HCC. The median expression levels of miR-495 in HCC tissues were regarded as cut-off. As shown in Table I, the low miR-495 expression level was correlated with tumour size (P=0.028), tumor-node-metastasis (TNM) stage (P=0.013) and lymph node metastasis (P=0.011). Conversely, miR-495 expression was not correlated with other clinical features, including age (P=0.642), sex (P=0.471), HBsAg (P=0.413), and differentiated (P=0.471). These results implied that miR-495 may be a prognostic indicator for patients with HCC.

Hep3B cells were transfected with miR-495 mimics or miR-NC to examine the effects of miR-495 on the biological characteristics of tumours. Transfection efficiency was determined through RT-qPCR, and the results indicated that miR-495 was markedly upregulated in Hep3B cells transfected with miR-495 mimics (P<0.05; Fig. 2A). CCK-8 assay was conducted to verify the effect of miR-495 overexpression on HCC cell proliferation. In Fig. 2B, upregulation of miR-495 inhibited Hep3B cell proliferation (P<0.05). Cell invasion assay was also performed to show the effect of miR-495 on HCC cell invasion abilities. The results demonstrated that the restored miR-495 expression reduced the invasive capabilities of Hep3B cells (P<0.05; Fig. 2C). These results illustrated the tumour-suppressive effects of miR-495 on HCC cell proliferation and invasion.

To investigate the mechanisms by which miR-495 plays its tumour-suppressing roles in HCC, bioinformatics analysis was conducted to predict the candidate targets of miR-495. IGF1R, which is highly expressed in HCC and possibly involved in HCC formation and progression (24,25), was predicted as a potential target of miR-495 (Fig. 3A) and consequently selected for further experimental validation. To confirm this hypothesis, we carried out a luciferase reporter assay in Hep3B cells co-transfected with wild-type or mutant 3′-UTR of IGF1R plasmid and miR-495 mimics or miR-NC. In Fig. 3B, ectopic miR-495 expression significantly decreased the luciferase activity of pMIR-IGF1R-3′-UTR Wt in Hep3B cells (P<0.05) but did not affect the luciferase activity of pMIR-IGF1R-3′-UTR Mut. To confirm whether IGF1R is a direct target of miR-495, we performed RT-qPCR and Western blot analysis and then determined the regulatory effects of miR-495 on endogenous IGF1R expression in HCC cells. RT-qPCR and Western blot analysis revealed that the restored miR-495 expression suppressed the IGF1R expression in Hep3B cells at mRNA (P<0.05; Fig. 3C) and protein (P<0.05; Fig. 3C) levels. These results suggested that IGF1R is a direct target of miR-495 in HCC.

The IGF1R level was examined in HCC tissues and matched adjacent non-tumour tissues to analyse the correlation between miR-495 and IGF1R. In Fig. 4A and B, the IGF1R expression levels were significantly higher in HCC tissues than in matched adjacent non-tumour tissues at mRNA (P<0.05) and protein (P<0.05) levels. Spearman's correlation analysis revealed that miR-495 was negatively associated with the mRNA expression level of IGF1R in HCC tissues (r=−0.6591, P<0.001; Fig. 4C).

Rescue experiments were performed to confirm whether the biological roles of miR-495 in HCC are mediated by IGF1R. Hep3B cells were transfected with miR-495 mimics with or without IGF1R overexpression plasmid (pcDNA3.1-IGF1R). Western blot analysis indicated that miR-495 overexpression decreased the IGF1R protein expression, whereas pcDNA3.1-IGF1R co-transfection could rescue the IGF1R expression in Hep3B cells (P<0.05; Fig. 5A). Subsequent functional assays demonstrated that the restored IGF1R expression rescued the suppressive effects of miR-495 on the proliferation (P<0.05; Fig. 5B) and invasion of Hep3B cells. These results implied that miR-495 plays its tumour-suppressing roles in HCC partly by downregulating IGF1R.

IGF1R likely performs its functions partly by participating in the regulation of AKT and ERK signalling pathways (26–28). To determine whether miR-495 is involved in the regulation of AKT and ERK pathways in HCC, we detected the expression levels of AKT, p-AKT, ERK and p-ERK in Hep3B cells transfected with miR-495 mimics or miR-NC. Western blot analysis demonstrated that miR-495 overexpression reduced the expression of p-AKT and p-ERK in Hep3B cells (Fig. 6). Conversely, the expression of total AKT and ERK in these two cell lines did not significantly change. These results suggested that miR-495 inactivates AKT and ERK signalling pathways in HCC.