MicroRNA ‐ 495 suppresses cell proliferation and invasion of hepatocellular carcinoma by directly targeting insulin ‐ like growth factor receptor ‐ 1

Hepatocellular carcinoma (HCC) is the fifth most common malignancy and second‐most frequent cause of cancer-associated deaths worldwide. Previously, increasing studies report that microRNAs (miRNAs/miRs) are abnormally expressed in various types of human cancers and may participate in the tumourigenesis and tumour development of HCC. miRNA‐based targeted therapy is effective against different molecular targets and may increase the sensitisation of cancer cells to therapy by several folds. Therefore, further validation of potentially important miRNAs involved in HCC initiation and progression may provide valuable insights into the treatment of patients with HCC. miR‐495 is abnormally expressed in multiple types of human cancers. However, the expression level and roles of miR‐495 in HCC have yet to be completely elucidated. In the present study, miR‐495 expression was frequently downregulated in HCC tissues and cell lines, and miR‐495 expression levels were significantly correlated with tumour size, tumor-node-metastasis (TNM) stage and lymph node metastasis in patients with HCC. Functional assays revealed that miR‐495 overexpression inhibited cell proliferation and invasion in HCC. Insulin‐like growth factor receptor‐1 (IGF1R) was identified as a direct target gene of miR‐495 in HCC. IGF1R was upregulated in HCC tissues and negatively correlated with miR‐495 expression level. The upregulation of IGF1R rescued the miR‐495‐induced tumour‐suppressive roles in HCC cell proliferation and invasion, and the restored miR‐495 expression inactivated the protein kinase B and extracellular regulated protein kinase signalling pathways in HCC. These results provide novel insights into the molecular mechanism underlying HCC progression, and suggest that miR‐495 may be investigated as a novel therapeutic target for patients with this disease.


Introduction
Hepatocellular carcinoma (HCC), the fifth-most common malignancy and second-most frequent cause of cancer-related deaths worldwide (1,2), accounts for approximately 780,000 new cases and 745,000 deaths attributed to HCC each year (3). Multiple risk factors, including hepatitis B virus or hepatitis C virus infection, dietary aflatoxin B1 contamination, chronic alcohol abuse and tobacco consumption, lack of dietary antioxidants, arsenic exposure, obesity and non-alcoholic fatty liver disease, involved in HCC formation and progression have been identified (2,4). Chronic infection with hepatitis B virus or hepatitis C virus causes approximately 75% of all HCC cases (5). Although advanced treatments for patients with HCC have been developed, the long-term prognosis for these patients remains poor, and their current 5-year survival rate is approximately 30% (6). In addition, molecular mechanisms underlying HCC development have yet to be fully elucidated (7). Therefore, the molecular mechanisms of HCC initiation and progression should be further investigated to establish novel prognostic biomarkers and therapeutic methods for patients with this disease.
MicroRNAs (miRNAs/miRs), an endogenous group of short (18-25 nucleotides), non-coding and single-stranded RNA molecules, have emerged as a major regulator of tumourigenesis and tumour development (8,9). miRNAs negatively regulate gene expression by typically binding to complementary sequences in 3'-untranslated regions (3'-UTRs) of their target genes and therefore stimulate mRNA degradation or translational inhibition (10). Altered miRNA expression has been described in various types of human malignancies, such as HCC (11), renal cell carcinoma (12), gastric cancer (13) and colorectal cancer (14). miRNA dysregulation has been

MicroRNA-495 suppresses cell proliferation and invasion of hepatocellular carcinoma by directly targeting insulin-like growth factor receptor-1
demonstrated to be involved in various cellular biological processes, including cell proliferation, cell cycle, apoptosis, angiogenesis, invasion, migration, epithelial-to-mesenchymal transition and metastasis (15)(16)(17). In human cancer, miRNAs may serve as tumour suppressors or oncogenes which mainly depend on the biological roles of their target genes (13). Highly expressed miRNAs act as oncogenes by inhibiting tumour suppressor gene function, whereas downregulated miRNAs function as tumour suppressor genes through oncogene downregulation (14,15). Therefore, the expression and molecular mechanisms of miRNAs in human cancers should be further investigated to propose therapeutic targets for anticancer treatments. miR-495, mapped in the 14q32.31 region, is abnormally expressed in multiple types of human cancers, such as upregulated in bladder cancer (18), and downregulated in osteosarcoma (19), melanoma (20), endometrial cancer (21), acute myeloid leukaemia (22). However, the expression level and roles of miR-495 in HCC have yet to be completely elucidated. In our study, the expression levels of miR-495 in HCC tissues and cell lines were examined, and the effects of this miRNA on cell proliferation and invasion were evaluated in vitro. The underlying mechanism of miR-495 in HCC cells was also determined. Our study might provide novel insights into HCC occurrence and development.

Materials and methods
Human tissue specimens. 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.
Cell lines and culture conditions. 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).
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR). 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).
Cell Counting kit-8 (CCK-8) assay. 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% CO 2 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. 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 (5x10 4 ) 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% CO 2 , 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 R E T R A C T E D microscope (magnification, x200; Olympus Corporation, Tokyo, Japan).
Luciferase reporter assay. 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 1.5x10 5 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.
Western blot analysis. 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.
Statistical analysis. 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.

miR-495 is downregulated in HCC tissues and cell lines.
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.
Low miR-495 expression is correlated with adverse clinical features of patients with HCC. The patients with HCC were subsequently divided into either miR-495 low-expression

miR-495 inhibits cell proliferation and invasion in 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.

IGF1R is a direct downstream target of miR-495 in HCC.
To investigate the mechanisms by which miR-495 plays its tumour-suppressing roles in HCC, bioinformatics analysis

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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.
IGF1R is upregulated in HCC tissues and negatively correlated with miR-495 expression levels. 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).

IGF1R upregulation rescues the tumour-suppressive roles induced by miR-495 overexpression in HCC.
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.
miR-495 attenuates AKT and ERK signalling pathways in HCC. IGF1R likely performs its functions partly by participating in

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the regulation of AKT and ERK signalling pathways (26)(27)(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

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cell lines did not significantly change. These results suggested that miR-495 inactivates AKT and ERK signalling pathways in HCC.

Discussion
Recently, increasing studies reported that miRNAs are abnormally expressed in various types of human cancers (29)(30)(31) and can participate in the tumourigenesis and tumour development of HCC (32,33). miRNA-based targeted therapy is effective against different molecular targets and can increase the sensitisation of cancer cells to therapy by several folds (34). Therefore, further validation of potentially important miRNAs involved in HCC initiation and progression may provide valuable insights into the treatment of patients with HCC. In our study, miR-495 was significantly downregulated in HCC tissues and cell lines. Low miR-495 expression levels were correlated with adverse clinical features of the patients with HCC. The restored expression of miR-495 inhibited the proliferation and invasion of HCC cells in vitro. IGF1R was identified as a direct target of miR-495 in HCC, and miR-495 upregulation attenuated the activation of AKT and ERK signalling pathways in HCC. Therefore, miR-495 might serve as a tumour suppressor in HCC by directly targeting IGF1R and regulating AKT and ERK signalling pathways and might be developed as a novel therapeutic target for the treatment of patients with this fatal malignancy. miR-495 is aberrantly expressed in numerous types of human cancer. For example, miR-495 is upregulated in bladder cancer tissues and cell lines. High miR-495 expression levels are correlated with tumour size, TNM stage and lymph node metastasis of patients with bladder cancer (18). However, miR-495 in medulloblastoma is downregulated in tumour tissues compared with that in normal cerebellum tissues.
Log-rank analysis demonstrated that the average survival time of patients with medulloblastoma and with low miR-495 levels is shorter than that of patients with high miR-495 expression levels. Multivariate analysis also demonstrates miR-495 as an independent predictor of the overall survival of patients with medulloblastoma (35). miR-495 is weakly expressed in prostate cancer and associated with prostate-specific antigen levels, lymph node invasion and Gleason scores (36). miR-495 is also downregulated in osteosarcoma (19), melanoma (20), endometrial cancer (21), acute myeloid leukaemia (22), renal cell carcinoma (37), breast cancer (38,39) and lung cancer (40,41). These findings suggested that miR-495 expression exhibits tissue specificity and may be a biomarker for these human cancers.
miR-495 plays tumour-suppressive roles in multiple types of human malignancy. For instance, Jiang et al revealed that miR-495 overexpression suppresses osteosarcoma cell proliferation, colony formation, invasion and increased apoptosis in vitro (19). Studies showed that miR-495 upregulation inhibits cell proliferation and invasion and induces a metabolic shift in glioma (42)(43)(44). Formosa et al (36) and Li et al (45) indicated that the restored miR-495 expression attenuates prostate cancer cell growth and metastasis and promotes apoptosis in vitro. Liu et al reported that ectopic miR-495 expression decreases cell proliferation and metastasis and triggers cell apoptosis of melanoma (20). Xu et al demonstrated that miR-495 re-expression represses endometrial cancer cell growth and migration, promotes apoptosis and reduces growth in vivo (21). Lv et al found that restored miR-495 expression inhibits cell proliferation and motility and induces G0/G1 phase arrest in renal cell carcinoma (37). In lung cancer, miR-495 is involved in the regulation of cell proliferation, migration, epithelial-mesenchymal transition, chemosensitivity and chemoresistance (40,41,46). However, Tan et al indicated that miR-495 serves as an oncogene in bladder cancer through the regulation of cell proliferation and invasion (18). These conflicting findings suggested that the functional roles of miR-495 in human malignancies may be multifaceted and mainly dependent on involved tissues and their target genes.

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downregulation inhibits HCC cell proliferation and invasion but increases apoptosis (25). Therefore, IGF1R could be developed as a novel therapeutic target in HCC because of its cancer-related functions.
In conclusion, this study is the first to demonstrate that miR-495 inhibited the proliferation and invasion of HCC cells by directly targeting IGF1R and regulating AKT and ERK signalling pathways. These results provide novel insights into the molecular mechanism underlying HCC progression and suggest that miR-495 may be investigated as a novel therapeutic target for patients with HCC.