MicroRNA-212 targets FOXA 1 and suppresses the proliferation and invasion of intrahepatic cholangiocarcinoma cells

MicroRNAs (miRNAs), which are a class of small RNAs, have been shown to negatively regulate the expression of their target genes by directly binding to the 3'-untranslated region (3'-UTR) of mRNA. miRNA dysregulation has been associated with the pathogenesis of numerous types of human cancer. However, the role of miRNAs in intrahepatic cholangiocarcinoma (ICC) has yet to be fully elucidated. The present study aimed to investigate the role of miR-212 in the growth and metastasis of ICC in vitro, as well as the underlying mechanism. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting were used to examine mRNA and protein expression. An MTT assay and transwell assay were conducted to determine cell proliferation and invasion rates. The results of the RT-qPCR demonstrated that miR-212 was downregulated in the majority of investigated ICC tissues, as compared with their matched adjacent non-tumor tissues. In addition, miR-212 expression was shown to be markedly downregulated in three ICC cell lines, as compared with human intrahepatic biliary epithelial cells. Furthermore, restoration of miR‐212 expression significantly suppressed the proliferation and invasion of ICC QBC939 cells. Forkhead box protein A1 (FOXA1) was predicted to be a putative target of miR-212 by bioinformatics analysis with TargetScan. Therefore, a luciferase reporter assay was conducted to confirm that miR‐212 was able to directly bind to the 3'-UTR of FOXA1 mRNA. In addition, using western blot analysis, the protein expression of FOXA1 was shown to be negatively regulated by miR-212 in ICC QBC939 cells. In conclusion, it was demonstrated that FOXA1 was frequently upregulated in various ICC tissues and cell lines. The results of the present study suggested that miR-212 inhibits the proliferation and invasion of ICC cells by directly targeting FOXA1, and thus may be considered a potential candidate for the treatment of ICC.


Introduction
Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer (1), and the most common neoplasm of the biliary tract (2). Due to its increasing incidence worldwide and relatively poor prognosis, ICC remains an often fatal disease (3). Therefore, elucidating the molecular mechanisms underlying ICC is required in order to develop novel therapeutic strategies for ICC.
MicroRNAs (miRNAs) are a class of endogenous small RNAs containing ~22 nucleotides that have been reported to be frequently dysregulated in various types of human cancer (4,5). By negatively regulating the protein expression levels of their target genes, miRNAs have key roles in cancer progression (6,7). Since miRNAs may act as oncogenes or tumor suppressors, they may be considered as potential molecular targets or candidates for the treatment of various human cancer types (8,9). Among the tumor-associated miRNAs, miR-212 has been shown to serve a suppressive role in human cancer (10). For instance, upregulation of miR-212 is closely associated with a poor prognosis in patients with esophageal cancer (11). In addition, Jiang et al (12) demonstrated that overexpression of the miR-212/132 cluster inhibited the proliferation of human lung cancer cells. Liang et al (13) also reported that miR-212 acts as a tumor suppressor in hepatocellular carcinoma. In particular, the authors demonstrated that overexpression of miR-212 resulted in the suppression of H3K4 demethylase retinoblastoma binding protein 2 expression, inhibition of cell proliferation and induction of cellular senescence in hepatocellular carcinoma cells (13). Conversely, another study reported that miR-212 promoted pancreatic cancer cell growth and invasion by targeting the hedgehog signaling pathway receptor patched-1 (14). Therefore, miR-212 may serve a dual role in cancer, and its role in different cancer types requires further investigation. To the best of our knowledge, the exact role of miR-212 in ICC has yet to be elucidated.
The present study aimed to investigate the expression levels of miR-212 in ICC tissues and matched normal adjacent tissues. Furthermore, the current study examined the role of miR-212 in the regulation of ICC cell proliferation and invasion, as well as the underlying regulatory mechanism of its action.

Materials and methods
Tissue collection. The present study was approved by the Ethics Committee of the Third Xiangya Hospital of Central South University (Changsha, China). ICC tissues and their matched adjacent non-tumor tissues were collected from 15 patients with ICC at the Third Xiangya Hospital of Central South University between October 2013 and June 2014. The ICC patients included 9 males and 6 females, with an age range of 48-74 years (mean age, 62.7 years). ICC was diagnosed with histopathological examination performed by a pathologist (3 patients at disease stage II, 8 at stage III and 4 at stage IV). The patients did not receive any treatment prior to surgery. Written informed consent was obtained from all patients. All tissue samples were snap-frozen in liquid nitrogen following surgical removal and stored at -80˚C until further use.
Cell culture. Human QBC939 ICC cell lines and human intrahepatic biliary epithelial cells (HIBEpiC) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.) and incubated at 37˚C in an atmosphere with 5% CO 2 .
Transfection. MISSION ® miR-212 mimics (cat. no. HMI0377) and MISSION ® negative control (miR-NC; cat. no. HMC0002) were provided by Sigma-Aldrich (St. Louis, MO, USA). The cells were seeded in 24-well plates (100,000 cells/well) and transfected with 100 nM miR-212 mimics or miR-NC using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The cells were then cultured in an incubator at 37˚C for 48 h, for use during reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis.
RT-qPCR. Following tissue homogenization and centrifugation (12,000 x g, 10 min, 4˚C), total RNA was extracted from the fresh tissues or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. The purity and concentration of the RNA was determined using a Thermo Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). The miR-212 expression levels were determined using the TaqMan ® MicroRNA Reverse Transcription kit (cat. no. 4366597; Applied Biosystems; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. U6 was used as a normalization control for miRNA expression. For the detection of the mRNA expression levels, 0.5 µg RNA was reverse transcribed to cDNA using the RevertAid First Strand cDNA Synthesis kit (cat. no. K1621; Thermo Fisher Scientific, Inc.). . Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a normalization control for gene expression. A negative control used H 2 O instead of cDNA. The PCR reaction was performed on an Applied Biosystems ® 7500 Real-Time PCR system (Thermo Fisher Scientific, Inc.). The PCR mixture included 0.33 µl cDNA solution, 10 µl of 1X TaqMan universal PCR master mix, 2 µl of 1X gene specific primer/probe set (all from Applied Biosystems; Thermo Fisher Scientific, Inc.), and 7.67 µl H 2 O was added to obtain a final reaction volume of 20 µl. The primer sequences were as follows: FOXA1 forward, 5'-GCA ATA CTC GCC TTA CGG CT-3' and reverse, 5'-TAC ACA CCT TGG TAG TAC GCC-3'; and GAPDH forward, 5'-CTG GGC TAC ACT GAG CACC-3' and reverse, 5'-AAG TGG TCG TTG AGG GCA ATG-3'. The PCR reaction conditions were as follows: Initialization at 95˚C for 5 min, followed by 40 cycles of denaturation at 95˚C for 15 sec and annealing/elongation at 60˚C for 1 min. The relative fold-changes of miR-212 and FOXA1 mRNA expression were calculated using the 2 -ΔΔCq method (15).
Western blot analysis. QBC939 cells were lysed using lysis buffer (Beyotime Institute of Biotechnology, Wuhan, China), and total protein (20 µl) was extracted. The protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. Subsequently, total protein was separated by 10% SDS-PAGE, followed by transfer to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with phosphate-buffered saline (PBS) and 5% non-fat milk at 4˚C. The membranes were incubated overnight at 4˚C with polyclonal rabbit anti-human FOXA1 antibody (1:800; cat. no. AV32630; Sigma-Aldrich) and polyclonal rabbit anti-human GAPDH antibody (1:800; cat. no. G9545; Sigma-Aldrich). After washing three times with PBS, the membrane was incubated with polyclonal horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. R4880; Sigma-Aldrich) and visualized using an enhanced chemiluminescence reagent (EMD Millipore). The results were quantified using ImageJ version 1.1 (National Institutes of Health, Bethesda, MA, USA).
MTT assay. MTT assays were performed to assess cell proliferation. QBC939 cells transfected with miR-212 mimics or miR-NC were seeded into 96-well plates (1x10 5 cells/well) and cultured for 48 h, after which 10 mg/ml MTT was added to the medium and the cells were incubated for a further 4 h. Subsequently, the supernatant was removed by centrifugation (1,000 x g, 5 min, room temperature) and 100 µl DMSO was added to each well for 30 min to dissolve the formazan product. The optical density (OD) was measured at 492 nm using an ELx808 plate reader (BioTek Instruments, Inc., Winooski, VT, USA). The relative cell proliferation was calculated using optical density values, with the value of the control group set at 1.
Transwell assay. Transwell assays were conducted to investigate cell invasion. QBC939 cells (1x10 5 cells/well) transfected with miR-212 mimics or miR-NC were seeded into the top chamber of Matrigel-coated polyethylene terephthalate membrane, after which 10% FBS was added into the lower chamber. After culturing for 24 h, QBC939 cells in the upper chamber were removed, and the cells in the lower chamber were stained with 0.1% crystal violet for 30 min, followed by counting under an optical microscope (Olympus Corporation, Tokyo, Japan).
Statistical analysis. Data are presented as the mean ± standard deviation. Significant differences among the groups were determined using one-way analysis of variance with SPSS version 17.0 software (SPSS, Inc., Chicago, Il, USA). P<0.05 was considered to indicate a statistically significant difference. All experiments were performed in triplicate.

miR-212 expression is downregulated in ICC tissues and cell lines.
In order to investigate the potential role of miR-212 in ICC, RT-qPCR was performed to determine the expression levels of miR-212 in ICC tissues and their matched adjacent non-tumor tissues. As shown in Fig. 1A, the expression levels of miR-212 were downregulated in the majority of ICC tissue samples, as compared with their matched adjacent tissues. The expression levels of miR-212 were upregulated in two stage II samples. In addition, miR-212 expression was significantly downregulated in ICC cell lines, as compared with HIBEpiC (P<0.05; Fig. 1B). These results suggest that miR-212 may act as a tumor suppressor in ICC. Since miR-212 expression in QBC939 cells was decreased to a greater extent (Fig. 1B), this cell line was used in the subsequent experiments.

Restoration of miR-212 expression suppresses the proliferation of ICC cells.
The role of miR-212 in the regulation of ICC cell proliferation was investigated following transfection of human ICC QBC939 cells with miR-212 mimics or miR-NC. RT-qPCR was performed to determine the miR-212 levels in the cells following transfection. As is shown in Fig. 2A, the levels of miR-212 were significantly increased in QBC939 cells transfected with miR-212 mimics, as compared with the control group (P<0.05). MTT assay was also conducted to examine the cell proliferation capacity. As shown in Fig. 2B, ICC QBC939 cells overexpressing miR-212 demonstrated a significant decrease in cell proliferation, as compared with the control group (P<0.05). These results suggest that miR-212 has a suppressive effect on ICC cell proliferation.

Overexpression of miR-212 inhibits ICC cell invasion.
ICC is typically associated with a high capacity for invasion and distal metastasis (1). Therefore, the role of miR-212 in the regulation of ICC cell invasion was investigated. As is shown in Fig. 3, overexpression of miR-212 significantly inhibited the invasion of ICC QBC939 cells, as compared with the control group (P<0.05; Fig. 3). These results suggest that miR-212 has a suppressive effect on ICC cell invasion.
FOXA1 is a target of miR-212. In order to further investigate the molecular mechanisms underlying the involvement of miR-212 in ICC, the targets of miR-212 were analyzed using Targetscan. FOXA1 was predicted to be a putative target of miR-212, and their association was shown to be evolutionarily conserved (Fig. 4A). To validate this, a luciferase assay was performed, in which QBC939 cells were co-transfected with pMIR-REPORT vector containing the WT or MT FOXA1 3'-UTR, and with miR-212 mimics or miR-NC (Fig. 4B). Transfection with miR-212 mimics significantly decreased the luciferase activity in the WT group, but not in the MT group (Fig. 4C). This suggests that miR-212 directly targets FOXA1 by binding to seed sequences in the 3'-UTR of FOXA1 mRNA. Furthermore, overexpression of miR-212 resulted in a significant decrease in the protein expression levels of FOXA1 in ICC QBC939 cells (P<0.05), although it did not alter the mRNA expression level of FOXA1 ( Fig. 5A and B). These results suggest that miR-212 negatively regulates the expression of FOXA1 in ICC QBC939 cells at a post-transcriptional level.
FOXA1 is upregulated in the majority of ICC tissues and cell lines. The mRNA expression levels of FOXA1 were upregulated in the majority of ICC tissue samples, as compared with their matched normal adjacent liver tissues (Fig. 6A). Furthermore, the protein expression levels of FOXA1 were significantly upregulated in the ICC cell lines, as compared with HIBEpiC (P<0.05; Fig. 6B). These results suggest that aberrant upregulation of FOXA1 may participate in the malignant progression of ICC.

Discussion
It has previously been demonstrated that miRNAs negatively regulate the expression of their target genes by binding to the 3'-UTR of their target mRNA (16). Furthermore, various miRNAs have been associated with the development and progression of numerous types of human cancer, including ICC (17)(18)(19). Tumor growth and metastasis are the two main characteristics of ICC that lead to a poor prognosis (20,21). Therefore, understanding the role of miRNAs in these processes may aid in the diagnosis and treatment of ICC.
Dysregulation of miR-212 expression has been reported in several types of human cancer. For example, Park et al (22) demonstrated that miR-212 was upregulated in pancreatic adenocarcinoma tissues, and that knockdown of miR-212 reduced cell proliferation and induced a G 2 /M cell cycle arrest in Panc-1 cells. Furthermore, high expression levels of miR-212 were shown to predict a shorter overall survival, independent of age, gender, calendar year of surgery, KRAS mutation status, tumor stage, American Society of Anesthesiologists score, and localization and differentiation of pancreatic cancer (23). Similarly, it has been reported that miR-212 was upregulated in oral carcinoma (24). However, miR-212 has been shown to be frequently downregulated in several other types of human cancer. For instance, miR-212 was downregulated in gastric cancer and was shown to inhibit the DNA methylation by suppressing the expression of methyl-CpG-binding protein 2 (25). In addition, the expression of miR-212 was found to be downregulated in lung cancer, which may be due to histone modifications rather than DNA hypermethylation (26).
The present study revealed that miR-212 was downregulated in the majority of ICC tissues investigated, as compared with their matched adjacent non-tumor tissues, thus suggesting that miR-212 may act as a candidate tumor suppressor in the pathogenesis of ICC. Therefore, the ability of miR-212 to regulate ICC growth and metastasis in vitro was investigated in the current study. The results suggested that miR-212 significantly inhibited ICC cell proliferation and invasion, indicating a tumor suppressive role of miR-212 in ICC. FOXA1, which is also known as HNF3α, encodes a member of the forkhead class of DNA-binding proteins, which are transcriptional activators of liver-specific genes, including albumin and transthyretin (27,28). Furthermore, FOXA1 exhibited increased expression in liver development and metabolism (29). Previous studies have reported that FOXA1 is involved in various human cancer types, including liver cancer (30-32). Zhao and Li (33) demonstrated that  A FOXA1 was essential for both estrogen and androgen signaling by acting as a central regulator of sexual dimorphism in liver cancer. However, the detailed role of FOXA1 in ICC remains unclear. The present study demonstrated that FOXA1 was upregulated in the majority of investigated ICC tissues and in ICC cell lines, as compared with their matched adjacent normal tissues and HIBEpiC, respectively. By performing bioinformatics analysis and a luciferase reporter assay, the present study demonstrated for the first time that FOXA1 was a direct target of miR-212 in ICC cells. In addition, FOXA1 was shown to be involved in miR-212-mediated ICC cell proliferation and invasion. These results suggested that miR-212 suppresses ICC growth and metastasis by targeting FOXA1.
In conclusion, the present study demonstrated that miR-212 was aberrantly downregulated in ICC tissues and cell lines. Restoration of miR-212 expression significantly suppressed the proliferation and invasion of ICC cells by inhibiting the protein expression of FOXA1, a novel identified target of miR-212. The present study is the first to suggest that miR-212 may be a potential therapeutic candidate for ICC. The potential seed sequences of miR-212 within the wild-type and mutant 3'-UTR of FOXA1 mRNA. (C) A luciferase assay was performed using the pMIR-REPORT vector containing the WT or MT FOXA1 3'-UTR, which were co-transfected into intrahepatic cholangiocarcinoma QBC939 cells with miR-212 mimics or miR-NC. In the control group, QBC939 cells were transfected with pMIR-REPORT vector containing WT or MT FOXA1 3'UTR only. Data are presented as the mean ± standard deviation. ** P<0.05 vs. the control. miR-212, microRNA-212; 3'-UTR, 3'-untranslated region; FOXA1, forkhead box protein A1; WT, wild-type; MT, mutant; miR-NC, microRNA-normal control.