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Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2016 Jan 29;142(5):969–980. doi: 10.1007/s00432-016-2121-8

Hepatocyte nuclear factor 6 inhibits the growth and metastasis of cholangiocarcinoma cells by regulating miR-122

Huaqiang Zhu 1, Yuetang Mi 2, Xian Jiang 3, Xu Zhou 1, Rui Li 2, Zheng Wei 3, Hongchi Jiang 3, Jun Lu 1, Xueying Sun 3,4,✉
PMCID: PMC11819340  PMID: 26825606

Abstract

Purpose

Hepatocyte nuclear factor 6 (HNF6) is a liver-enriched transcription factor and highly expressed in mature bile duct epithelial cells. This study sought to investigate the role of HNF6, particularly the molecular mechanisms for how HNF6 is involved in the growth and metastasis of cholangiocarcinoma (CCA) cells.

Methods

The expression of HNF6, miR-122 and key molecules was examined by Western blot analysis and real-time RT-PCR. Stable transfectants, HCCC-HNFlow and RBE-HNFhigh, were generated from human CCA HCCC-9810 and RBE cells, respectively. The regulatory effect of HNF6 on miR-122 was evaluated by luciferase reporter assay. Cell proliferation, cycle distribution, migration and invasion were analyzed. The xenograft model was used to assess the effects of HNF6 overexpression on tumorigenesis, growth, metastasis and therapeutic potentials.

Results

Human CCA tissues and cells expressed lower levels of HNF6, which positively correlated with miR-122. HNF6 regulated the expression of miR-122 by stimulating its promoter. HNF6 overexpression inhibited cell proliferation by inducing cell cycle arrest at G1 phase through regulating miR-122, cyclin G1 and insulin-like growth factor-1 receptor. HNF6 inhibited the migration and invasion of CCA cells by regulating matrix metalloproteinase-2 and metalloproteinase-9, reversion-inducing-cysteine-rich protein with kazal motifs, E-cadherin and N-cadherin. Co-transfection of anti-miR-122 abrogated the effects of HNF6. HNF6 overexpression inhibited the ability of cells to form tumors and to metastasize to the lungs of mice, and the growth of established tumors.

Conclusions

The results indicate that HNF6 may serve as a tumor suppressor by regulating miR-122, and its overexpression may represent a mechanism-based therapy for CCA.

Keywords: Hepatocyte nuclear factor 6, Cholangiocarcinoma, MicroRNA-122, Proliferation, Metastasis

Introduction

Cholangiocarcinoma (CCA), derived from the epithelial cells of biliary ducts, is the second most common type of primary liver cancer (Bridgewater et al. 2014). CCA is characterized by early lymph and distant metastases, and as a result, only 10 % of patients have an opportunity for curative surgical resection (Bridgewater et al. 2014). The combination of gemcitabine and cisplatin is the standard adjuvant therapy for advanced CCA, but offers unsatisfied survival benefits. CCA patients have a poor prognosis with a median survival of approximately 1 year (Valle et al. 2010). Therefore, it is urgently required to seek novel molecular targets for combating CCA.

Hepatocyte nuclear factor 6 (HNF6, also called ONECUT1) is one of the liver-enriched transcription factors (LETFs), which control hepatic gene expression (Wang and Holterman 2012). HNF6 plays an important role in regulating a wide range of genes, which are associated with important biological functions including cell proliferation, differentiation, migration and cell–matrix adhesion, and bile homeostasis (Wang and Holterman 2012). HNF6 is required for the development of biliary system (Kyrmizi et al. 2006) and highly expressed in mature bile duct epithelial cells (Sasaki et al. 2013). Loss of HNF6 expression correlates with the progression of human pancreatic cancer (Pekala et al. 2014). Recovery of HNF6 inhibits the growth of colon cancer and hepatocellular carcinoma (HCC) cells (Lehner et al. 2010), and liver metastasis of colorectal carcinoma (Lehner et al. 2007). HNF6 inhibits epithelial-mesenchymal transition, and migration and invasive growth of lung cancer cells (Yuan et al. 2013). However, the role of HNF6 in CCA, particularly the molecular mechanisms for how HNF6 is involved in the growth and metastasis of CCA cells, has not been reported.

MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate the expression of target genes. MiR-122 is one of the most abundant miRNAs in the liver, accounting for 52 % of the whole miRNome in adult human (Bandiera et al. 2015). MiR-122 has been characterized for its multiple functions in liver physiology and diseases (Bandiera et al. 2015). MiR-122 is identified as a tumor suppressor microRNA that regulates intrahepatic metastasis of HCC (Coulouarn et al. 2009). Lower levels of miR-122 are observed in HCC cells compared with normal hepatocytes and correlate with poor prognosis of HCC patients (Hsu et al. 2012). Restoration of miR-122 shows a strong inhibition of hepatocarcinogenesis and tumor growth (Tsai et al. 2009, 2012). However, the expression of miR-122 in CCA has been rarely studied, though one study provides a clue that miR-122 is downregulated in intrahepatic CCA tissues (Karakatsanis et al. 2013). It has been reported that HNF6 regulates hepatocyte-specific genes in controlling hepatocyte differentiation by stimulating the expression of miR-122 (Laudadio et al. 2012; Xu et al. 2010).

In the present study, we firstly demonstrated that the expression of HNF6 was significantly lower in clinical CCA tissues compared with adjacent noncancerous tissues and positively correlated with miR-122. Based on these observations, we hypothesized that HNF6 might play a role in the progression of CCA. To address this, we examined the proliferation and metastasis of CCA cells, which have been genetically modified to overexpress or depleted of HNF6, in culture and experimental animals. In exploring the underlying mechanisms, we investigated the regulatory effect between HNF6 and miR-122, and the alterations of genes involved in cell proliferation, migration and invasion.

Materials and methods

Patient tissues

A total of 21 CCA tissues and the corresponding adjacent noncancerous tissues were obtained from Provincial Hospital Affiliated to Shandong University (12 cases of perihilar cholangiocarcinoma and 3 cases of intrahepatic cholangiocarcinoma, Jinan, China) and Liaocheng People’s Hospital (6 cases of perihilar cholangiocarcinoma, Liaocheng, China). We did not include cholangiocarcinoma of Vater’s papilla, which is viewed as an independent entity because of the distinct biology (Blechacz et al. 2011). Fresh tissue samples were collected and snap-frozen in liquid nitrogen.

Cell culture

Human CCA cell lines (HCCC-9810, RBE) were obtained from Chinese Academy of Sciences Cell Bank (Shanghai, China), and QBC939 and CC262 were gifted from Professor Jie Li, Shandong University. Normal human biliary epithelial HIBEC cells were from ScienCell (San Diego, CA, USA). Cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum.

HNF6 expression vectors

The shRNA vector targeting human HNF6 was generated by using two oligonucleotides 5′-tcgaGCCUCCAUGAAUAACCUCUAUgaguacugAUAGAGGUUUUCAUGGAGGCUUUUU-3′and 5′-ctagAAAAAUGGAAGUAAUUCAGGGCAGAUcaguacucACCUUCAUUAAGUCCCGUCUA-3′, which were annealed and introduced into the pSuppressorNeo vector as described previously (He et al. 2015a; Yuan et al. 2013). The first capitalized region corresponds to nucleotides 603–623 of human HNF6 (GenBank U96173.1) (Yuan et al. 2013). An Sc shRNA vector was used as a control. The full-length complementary DNA encoding human HNF6 (GenBank U96173.1) was generated by RT-PCR, sequenced and subcloned into pcDNA3.1 vector (Invitrogen).

Establishment of stable transfectants

Cells were seeded in 10-cm plastic dishes and grown to 67 % confluence at which point they were transfected with 4 μg of each vector using Lipofectamine 2000 (Invitrogen, Beijing, China). They were detached by trypsinization after transfection for 48 h and seeded in selection media containing geneticin (G418) (500 μg/ml). Stable transfectants were selected at 4 weeks of culture. HCCC-9810 and RBE cells stably transfected with HNF6 shRNA or Sc vectors were termed HCCC-HNFlow and HCCC-Sc, respectively. RBE cells stably transfected with HNF6-pcDNA3.1 or pcDNA3.1 empty vectors were termed RBE-HNFhigh and RBE-vec, respectively.

Transfection of oligonucleotides

The double-stranded miR-122 mimics (5′-CCUUAGCAGAGCUGUGGAGUGUGACAAUGGUGUUUGUGUCUAAACUAUCAAACGCCAUUAUCACACUAAAUAGCUACUGCUAGGC-3′), anti-miR-122 (5′-AACGCCAUUAUCACACUAAAUA-3′) and the negative control oligonucleotides (5′-CAGUACUUUUGUGUAGUACAA-3′) were purchased from GenePharma Co., Ltd., Shanghai, China). Cells were grown to 60–70 % confluence and incubated with RNAs at a final concentration of 0.1 μM by using Lipofectamine™ 2000 (Invitrogen) in serum-free media for 48 h and then subjected to assays.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)

The methods have been described in detail previously (He et al. 2015a; Sun et al. 2014; Wei et al. 2013). The primers were designed for HNF6 mRNA (forward: 5′-TGCGTTCATGAAGAAGTTGC-3′ and reverse: 5′-TGCTATCTTGAGGTCCTGGTC-3′) (Lehner et al. 2007), and an internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (forward: 5′-CACCCATGGCAAATTCCATGGCA-3′ and reverse: 5′-TCTAGACGGCAGGTCAGGTCCACC-3′). The expression of mature miR-122 and U6 (an inner control) was examined by using a TaqMan MiRNA Reverse Transcription Kit (Applied Biosystems), individual TaqMan MiRNA assay and MX3000P Real-time PCR system. Experiments were performed in triplicate, and data were calculated by ΔΔC t methods.

Luciferase reporter assay

The miR-122 promoter containing HNF6-targeting sequence (CAATCGATA) (Laudadio et al. 2012) was cloned into a pMIR-REPORT luciferase reporter vector (Ambion). The assay was conducted as described previously (He et al. 2015b). Briefly, the reporter vector plasmid was transfected into cells using Lipofectamine 2000. To correct transfection efficiency, a luciferase reporter vector without the HNF6 target sequence was transfected in parallel. Luciferase activities in cells were measured by using a luciferase assay kit (Promega, Madison, WI), and relative luciferase activity was expressed as percentage of the luciferase activity of the reporter vector with HNF6 target sequence over one without the HNF6 target sequence.

Cell proliferation analysis

Cells were seeded into a 96-well plate (3 × 103/well) in triplicate and cultured. Cell viability was measured with a Cell Counting Kit-8 (CCK-8) kit (Dojindo Molecular Technologies, Gaithersburg, MD, USA) and represented by optical density (OD) at 450 nm.

Assessment of cell cycle

Cells were seeded at 5.0 × 105 cells/well in 6-well plates, cultured for 48 h and then harvested. The percentage of cells at G2/M, S and G0/G1 phases was determined with a cell cycle detection kit (BD Biosciences, Beijing, China) by a Beckman Coulter Epics Altra II cytometer (Beckman Coulter, California, USA).

Migration and invasion assays

The methods have been described previously (Wei et al. 2013).

Western blot analysis

The methods have been described previously (He et al. 2015a; Sun et al. 2014; Wei et al. 2013). Cells and tissues were homogenized in protein lysate buffer, and debris was removed by centrifugation. Protein concentrations were determined. Lysates were resolved on sodium dodecyl sulfate–polyacrylamide gels, electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were blocked and incubated overnight with primary Abs against HNF6 (Abcam, Cambridge, MA, USA), matrix metalloproteinase (MMP)-2, MMP-9, reversion-inducing-cysteine-rich protein with kazal motifs (RECK), metallopeptidase inhibitor 2 (TIMP-2) (Santa Cruz Biotechnology, CA, USA), cyclin G1, IGF-1R (insulin-like growth factor 1 receptor), E-cadherin and N-cadherin and GAPDH (Cell Signaling Technology, Danvers, USA). The membranes were subsequently incubated with secondary horseradish peroxidase-conjugated Abs (Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China). They were developed with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) (Tiangen Biotech Co. Ltd., Beijing, China). The density of each band was measured using a densitometric analysis program (FR200, Shanghai, China). In preliminary experiments, serial dilutions of lysates (containing 2.5, 5, 10, 20, 40 or 80 µg protein) were blotted; band intensities were measured and plotted against protein amounts to generate a standard curve, and the amount of protein for each blot was determined.

Animal experimental protocols

Six- to eight-week-old male nude BALB/c mice (H-2b) were obtained from the Animal Research Center, The First Affiliated Hospital of Harbin Medical University, China.

Tumorigenicity study

Cells (2 × 106) were injected subcutaneously into both sides of the flanks of group of 6 mice. The animals were monitored for tumor appearance every week. Six weeks later, mice were killed and tumors were measured. Tumor volume was estimated by the formula: π/6 × a 2 × b, where a is the short axis and b the long axis.

Metastasis study

Cells (1 × 106) were injected into groups of 6 mice via the lateral tail vein. The mice were killed 7 weeks later, and their lungs removed. The lungs were weighed and subjected to hematoxylin and eosin (HE) staining.

Therapeutic effect study

Cells (1 × 106) were injected subcutaneously into the left flanks of mice. When tumors reached ~100 mm3, mice were assigned to 4 groups of 6 mice, which received an intratumoral injection of pcDNA3.1 empty vector, HNF6-pcDNA3.1, HNF6-pcDNA3.1 + negative control oligonucleotides or HNF6-pcDNA3.1 + anti-miR-122 oligonucleotides, respectively. The gene transfection solution was prepared by mixing plasmids and/or oligonucleotides, Lipofectamine 2000 and serum-free medium. Each tumor received an injection of 50μl transfection solution containing 100 µg plasmids + 100 µg oligonucleotides. The tumors were measured every 4 days and the mice killed 20 days later.

In situ Ki-67 proliferation index

The methods have been described previously (He et al. 2015a; Sun et al. 2014; Wei et al. 2013).

Statistical analysis

The correlation between HNF6 expression and miR-122 was analyzed by Pearson test. Other data are expressed as mean values ± standard deviation. Comparisons were made with a one-way analysis of variance (ANOVA) followed by a Dunnet’s test. P < 0.05 was considered statistically significant.

Results

HNF6 expression correlates with miR-122 in CCA tissues

The expression of HNF6 mRNA and miR-122 was detected in 21 cases of CCA tissues and the corresponding adjacent noncancerous tissues by qRT-PCR analyses. The expression of both HNF6 mRNA (Fig. 1a) and mature miR-122 (Fig. 1b) was significantly lower in CCA tissues than adjacent noncancerous tissues. Moreover, HNF6 mRNA expression was positively correlated with that of miR-122 in CCA tissues by using a Pearson test (Fig. 1c).

Fig. 1.

Fig. 1

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Expression of HNF6 positively correlates with miR-122 in CCA tissues. The expression of HNF6 mRNA (a) and mature miR-122 (b) in 21 human CCA tissues and the corresponding adjacent noncancerous tissues was detected by qRT-PCR. c The correlation between HNF6 mRNA and miR-122 CCA tissues was analyzed using a Pearson test. n Number of samples examined

HNF6 and miR-122 expression in CCA cells

Human biliary epithelial HIBEC cells expressed a higher level of HNF6 protein than any of the four CCA cell lines, which expressed different levels of HNF6 protein (Fig. 2a). The order of cell lines with the highest to lowest HNF6 expression was HCCC-9810, QBC939, CC262 and RBE cells (Fig. 2a). The levels of HNF6 mRNA were consistent with that of HNF6 protein, and positively correlated with that of miR-122, in CCA cells (Fig. 2b). The levels of HNF6 protein were also positively correlated with that of miR-122 (Fig. 2c).

Fig. 2.

Fig. 2

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Expression of HNF6 and miR-122 in CCA cells. a Lysates of HCCC-9810, QBC939, CC262, RBE and HIBEC cells were Western blotted to detect the expression of HNF6 protein. b The expression of HNF6 mRNA and miR-122 was analyzed by qRT-PCR. c The expression of HNF6 protein in a was quantified and paralleled with miR-122. d Lysates of parental HCCC-9810 cells, and HCCC9810 cells stably transfected with Sc (HCCC-Sc) and HNF6 shRNA vectors (HCCC-HNFlow) were subjected to Western blotting. e Lysates of parental RBE cells, and RBE cells stably transfected with empty pcDNA3.1 vector (RBE-vec) and the HNF6/pcDNA3.1 vector (RBE-HNFhigh) were subjected to Western blotting. The density of each HNF6 protein band was measured and normalized to that of GAPDH, respectively. f, g The expression of miR-122 in the above cells in d, e was detected by qRT-PCR. *P < 0.05 and **P < 0.001 indicate a significant difference from the respective parental cells

We subsequently genetically modified HCCC-9810 and RBE cells, which had the highest and lowest levels of HNF6 expression, respectively. HNF6 expression by HCCC-9810 cells was significantly reduced by transfection with an shRNA vector, generating HCCC-HNFlow cells (Fig. 2d). Conversely, HNF6 expression by RBE cells was significantly increased by transfection with an HNF6 expression vector (HNF6-pcDNA3.1), generating RBE-HNFhigh cells (Fig. 2e). HCCC-9810 and RBE cells were also transfected with the scrambled control (Sc) shRNA vector, or pcDNA3.1 empty vector, to generate control HCCC-Sc and RBE-vec cells, which were shown to express a similar level of HNF6, compared with their respective parental cells (Fig. 2d, e). The level of miR-122 was significantly downregulated in HCCC-HNFlow cells (Fig. 2f) and upregulated in RBE-HNFhigh cells (Fig. 2g), whereas the level of miR-122 remained unchanged in HCCC-Sc and RBE-vec cells, compared with their respective parental cells (Fig. 2f, g). The results indicate a positive correlation between HNF6 and miR-122 in CCA cells, in consistence with the results in CCA tissues.

The regulatory effect between HNF6 and miR-122 in CCA cells

The miR-122 promoter contains a conserved sequence matching the HNF6 binding consensus upstream of the transcription start site (Laudadio et al. 2012; Xu et al. 2010). To verify whether HNF6 could upregulate the expression of miR-122 by stimulating its promoter, we transfected CCA cells with a luciferase reporter containing miR-122 promoter with a HNF6 binding site (Fig. 3a). Luciferase activities were significantly reduced in HCCC-HNFlow cells than HCCC-9810 or HCCC-Sc cells and significantly elevated in RBE-HNFhigh cells than RBE or RBE-vec cells (Fig. 3b). It has been reported that miR-122 stimulates the expression of hepatocyte-specific genes including HNF6 in bipotent murine embryonic liver (BMEL) cells (Laudadio et al. 2012). Interestingly, transfection of either anti-miR-122 or miR-122 mimics into HCCC-9810 or RBE cells, respectively, did not induce significant alterations in the expression of HNF6 mRNA (Fig. 3c) or protein (Fig. 3d). The results indicate that the regulatory effect between HNF6 and miR122 may be in a one-way manner in CCA cells.

Fig. 3.

Fig. 3

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HNF6 regulates the expression of miR-122 in CCA cells. a Predicted paring of HNF6 binding site to the promoter region of miR-122, and the diagram of a pMIR-REPORT luciferase reporter vector containing the promoter region of miR-122 with HNF6 binding seed site (pMIR-miR-122/prom/luc). b Cells as indicated were transfected with pMIR-miR-122/prom/luc or a control vector. Relative luciferase activity was calculated as the percentage of luciferase activity in pMIR-miR-122/prom/luc-transfected cells over those with the control vector. c, d HCCC-9810 and RBE cells were transfected with negative control, anti-miR122 or miR122 mimics as indicated. c The expression of HNF6 mRNA was analyzed by qRT-PCR. d The expression of HNF6 protein was detected by Western blotting. **P < 0.001 indicates a significant difference; NS no significant difference

HNF6 inhibits cell proliferation by inducing cell cycle arrest at G1 phase

HNF6 inhibits the proliferation of colon carcinoma and HCC cells (Lehner et al. 2010); thus, we investigated whether HNF6 could have an inhibitory effect on the proliferation of CCA cells. The viability of HCCC-HNFlow cells was slightly higher than that of HCCC-9810 or HCCC-HNF-Sc cells (Fig. 4a). This is possibly due to the low basal level of HNF6 in HCCC-9819 cells. However, the viability of RBE-HNFhigh cells was significantly lower than that of RBE or RBE-HNF-vec cells (Fig. 4b). The above results showed that HCCC-HNF-Sc and RBE-HNF-vec cells did not differ in gene expression and cell proliferation from their respective parental cells; thus, only parental cells were used as controls in the following experiments. A slight reduction in cell proportion arrested at G0/G1 phase was observed in HCCC-HNFlow cells compared with HCCC-9810 cells (Fig. 4c). However, RBE-HNFhigh cells had a marked increase in the proportion of cells arrested at G0/G1 phase compared with RBE cells (Fig. 4d).

Fig. 4.

Fig. 4

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HNF6 inhibits cell proliferation by regulating miR-122. a, b Cells were cultured for 7 days, and their viability measured at indicated time points. Cell viability is represented by optical density (OD) at 450 nm. c HCCC-9810 and HCCC-HNFlow cells, and d RBE and RBE-HNFhigh cells were cultured for 72 h and subjected to flow cytometry to measure cell cycle distribution. e HCCC-9810 and HCCC-HNFlow cells were incubated with negative control or miR-122 mimics for 48 h, and cell cycle distribution was measured. f RBE and RBE-HNFhigh cells were incubated with negative control or anti-miR-122 for 48 h, and cell cycle distribution was measured. g, h Lysates of the above cells in e, f were subjected to Western blotting. The density of each band was measured and normalized to that of GAPDH, respectively. *P < 0.05 and **P < 0.001 indicate a significant difference

MiR-122 affects the influence of HNF6 on cell cycle distribution

MiR-122 induces cell cycle arrest at G1 phase by downregulating the expression of cyclin G1 and insulin-like growth factor 1 receptor (IGF-1R) (Gramantieri et al. 2007; He et al. 2015b). Therefore, we hypothesized that HNF6 may display its activity on cell cycle distribution through regulating miR-122 in CCA cells. We first examined whether miR-122 mimics could restore the ability of NNF6 in regulating cell cycle in HCCC-HNFlow cells. As shown in Fig. 4e, transfection of miR-122 mimics resulted in a significant increase in the proportion of cells arrested at G0/G1 phase in HCCC-HNFlow cells. We next examined whether anti-miR-122 could abolish the effects of NNF6 overexpression in regulating cell cycle. As shown in Fig. 4f, transfection of anti-miR-122 resulted in a significant reduction in the proportion of cells arrested at G0/G1 phase in RBE-HNFhigh cells. Western blot analysis showed that HCCC-HNFlow cells expressed slightly higher levels of cyclin G1 and IGF-1R than HCCC-9810 cells; miR-122 mimics slightly reduced the expression of the two proteins in HCCC-HNFlow cells (Fig. 4g). However, RBE-HNFhigh cells expressed significantly lower levels of cyclin G1 and IGF-1R than RBE cells, and anti-miR-122 abrogated the effects of HNF6 overexpression in RBE-HNFhigh cells (Fig. 4h). The above results indicate that HNF6 downregulation had slight effects on the proliferation of HCCC-9810 cells, but HNF6 overexpression markedly inhibited the proliferation of RBE cells. This could possibly be explained by that the basal level of HNF6 was very low in CCA cells. Therefore, we further investigated the effects of HNF6 overexpression in the following experiments.

Overexpression of HNF6 inhibits cell migration and invasion

HNF6 inhibits the cellular ability to migrate and invade, thus suppressing tumor metastasis (Lehner et al. 2007; Yuan et al. 2013). We demonstrated that the numbers of RBE-HNFhigh cells migrated and invaded through the transwells were significantly lower than RBE cells, and transfection of anti-miR-122 abrogated the effects of HNF6 overexpression (Fig. 5a, b). The gelatin zymography assay showed that the activities of MMP-2 and MMP-9, which participate in cell migration (Bourboulia and Stetler-Stevenson 2010), were significantly reduced in RBE-HNFhigh cells, and transfection of anti-miR-122 abrogated these effects of HNF6 overexpression (Fig. 5c). The results of MMP-2 and MMP-9 activities were supported by their protein expression; thus, RBE-HNFhigh cells expressed lower levels of MMP-2 and MMP-9 proteins compared with RBE cells, and transfection of anti-miR-122 abrogated this effect of HNF6 overexpression (Fig. 5d). RECK, a negative regulator of MMP-2 and MMP-9 (Oh et al. 2001), was significantly upregulated by HNF6 overexpression, whereas TIMP2, a tissue inhibitor of MMP-2 and MMP-9 (Bourboulia and Stetler-Stevenson 2010), remained unchanged upon HNF6 overexpression (Fig. 5d, e). E-cadherin and N-cadherin are important transmembrane proteins mediating cellular migration and invasion (Nelson and Nusse 2004). Here we showed that RBE-HNFhigh cells expressed a higher level of E-cadherin and a lower level of N-cadherin than RBE cells, and transfection of anti-miR-122 abrogated these effects of HNF6 overexpression (Fig. 5d, e).

Fig. 5.

Fig. 5

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Overexpression of HNF6 inhibits cell migration and invasion by regulating miR-122. RBE and RBE-HNFhigh cells were incubated for 48 h with negative control or anti-miR-122. a Cells were subjected to migration and invasion assays. Migrated or invaded cells were visualized using Giemsa staining (× 200 magnification). Bar 200 µm. b Numbers of migrated and invaded cells were counted, respectively. c The conditioned media from cell culture were subjected to a gelatin zymography assay to measure the gelatinolytic activity of pro-MMP-9 (upper line, ~92 kDa) and active MMP-2 (lower line, ~66 kDa). d Cell lysates were subjected to Western blot analysis. e The density of each band was measured and normalized to that of GAPDH, respectively. *P < 0.05 and **P < 0.001 indicate a significant difference from parental cells. # P < 0.05 and ## P < 0.001 indicate a significant increase from RBE-HNFhigh cells transfected with negative control

Overexpression of HNF6 inhibits tumorigenesis and lung metastasis

RBE-HNFhigh cells were subcutaneously injected into mice to investigate whether their ability to form tumors in vivo had been changed compared with RBE cells. Four out of 6 mice had 8 palpable subcutaneous tumors on their two sides of flanks 6 weeks after inoculation of RBE-HNFhigh cells, while all the 6 mice injected with RBE cells had 12 tumors on their both side of flanks. In addition, 6 weeks after cell inoculation, RBE-HNFhigh tumors grew to only 814.6 ± 125.7 mm3 in size, which was highly significantly smaller than RBE tumors of 2146.5 ± 327.8 mm3 (Fig. 6a). RBE and RBE-HNFhigh cells were intravenously injected into the mice, which were killed 7 weeks later, and their lungs were harvested to assess the formation of lung metastases. As shown in Fig. 6b, mice injected with RBE-HNFhigh cells had an average lung weight of 219.2 ± 45.3 mg, which was significantly lighter than that (374.9 ± 81.6 mg) of mice receiving RBE cells. Representative images were taken from HE-stained lung sections from the mice injected with RBE or RBE-HNFhigh cells (Fig. 6c). We next examined whether overexpression of HNF6 could have a therapeutic effect on the growth of tumors, which were established by subcutaneous injection of RBE cells into mice. When tumors reached ~100 mm3, the mice were assigned to different treatments, namely pcDNA3.1 empty vector, HNF6-pcDNA3.1, HNF6-pcDNA3.1 + negative control oligonucleotides or HNF6-pcDNA3.1 + anti-miR-122. Gene transfer of HNF6-pcDNA3.1 led to a significant inhibition of tumor growth compared with pcDNA3.1, while anti-miR-122 could abrogate this inhibition (Fig. 6d).

Fig. 6.

Fig. 6

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Overexpression of HNF6 inhibits tumorigenesis, metastasis and tumor growth in mice. a RBE and RBE-HNFhigh cells were subcutaneously inoculated into both flanks of mice. Tumor volumes were measured 6 weeks later. b, c RBE and RBE-HNFhigh cells were intravenously injected into the mice. The mice were killed 7 weeks later to harvest lungs, which were weighed (b). c Representative photographs of HE-stained sections showed the metastatic lesions in lungs. d Tumors were established in mice by subcutaneous inoculation of RBE cells. Tumors were injected with pcDNA3.1 empty vector, HNF6-pcDNA3.1 vector or in combination with negative control or anti-miR-122 oligonucleotides when they reached ~100 mm3 in volume. The sizes of tumors were measured. n number of tumors examined. *P < 0.05 and **P < 0.001 a significant difference

Immunohistochemistry demonstrated that gene delivery of HNF6-pcDNA3.1 resulted in HNF6 overexpression in tumors, while transfection of either negative control or anti-miR-122 did not change HNF6 expression (Fig. 7a). There were fewer Ki-67-positive cells in tumors treated with HNF6-pcDNA3.1, compared with tumors injected with pcDNA3.1 empty vector (Fig. 7a, b). Gene delivery of negative control had no effect, but anti-miR-122 significantly increased the number of Ki-67-positive cells in tumors treated with HNF6-pcDNA3.1 (Fig. 7a, b). Western blot analyses of tumor tissues demonstrated the similar trend in the expression of cyclin G1, IGF-1R, MMP-9, MMP-2, RECK, E-cadherin and N-cadherin proteins (Fig. 7c), compared to the in vitro results (Figs. 4h, 5d).

Fig. 7.

Fig. 7

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Overexpression of HNF6 regulates gene expression and inhibits cell proliferation in situ. Tumors were harvested from the mice in Fig. 6d at day 20. a Illustrated are representative tumor sections immunostained with antibodies against HNF6 and Ki-67. b The tumor cell proliferation index was quantified. c Lysates of tumor tissues were Western blotted to detect the expression of proteins as indicated. **P < 0.001 a significant difference

Discussion

HNF6 was initially identified as a regulator in the development of hepatobiliary and pancreatic system. Recent studies have revealed that loss of HNF6 expression correlates with the progression of HCC (Lehner et al. 2010), and pancreatic (Pekala et al. 2014), colorectal (Lehner et al. 2010) and lung (Yuan et al. 2013) cancers, and contributes to cell proliferation and metastasis (Lehner et al. 2007, 2010). HNF6 has been shown to be essential for the development of biliary system (Kyrmizi et al. 2006) and highly expressed in mature bile duct epithelial cells (Sasaki et al. 2013). However, the role of HNF6 in CCA has not been reported, and the function of HNF6 in the proliferation and metastasis of CCA cells remain unclear. The present study has for the first time, to our knowledge, demonstrated that HNF6 is downregulated in CCA tissues compared with adjacent noncancerous tissues. The expression of HNF6 positively correlates with miR-122 in CCA tissues and cells. HNF6 is shown to upregulate the expression of miR-122 by stimulating the miR-122 promoter. Overexpression of HNF6 inhibits the proliferation of CCA cells by inducing cell cycle arrest at G1 phase through regulating miR-122, and its downstream factors, cyclin G1 and IGF-1R. Overexpression of HNF6 inhibits the migration and invasion of CCA cells by regulating MMP-2 and MMP-9, RECK, E-cadherin and N-cadherin. Overexpression of HNF6 inhibits the ability of CCA cells to form tumors and to metastasize to the lungs, and the growth of established tumors, in experimental animals.

MiR-122 is the most abundant liver-specific miRNA (Landgraf et al. 2007) and has been shown to be positively correlated with the levels of LETFs (Zeng et al. 2010). Moreover, the promoter of miR-122 is stimulated by LETFs, such as HNF4α (Wu et al. 2015; Xu et al. 2010). A recent study has also provided a link between HNF6 and miR-122 that HNF6 stimulates the expression of miR-122 in controlling hepatocyte differentiation (Laudadio et al. 2012). MiR-122 plays a crucial role in controlling diverse aspects of hepatic function and dysfunctions including hepatocarcinogenesis (Tsai et al. 2009). In general, miR-122 is regarded as a tumor suppressor miRNA that inhibits cancer cell proliferation, metastasis and tumor angiogenesis (Coulouarn et al. 2009; He et al. 2015b). Accordingly, we showed herein that HNF6 is positively correlated with miR-122 in CCA tissues and cells. The function of HNF6 in mediating cell proliferation and metastasis may at least partially rely on its effect on regulating miR-122 in CCA. It has been reported that miR-122 also stimulates the expression of some LETFs including HNF6 to induce differentiation of hepatocytes (Laudadio et al. 2012). However, the stimulation of HNF6 expression by miR-122 was not observed in CCA cells. Thus, overexpression of HNF6 increased, while depletion of HNF6 inhibited, the expression of miR-122; but transfection of either miR-122 mimics or anti-miR-122 did not significantly alter the expression of HNF6 in CCA cells. The results indicate the regulation between HNF6 and miR-122 may be in a one-way manner in CCA.

The inhibitory effects of HNF6 on cell proliferation have been demonstrated in lung cancer (Yuan et al. 2013) and colon carcinoma and HCC (Lehner et al. 2010). Particularly, overexpression of HNF6 induced cell cycle arrest at the G2/M and G1 phase in HCC cells (Lehner et al. 2010). The present results showed that HNF6 overexpression inhibited the proliferation of CCA cells by inducing cell cycle arrest at G0/G1 phase. However, depletion of HNF6 had little effects on cell proliferation, possibly because of the low basal level of HNF6 in CCA cells. In exploring the mechanisms, we found that HNF6 overexpression downregulated the expression of cyclin G1 and IGF-1R. As a tumor suppressor miRNA, miR-122 has been shown to induce cell cycle arrest at G1 phase by downregulating the expression of cyclin G1 and IGF-1R in HCC cells (Gramantieri et al. 2007; He et al. 2015b) and also in cancer cells of lung (Ma et al. 2010; Zhao et al. 2015), cervix (Ma et al. 2010) and breast (Wang et al. 2012). Therefore, anti-miR-122 abrogated the effects of HNF on cell cycle progression and the expression of cyclin G1 and IGF-1R in CCA cells and xenografts in mice.

HNF6 has been demonstrated to inhibit cell migration and invasion, thus suppressing metastasis of colorectal (Lehner et al. 2007) and lung (Yuan et al. 2013) cancers. Here we have shown that HNF6 overexpression inhibited the migration and invasion of CCA cells through downregulating the expression of MMP-2, MMP-9 and N-cadherin and upregulating the expression of RECK and E-cadherin. These proteins are involved in tumor metastasis and regulated by miR-122 (Tsai et al. 2009). MMPs are known to play an important role in cell migration during cancer invasion by degrading extracellular matrix proteins (Bourboulia and Stetler-Stevenson 2010). RECK is a negative regulator of MMP-2 and MMP-9 and an inhibitor of tumor metastases (Oh et al. 2001). Loss of E-cadherin enhances cancer metastasis by modulating growth inhibition at cell–cell contacts, while increase of E-cadherin expression prevents tumor invasion and metastasis (Nelson and Nusse 2004). N-cadherin is a similar transmembrane protein to E-cadherin, but has an opposite role in tumor metastasis (Nelson and Nusse 2004). Our results are also supported by a previous study, where HNF6 inhibits epithelial-mesenchymal transition, cell migration and invasive growth through a mechanism involving E-cadherin upregulation and N-cadherin downregulation (Yuan et al. 2013).

MiR-122 has been identified to be one of the metastasis-related microRNAs in HCC (Budhu et al. 2008). Loss of miR-122 expression is strongly associated with increased invasion and metastasis of HCC cells (Wang et al. 2014). Overexpression of miR-122 has been shown to upregulate the expression of E-cadherin and trigger mesenchymal-epithelial transition in HCC cells (Wang et al. 2014) and upregulate the expression of E-cadherin in embryonic stem cells (Deng et al. 2014). Accordingly, our results showed that anti-miR-122 abrogated the inhibitory effects of HNF6 on cell migration and invasion and its regulatory effects on the expression of the above metastasis-related genes.

The proposed mechanism by which HNF6 exhibits anti-tumor activities against CCA is summarized in Fig. 8. HNF6 is able to upregulate the expression of miR-122 by stimulating its promoter, but miR-122 does not regulate HNF6 expression, in CCA cells. HNF6 inhibits cell proliferation by inducing cell cycle arrest at G1 phase by downregulating cyclin G1 and IGF-1R through miR-122. HNF6 inhibits the migration and invasion of CCA cells by regulating MMP-2 and MMP-9, RECK, E-cadherin and N-cadherin, through its effects on miR-122. However, it must be mentioned that the regulatory role of miR-122 on these molecules has not been validated in CCA cells by luciferase assays. This is a limitation of the current study and needs further investigation in the future.

Fig. 8.

Fig. 8

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Proposed mechanisms by which HNF6 regulates genes involved in the proliferation and metastasis of cholangiocarcinoma cells through miR-122. “→” indicates promotion, positive regulation or activation; “⊥” inhibition, negative regulation or blockade. HNF6 hepatic nuclear factor 6, IGF-1R insulin-like growth factor-1 receptor; MMP matrix metalloproteinase, RECK reversion-inducing-cysteine-rich protein with kazal motifs

In summary, the present results suggest that HNF6 may serve as a tumor suppressor for CCA. HNF6 inhibits the proliferation and metastasis of CCA cells in vitro and in vivo through its regulatory effects on miR-122 and related genes, indicating that overexpression of HNF6 may represent a mechanism-based therapy for CCA.

Acknowledgments

This study was supported by grants from the National Natural Scientific Foundation of China (81272467 and 81472321), Shandong Provincial Natural Scientific Research Foundation (BS2015YY024), Shandong Provincial Scientific and Technology Development Program (2014GGH218039) and Heilongjiang Provincial Scientific Fund for Youths (QC2011C089). Huaqiang Zhu and Yuetang Mi contributed equally to this work.

Compliance with ethical standards

Conflict of interest

We declare that we have no conflict of interest.

Ethical standards

The study has been approved by the ethic committees of Provincial Hospital Affiliated to Shandong University and Liaocheng People’s Hospital in China. Preoperative informed consent was obtained from each patient registered in this study in accordance with institutional guidance. The pathological samples were taken from the surgical resection specimens, which would not result in any disadvantages to health and prognosis of patients. We are committed to maintain the privacy of patients’ information. The animal study has been approved (permit SYXK20020009) by the Animal Ethics Committee of Harbin Medical University, in compliance with the Experimental Animal Regulations by the National Science and Technology Commission, China.

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