Cholangiocarcinoma (CCA) is a highly malignant cancer with a poor prognosis. Radical resection provides the only option for cure. However, only a small proportion of CCA patients can receive surgery because of the metastatic nature of the disease [
]. Metastasis is a multistep process by which primary tumour cells invade adjacent tissues, enter the bloodstream, survive in circulation, extravasate into the surrounding tissue parenchyma, and finally form clinically detectable metastases [
]. This multistep process is initiated and regulated through the alteration of numerous molecules acting as oncogenes or suppressors. Thus far, a number of altered molecules and the related signalling pathways have been reported [
]. However, the invasion and metastasis of CCA might be regulated by a molecular network that is far from completely understood.
Among the molecules that are altered during CCA progression, our previous study found that GATA6, a member of an evolutionarily conserved family of zinc finger transcription factors, is aberrantly upregulated [
]. In addition, GATA6 promoted CCA invasion and metastasis. These results indicate that GATA6 acts as a potential oncogene in CCA. While the mechanism of GATA6 upregulation in CCA is unclear, the upregulation might be attributed to amplification of promoting factors and inhibition of blocking factors. Kwei et al. [
] reported that amplification of the GATA6 gene might contribute to the aberrant expression of GATA6, which is one amplification promoting factor. However, there are few reports that mention the inhibition of blocking factors of GATA6 in CCA.
MicroRNAs (miRs) are small noncoding RNA oligonucleotides that regulate a large number of genes. They perform the negative regulation through complementary binding to mRNAs 3′-untranslated regions (UTRs). A growing body of evidence suggests that some miRNAs function as onco-miRs or tumour-suppressor miRs by targeting known oncogenes or tumour-suppressor genes [
]. Thus, we hypothesized that miRs might participate in negative regulation of GATA6 in CCA. Among the numerous miRNAs, we focused on miR-124 based on the following observations: (1) Recently, mir-124 was reported to be downregulated and to affect metastasis in several types of cancer, including hepatocellular carcinoma, pancreatic cancer, breast cancer, prostate cancer, glioma and lung cancer [
]. However, its role in CCA is uncertain. (2) Bioinformatics analysis has shown that the 3′-UTR of GATA6 gene contains a potential miR-124 binding site. (3) Our preliminary experiments indicated that the level of miR-124 is negatively associated with GATA6 in a CCA cell line, QBC939. Thus, we postulate that miR-124 might participate in the regulation of GATA6 in CCA.
In the present study, the expression profile of miR-124 in CCA samples was investigated. The role of miR-124 on CCA cell migration, invasion and metastasis was also investigated. Finally, we examined the mechanism of miR-124 in regulating GATA6 in CCA cells. Based on the present study, we may abandon the mechanism of CCA metastasis. In addition, miR-124 may potentially be a new prognostic indicator and molecular target for treatment of CCA.
In total, 57 frozen cancerous samples from CCA patients undergoing surgery from 2005 to 2010 at our department were collected in this study. In addition, 38 matched paracancerous samples were collected. The clinical features were obtained.
Overall survival was defined from surgery date to until the date of last contact. Recurrence-free survival was calculated from the date of surgery until the date of tumour recurrence.
QBC939 and RBE, two human cholangiocarcinoma cell lines, were cultured in RPMI 1640 medium with 10% foetal bovine serum (HyClone). Primary biliary epithelial cells were bought from ScienCell Research Laboratories (San Diego, CA, USA) and cultured using the media comprising DMEM/F12 (1:1) with 10% foetal bovine serum (FBS), 25 ng/mL epidermal growth factor and 393 ng/mL dexamethasone.
GATA6 transfection was performed as previously described [
]. In brief, The CDS template of GATA6 without the miR-124 binding site was synthesized chemically and amplified by PCR. Then, DNA was validated by sequencing and cloned into a pCMX plasmid. A total of 10 μg of GATA6 plasmid (ExGATA6) or empty plasmid (ExControl) were transfected into cells using Lipofectamine LTX and Plus Reagent (Invitrogen, USA).
Cells were transfected with 100 nM miR-124 mimic (ExmiR-124) or 200 nM miR-124 inhibitor (InmiR-124) (Ribobio, Guangzhou, China) in the six-well plate using Lipofectamine 2000 (Invitrogen). After 24 and 48 h, the expression was evaluated by real-time PCR.
Xenotransplantation of CCA cells into nude mice
CCA cell metastasis was evaluated following xenotransplantation into nude mice by intrasplenic injection as previous described [
]. In brief, Cells (5 × 10
) were injected into the spleen of 4-week-old male nude mice (Laboratory Animal Centre, Third Military Medical University). The mice were sacrificed after 1 month. Tumour masses were primarily found in the spleen, liver and bowel grossly at autopsy. All masses except those in the spleen were considered distant metastases. Distant masses were embedded in paraffin, stained with haematoxylin and eosin (HE), and examined under a microscope.
QBC939 cells were transfected with a miR-124 agomir (200 nM) or an negative control for 48 h (200 nM) (Ribobio, Guangzhou, China). Intraperitoneal injection was performed with miR-124 agomir (5 nmol each) or negative control agomir (5 nmol each) twice a week for 2 weeks, which began at week 3 after xenotransplantation.
Luciferase reporter assays
CCA cells (5 × 10
4) were seeded in a 48-well plate. The cells were co-transfected with 10 nM miR-124 mimics or NC and 10 ng of firefly luciferase reporter construct. The reporter contained either a wild-type or mutant GATA6 3′-UTR. Luciferase activities were analysed 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega) in an M200 microplate fluorescence reader (Tecan, Vienna, Austria).
Cell invasion (Transwell) or migration (wound healing) assay
Cell invasion (Transwell) assay was performed as previously described [
]. Briefly, 2 × 10
cells were suspended in 400 μL of serum-free RPMI 1640 medium and seeded in the top chamber that had been coated with a layer of extracellular matrix (BD Biosciences, USA). Complete medium with serum (500 μl) was added to the bottom chamber. After 48 h of incubation, the cells that had invaded through the extracellular matrix layer to the lower surface of the filters were stained. Photographs of three randomly selected fields of the fixed cells were captured, and cells were counted. Experiments were repeated independently three times.
Cells were seeded in a 6-well plate, grown until confluence, and then starved for 24 h. A linear wound was made by scraping a pipette tip through the confluent cells. The cell motility was measured in terms of wound closure by photographing three random fields 72 h after the wound was made. Experiments were repeated independently three times.
Total RNA extraction was performed using TRIzol reagent (Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. The RNAs were mixed with oligo (dT) or miRNA-specific stem-loop RT primers and reverse transcribed to cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). These cDNAs were used to analyse the expression of miR-124 and GATA6 by qPCR using a SYBR premix Ex Taq kit (TaKaRa, Dalian, China). The expression levels were normalized against endogenous β-actin or U6 mRNA as controls. All reactions were performed in an ABI 7500 system (Applied Biosystems, Foster, CA, USA) in triplicate, and the levels of gene expression were calculated using the 2
method. All primers are shown in Additional file
: Table S1.
Western blot analysis
Western blot analysis was performed as previously described [
]. The total protein in CCA cells was isolated using RIPA Lysis Buffer (Beyotime, China). For immunoblotting, equal amounts of proteins were separated on a 5–8% SDS-PAGE gel and electrophoretically transferred onto nitrocellulose membranes (Millipore), which were blocked in TBST containing 5% milk for 2 h at RT and blotted with antibody overnight at 4 °C using anti-GATA6 (1:500, Abcam) or β-actin (1:500, Biotechnology). After washing membranes with TBST and incubating them with either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (Biosynthesis Biotechnology, China) for 2 h at room temperature, immunocomplexes were visualized using chemiluminescence (GE, USA) following the manufacturer’s protocol.
Data were analysed using SPSS 17.0 software. Continuous data were measured with a
t-test. For categorical data, chi-square analysis or Fisher’s exact test was used. Kaplan-Meier analysis was applied for overall survival and recurrence-free survival. Statistical significance was set at
P < 0.05.
Deregulation of miRs has been reported in various cancer tissues from numerous profiling data [
]. Moreover, these small non-coding RNAs can function as oncogenes or tumour-suppressor genes that contribute to tumourigenesis and progression [
]. In the present study, we found miR-124 was downregulated in cancerous samples from CCA patients. Downregulation of miR-124 contributed to CCA invasion and metastasis, indicating its tumour-suppressor function. In addition, we demonstrated that GATA6 is a new potential target of miR-124. miR-124 downregulated GATA6 by targeting the 3′-UTR. Moreover, miR-124 inhibited CCA cell invasion and metastasis by downregulating GATA6. Taken together, these findings suggest an important role of miR-124 in CCA invasive and metastatic potential.
miR-124 has been reported to be downregulated by the hepatitis C virus (HCV) core protein in HCV-related intrahepatic CCA [
]. Here, we found that miR-124 was downregulated in both intrahepatic and extrahepatic CCA. Furthermore, many miR-124-decreased CCA patients did not have HCV infections, indicating that there might be other mechanisms regulating miR-124 expression during CCA progression. Recent studies have shown that aberrant DNA methylation plays important roles in miR-124 downregulation in several types of cancers, including hepatocellular carcinoma, gastric cancer, colon cancer, lung cancer, lymphoma, and pancreatic cancer [
]. Whether DNA methylation also participates in the downregulation of miR-124 in CCA, especially HCV-negative CCA, needs to be investigated further.
Dysregulation of GATA6 has been reported in several types of cancers. It functions as a promoter and suppressor according to the tumour origin. Our previous study showed that GATA6 is aberrantly upregulated in CCA, which indicates that it functions as a potential oncogene [
]. Kwei et al. [
] reported that an increased gene copy number contributes to the aberrant expression of GATA6. Wang et al. [
] reported that DeltaNp63alpha, the oncogenic isoform of the p63 protein, regulates GATA6 expression in gastric cancer. Here, we showed that a downregulation of miR-124 expression might contribute to the aberrant expression of GATA6 in CCA. Both of the above studies suggest that the dysregulation of GATA6 expression during CCA progression is controlled by multiple pathways, including the enhancement of positive regulation pathways and the inhibition of negative regulation pathways.
GATA6 participates in cancer progression by regulating certain key molecules. Among these key molecules, 67LR is an important protein that is regulated by GATA6 through binding to its prompter region in CCA [
]. 67LR is a cell surface non-integrin receptor expressed in epithelial cells that mediates cell attachment to laminin. An increase in 67LR expression has been found in a variety of cancers including CCA [
]. 67LR promotes CCA cell invasion and metastasis through various mechanisms, including facilitating tumour cell invasion into adjacent tissue by upregulating proteolytic enzymes [
] and assisting tumour cell survival in the circulatory system by promoting immune privilege [
]. By targeting GATA6, miR-124 may also downregulate 67LR expression and play important roles in these processes during CCA cell metastasis. The present results, together with the above evidence, indicate the important role of the miR-124-GATA6-67LR pathway in CCA cell invasion and metastasis.
Although our results showed a potential new target of miR-124 and the important role of this pathway in CCA cell metastasis, there must be other pathways by which miR-124 exerts its effects. Previous studies have shown that miR-124 inhibits cancer cell invasion and metastasis by targeting other molecules, including slug [
], Rac-1 [
], SMYD3 [
], SphK1 [
], and ROCK1 [
]. GATA6 is also reported to participate in cancer cell invasion and metastasis by regulating other proteins, such as urokinase plasminogen activator [
], slug [
] and BMP4 [
]. All of this evidence suggests that invasion and metastasis of cancers including CCA is regulated by a molecular network. Our previous and present results suggest that miR-124 and GATA6 play important roles in this network.
In addition to metastasis, GATA6 is reported to play important roles in tumourigenesis, self-renewal of cancer stem cells, and proliferation and apoptosis of various types of cancer [
]. miR-124 is also reported to participate in regulating angiogenesis, chemosensitivity and proliferation of cancers [
]. Whether miR-124 participates in regulating other hallmarks of CCA by targeting GATA6 needs to be investigated further.
This study was supported by the National Natural Science Foundation of China (grant numbers 81301839, 81402406). The fundings mainly contribute to the cost of the reagents and consumables used in this study.
Availability of data and materials
The dataset supporting the conclusions of this article is available from the corresponding author on reasonable request.
FT, JC and JL participated in research design, writing of the manuscript, performance of the research, and data analysis. SZ, DL, XZ and PJ participated in performing the research. SW participated in the research design and the writing of the manuscript. All authors have read and approved the manuscript, and ensure that this is the case.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
All procedures involving the clinical samples were approved by the ethics committee of Southwest Hospital. All patients provided informed consent which was written. All of the procedures involving mice were approved by the Animal Care and Use Committee of Third Military Medical University.