Background
Cholangiocarcinoma (CC) is a bile duct cancer, and is classified anatomically as intrahepatic CC (IHCC) or extrahepatic CC (EHCC). EHCC is a highly malignant cancer of the biliary tract [
1,
2]. The incidence and mortality of EHCC is rising worldwide. Despite advances in surgical techniques, chemotherapies and radiotherapies, median survival of EHCC remains less than 24 months because of the patients are usually diagnosed at the advanced stage as the tumor has metastasized to regional lymph nodes or liver sites, which are the main prognostic factors in EHCC patients [
3]. Exploring the molecular mechanisms underlying the initiation, progression, invasion and metastasis of EHCC is vital as it may provide new therapeutic targets, leading to improvements in the long-term survival of patients with EHCC.
MicroRNAs (miRNAs) are small noncoding RNAs of 20–22 nucleotides involved in the regulation of gene expression at a post-transcriptional level by binding to the target sites of messenger RNAs (mRNAs). miRNAs act as important post-transcriptional regulators of gene expression, and have recently emerged as key regulatory molecules in various cellular processes, including differentiation, self-renewal, proliferation and apoptosis [
4]. It has been found that miRNAs regulate the expression of target genes by interacting with complementary sites in the 3'-untranslated region (UTR) of target mRNAs [
5], and more than 30 % of human genes are regulated post transcriptionally by miRNAs [
6]. miRNAs may function as oncogenes or tumor suppressors by targeting many cancer-associated genes in the progression of EHCC. The miR-34 family members share high sequence homology [
7]. Among these, miR-34a is one of the earliest known tumor suppressors and is commonly deleted in various types of cancers. As a direct transcriptional target of p53, decreased expression of miR-34a is partly due to the mutations of p53 in tumors [
8]. Recent research has found that down-regulation of miR-34a leads to a switch from Mnt (MAX network transcriptional repressor) to c-Myc expression during cholestatic cholangiocarcinogenesis in a mouse model [
9]. Moreover, miR-34a can suppress tumor metastasis and invasion through a variety of signaling pathways in several cancers [
10‐
14]. However, the anti-tumor function of miR-34a in EHCC is still not clear yet.
Transforming growth factor-β (TGF-β), which is a secreted homodimeric protein, belongs to a large family of pleiotropic factors that signal via heterotetrameric complexes of type I and type II serine/threonine kinase receptors. Important intracellular mediators of TGF-β signaling are members of the Smad family [
15]. Smad4 is the common-mediator which cooperates with other transcription factors to regulate TGF-β signaling pathway [
16]. The TGF-β signaling pathway has been shown to involve in various cellular responses in carcinogenesis of EHCC including cell proliferation and differentiation, migration and epithelial-mesenchymal transition (EMT) [
17‐
19].
In the present study, miR-34a expression levels were detected in EHCC tissues, and CC cell lines. Relationships of miR-34a with clinical characteristics of EHCC patients were further examined. Moreover, Smad4 was demonstrated as a direct transcriptional target of miR-34a in CC. We identify miR-34a could mediate TGF-β/Smad4 signaling pathway induced EMT in the progression of cholangiocarcinoma.
Methods
Patients and tissue samples
EHCC tissues and adjacent non-tumor tissues used for qRT-PCR and/or immunohistochemistry (IHC) were collected from 27 EHCC patients who underwent potentially curative surgery between 2010 and 2011 at the Second Affiliated Hospital of Harbin Medical University (Harbin, China) and were verified by a pathologist. Seven primary normal bile duct (NBD) specimens were also collected from surgical resections performed for pancreatic cancer. These patients underwent a Whipple’s procedure. The hard and firm tumor tissues were trimmed free of normal tissue and snap frozen in liquid nitrogen immediately after resection. No patient in the current study received chemotherapy or radiation therapy before the surgery. The tumor stage was classified according to the 7th tumor-node-metastasis classification of the International Union against Cancer (UICC). All the patients signed informed consent forms according to our institutional guidelines, and the study was approved by Institutional Review Board (IRB) protocols of Harbin Medical University. Information on gender, age, stage of disease, and histological factors was extracted from medical records.
Immunohistochemistry (IHC)
Immunohistochemical staining of sections for Smad4 expression was performed by a standard streptavidin-biotin peroxidase complex method [
20]. Each 4-mm section was deparaffinised, rehydrated, and incubated with fresh 0.3 % hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity. After rehydration through a graded series of ethanol solutions, the sections were autoclaved in 10 mM citrate buffer (pH 6.0) at 95 °C for 20 min and then cooled to 30 °C. After rinsing in 0.1 M phosphate buffer saline (PBS, pH 7.4), non-specific binding sites were blocked by incubation with 10 % normal rabbit serum for 30 min. The sections were then incubated with anti-Smad4 primary antibodies (Santa Cruz Biotechnology, USA) at a dilution of 1:100 in PBS containing 1 % bovine serum albumin at 4 °C overnight. The sections were washed in PBS, incubated with biotinylated anti-mouse IgG for 30 min at room temperature, and finally incubated in a streptavidin-biotin peroxidase complex solution (Nichirei Co., Tokyo, Japan). The chromogen, 3, 3′-diaminobenzidine tetra-hydrochloride, was applied as a 0.02 % solution containing 0.005 % H
2O
2 in 50 mM ammonium acetate-citrate acid buffer (pH 6.0). The sections were lightly counterstained with Mayer’s hematoxylin and mounted. Negative controls were established by replacing the primary antibody with normal rabbit serum. No detectable staining was evident in the negative controls.
RNA extraction and quantitative real-time PCR (qRT-PCR)
qRT-PCR was used to confirm the expression levels of mRNAs and miRNAs. For mRNAs detection, total RNA from cultured cells and fresh surgical tissues was extracted using Trizol (Invitrogen, USA) according to the protocol. Reverse transcription was performed according to the protocol of High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). For miRNAs detection, total miRNA from cultured cells and fresh surgical tissues was extracted using the mirVana miRNA Isolation Kit (Ambion, USA), according to the manufacturer’s protocol. Complimentary DNA was synthesized from 2 μg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). The expression level of miRNA and mRNA were assessed with qRT-PCR using Power SYBR® Green (Applied Biosystems, USA) by an Applied Biosystems 7500 Sequence Detection system. The expression level of mRNA and miRNA was defined based on the threshold cycle (Ct), and relative expression levels were calculated using the 2
-∆∆Ct method, using the expression level of β-actin mRNA and U6 small nuclear RNA as a reference gene. The names of the genes and the primers are listed in Additional file
1: Table S1.
Cell culture and quick transfection
The human EHCC cell lines QBC939 and HuCCT1 used in this study were purchased from American Type Culture Collection (Manassas, USA) and the human IHCC cell line RBE and HCCC9810 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human intrahepatic biliary epithelial cells (HiBECs) were purchased from PriCells Biomedical Technology Co., Ltd. (Wuhan, China). All the cells were cultured according to the manufacturer’s instructions. A chemically modified antisense oligonucleotide and a synthetic miR-34a mimic (GenePharm Co. Ltd, China) were used to inhibit and increase miR-34a expression respectively. A scrambled oligonucleotide (GenePharm Co. Ltd, China) was used as a control. The transfections were performed using Lipofectamine TM 2000 transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. A mixture of Lipofectamine 2000 and RNA was added to CC cells, which were 70 % confluent, for 4–6 hrs, and the cells were then incubated for 24 hrs in fresh medium. After that, the cells were harvested using lysis buffer for luciferase assay. Total RNAs and protein were prepared 48 hrs after transfection and used for qRT-PCR or western blot analysis.
The fragment containing miR-34a binding sites in the Smad4 3′-UTR was amplified by PCR and inserted downstream of the firefly luciferase gene in a pGL3-promoter vector (Promega, Madison, WI, USA). The mutant reporter plasmids were constructed using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA, USA). These constructed plasmids were all sequenced to confirm their orientation. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) as mentioned before [
21,
22]. Promoter activities were expressed as the ratio between Firefly luciferase and Renilla luciferase activities.
Western blot
Protein lysates were separated using 8 % or 10 % SDS-PAGE gel electrophoresis and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, USA). The membrane was probed with the following antibodies: anti-Smad4, anti-Snail, anti-E-cadherin, anti-N-cadherin (Santa Cruz Biotechnology, USA). Finally, the membrane was probed with Alexa Fluor® 680 donkey anti-mouse IgG (H + L) (1:5000) (Invitrogen, USA). Antibody binding was detected by Odyssey™ Infrared Imaging System (Li-Cor, Lincoln, NE). The names of the antibodies are listed in Additional file
2: Table S2.
Cell migration and invasion assays
The invasive potential of cells was measured in 6.5 micrometers Transwell with 8.0 micrometers Pore Polycarbonate Membrane Insert (Corning, USA) according to the manufacturer’s instructions. The filter of top chamber was matrigel-coated with 50 μl of diluted matrigel following the standard procedure and incubated at 37 °C for 2 hrs. The lower chambers were filled with 600 μl of DMEM medium with or without TGF-β (5 ng/mL) (R&D Systems Inc., USA) containing 5 % FBS as chemoattractant for a further 24 hrs [
19]. Cells were serum-free-starved overnight, and then harvested and resuspended in migration medium (DMEM medium with 0.5 % BSA). Then the suspension of 5,000 cells in 100 μl migration medium was added into each top chamber. After the cells were incubated for 16 hrs, the non-invading cells that remained on the upper surface were removed with a cotton swab. The invasive cells on the lower surface of the membrane insert were fixed with 4 % paraformaldehyde for 30 min, permeabilized with 0.2 % Triton X-100 at room temperature for 15 min, and then stained with 0.1 % crystal violet for 5 min. The number of cells on the lower surface, which had invaded through the membrane, was counted under a light microscope in five random fields at a magnification of 100×. The experiments were repeated three times independently and results were given as means ± SD. The procedure for transwell migration assays were the same as the transwell invasion assay except that the filter of top chamber was not coated with matrigel.
Statistical analysis
All the presented data were expressed as the mean ± SD and representative results were from at least three independent experiments. Statistical comparisons were calculated by Student’s two-tailed
t-test. When multiple comparisons were possible, ANOVA coupled with Tukey correction was used. Multivariate logistic regression analysis and Cox regression analysis were performed to analyze all factors in the Table
1 by backward variable selection. Survival curves for the patients were calculated using the Kaplan-Meier method, and analyzed using the Log-rank test. Correlation analysis between relative expressions of Smad4 and miR-34a was examined by logistic regression analysis. P < 0.05 was considered statistically significant. Statistical analysis was carried out using SPSS 21 (IBM Corporation Software Group, USA) or the GraphPad Prism 5.0 software package (GraphPad Software, Inc., USA).
Table 1
Relationship between miR-34a expression and clinicopathological features in EHCC patients
Age (yr) | | | | 0.695 |
<60 | 14 | 4 | 10 | |
≥60 | 13 | 5 | 8 | |
Gender | | | | 1.000 |
Male | 16 | 8 | 8 | |
Female | 11 | 6 | 5 | |
Tumor size (cm) | | | | 0.449 |
<2 | 12 | 5 | 7 | |
≥2 | 15 | 9 | 6 | |
Pathological type | | | | 0.222 |
Adenocarcinoma | 24 | 11 | 13 | |
Mucocellulare carcinoma | 0 | 0 | 0 | |
Adenosquamous carcinoma | 3 | 3 | 0 | |
Squamous carcinoma | 0 | 0 | 0 | |
Undifferentiated carcinoma | 0 | 0 | 0 | |
Cell differentiation | | | | 0.326 |
Well | 4 | 3 | 1 | |
Moderately | 8 | 5 | 3 | |
Poorly | 15 | 8 | 7 | |
Bismuth classification | | | | 0.385 |
Bismuth I | 7 | 1 | 6 | |
Bismuth II | 13 | 10 | 3 | |
Bismuth III | 6 | 2 | 4 | |
Bismuth IV | 1 | 1 | 0 | |
Lymphatic node metastasis | | | | 0.004 |
Absent | 9 | 1 | 8 | |
Present | 18 | 13 | 5 | |
Clinical stages | | | | <0.001 |
I + II | 13 | 2 | 11 | |
III + IV | 14 | 12 | 2 | |
Discussion
miR-34a is one of the most prominent miRNAs implicated in the development and progression of human cancers [
7]. It is down-regulated in many human cancers including hepatocellular, ovarian, prostate and urothelial carcinoma, as well as colon, head and neck, gastric, breast, lung and pancreatic cancers [
8,
10‐
14,
26‐
29]. In the present study, we found that miR-34a expression was significantly decreased in human EHCC tissues and CC cell lines when compared with the adjacent non-tumor tissues, NBD tissues and the HiBECs. Smad4 is further demonstrated as one of the targets of miR-34a, which was involved in the migration and invasion in EHCC cells. We also found activation of miR-34a suppresses the invasion and migration through TGF-β/Smad4 signaling pathway
in vitro.
Previous studies have provided evidence for the important roles of deregulated expression of miRNAs in the pathogenesis of CC, including miR-29, miR-122, miR-124, miR-145, miR-146a, miR-200c, miR-370, miR-373, miR-376c and miR-494 [
30‐
36]. Some reports have demonstrated that miR-34a to be an important anti-oncogene by regulation of different downstream targets in various types of cancers [
7,
8,
10‐
14,
26‐
29]. Down-regulation of miR-34a has been found in left and median bile duct ligation (LMBDL) mouse livers [
9]. However, the role of miR-34a in EHCC has yet to be elucidated. In the present study, we showed that the expression of miR-34a was down-regulated in EHCC tissues. These data are consistent with the decreased expression levels of miR-34a in the digestive system cancers which has been demonstrated by several groups [
9,
26‐
29]. miR-34a functions as a tumor-suppressor miRNA, and can target many downstream genes in the development and progression of carcinogenesis. Our clinical analysis showed that down-regulation of miR-34a is correlated with lymphatic metastasis and advanced clinical stages. Moreover, lower expression of miR-34a correlated with the decreased disease-free survival in these EHCC patients. These data suggest that miR-34a is more likely involved in the metastasis or invasion during the progression of EHCC. Recent studies have found that miR-34a represses RhoA, a regulator of cell migration and invasion, by suppressing c-Myc–Skp2–Miz1 transcriptional complex that activates RhoA in human prostate cancer cells [
37]. miR-34a was also found to reduce cell proliferation and invasiveness partially through its inhibitory effect on Delta-like 1 (DLL1) in choriocarcinoma [
11]. Thus, miR-34a could also regulate some downstream targets in the progress of metastasis or invasion of EHCC.
Since miRNAs are generally involved in the pathogenesis of cancer by directly regulating the expression of their targets at a post-transcriptional level, we applied bioinformatic methods to predict the potential targets of miR-34a. Further investigation showed that miR-34a suppresses the activity of a luciferase reporter gene fused with the 3′-UTR of Smad4 mRNA, which is dependent on the miR-34a binding sequence. Our data revealed that miR-34a directly targets the 3′-UTR of Smad4, and that ectopic expression of miR-34a represses Smad4 protein level in CC cell lines. Interestingly, recent report suggested that miR-34a plays a critical role in the progression of cardiac fibrosis by increasing Smad4 expression to activate TGF-β1 [
38]. These controversial results may due to the different experimental models and/or the various functions of miR-34a in different diseases. Besides, over-expression of miR-34a also inhibited the expression of TGF-β/Smad4 downstream targets N-cadherin and induced the expression level of E-cadherin. N-cadherin and E-cadherin are highly involved in the EMT during carcinogenesis [
20]. EMT is a complex process in which epithelial cells acquire the characteristics of invasive mesenchymal cells. EMT has been implicated in cancer progression and metastasis. Several oncogenic pathways such as TGF-β, Wnt, and Notch signaling pathways have been shown to induce EMT [
39]. The role of TGF-β in EMT, tumor invasiveness and metastasis has been firmly established
in vitro and
in vivo studies [
28,
40], including in human EHCC. More importantly, TGF-β-induced activation of Smad complexes has been shown to play a crucial role during the induction of EMT [
19]. Several reports have also shown that the levels of transcription factors driving EMT are controlled by miRNAs including miR-34a [
26,
41‐
44]. Thus, our data showed that activation of miR-34a could antagonize Smad4-mediated TGF-β induction of EMT process through regulation of E-cadherin and N-cadherin expression. Snail, which is a downstream target of TGF-β/Smad4 signaling pathway, was also decreased by increasing miR-34a expression but increased by using miR-34a inhibitor in EHCC cells.
Moreover, the expression levels of miR-34a and Smad4 are inversely correlated in human clinical specimens of EHCC. Although there are a few samples with both miR-34a down-regulation and negative staining for Smad4 proteins by IHC, the protein level of Smad4 was increased in most of our EHCC tissues compared with NBD tissues. For those EHCC specimens, which did not have inverse correlation of miR-34a and Smad4 expression, we speculate that other factors might antagonize or interfere with the effect of miR-34a on Smad4. The expression pattern of individual miRs with strict tissues, the clinical-feature-specificity or the different target genes involved in the unique regulation network of EHCC may all involved in the effect of miR-34a on Smad4 [
35,
45]. These speculations need further investigations in the future. Our results showed forced up-regulation of miR-34a significantly inhibited the protein expression of Smad4, and inhibited CC cells invasion and migration. The contrast results were observed when the CC cells were treated with the miR-34a inhibitor. These results identified Smad4 as a novel target of miR-34a in the EMT process of EHCC. Based on the contrasting expression patterns of miR-34a and Smad4 in most of the EHCC tissues and our
in vitro data, we proposed that miR-34a is involved in the pathogenesis of EHCC by directly inhibiting the protein expression of Smad4. Therefore, our studies demonstrated that miR-34a functions as a tumor-suppressor miRNA by inhibiting TGF-β/Smad-induced EMT in CC cells.
Conclusion
In summary, our results have identified miR-34a as a tumor suppressive miRNA in human EHCC, which acts at least in part through the repression of Smad4. Decreased expression of miR-34a in EHCC patients is correlated with lymphatic metastasis, advanced clinical stages and overall survival rate. Loss of the miR-34a expression leads to an induction of Smad4 and activation of TGF-β/Smad4 signaling pathway, which accelerate CC cells invasion and migration via EMT. Taken together, our data provide new insights into the potential contribution of miR-34a in inhibition the progression of EHCC, and suggest miR-34a is a useful molecule target for developing new therapeutic method against EHCC.
Acknowledgments
This study was supported by the National Natural Science Foundation of China (Grant No. 81302059), Natural Science Foundation of Heilongjiang Province of China (Grant No. LC2013C35), the Foundation of Educational Committee of Heilongjiang Province of China (Grant No. 12541300), the Scientific Research Foundation for Returned Scholars, Ministry of Education of China, and the Youth Foundation of the Fourth Affiliated Hospital of Harbin Medical University.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
GL, PQ and DW designed the study and drafted the manuscript. GL and PQ contributed equally as first author. GL and DW contributed equally as corresponding author. PQ, GL, WB, LY, XZ and LY have carried out all the experiments. All the authors read and approved the final version of the manuscript.