Background
Human cholangiocarcinoma (CCA), an epithelial cell malignancy arising from varying locations in the biliary tree, is the second most common primary hepatic cancer worldwide [
1]. Currently, surgical resection is the only curative treatment. However, the resectability and curability remain low. Therefore, it is critical to elucidate the molecular mechanisms regulating CCA tumor progression to find potential therapeutic strategies.
Tetraspanin 1 (TSPAN1) is a member of the TSPAN family of proteins whose important feature is their ability to aggregate with one another or various other transmembrane receptors, to become TSPAN-enriched microdomains (TEMs). TEMs are essential in determining the fundamental biological activities such as cell adhesion, proliferation and cell motility [
2]. Recently, TSPAN1 was reported to accelerate many kinds of cancer progression, especially digestive malignancies such as hepatocellular carcinoma (HCC), pancreatic, gastric, colorectal, and esophageal cancers [
3‐
7], and some other non-digestive cancers such as osteosarcoma and cervical cancer [
8,
9]. In these studies, TSPAN1 was treated as an oncogene promoting cancer process. However, its exact mechanism is unknown. Therefore, for the first time, we previously demonstrated that TSPAN1 was a critical promoter in human CCA and explored its mechanism. Coincidently, Subrungruanga et al. [
10] conducted a whole-transcript expression array study using microarray profiling of 15 pairs of intrahepatic CCA tumors and corresponding normal liver tissue samples, and TSPAN1 was one of the 42 upregulated genes in intrahepatic CCA.
MicroRNAs (miRNAs), are small non-coded strands of RNAs that regulate post-transcriptional gene expression and silence an extensive range of target genes. Oncogenes or tumor suppressor genes regulated by miRNAs have a wide range of functions in cancer, indicating that they could act as therapeutic targets or biomarkers for the diagnosis of cancer [
11]. To date, miR-194-5p has been studied in several distinct cancer types such as HCC, gallbladder cancer, glioblastoma, and acute myeloid leukemia [
12‐
16]. In our study, for the first time, we investigated the tumor suppressor role of miR-194-5p in CCA.
Accumulating evidence shows that epithelial-mesenchymal transition (EMT) has a critical role in the dissemination of malignant oncocytes during CCA progression [
17,
18]. Integrins are a family of cell adhesion receptors that are transmembrane α-β heterodimers, and 18α and 8β subunits have been currently identified [
19]. This family of proteins recognizes extracellular matrix (ECM) ligands, cell surface molecules, and growth factors to activate further several intracellular signaling pathways, which regulate different pathological progress including EMT [
20]. TSPANs are peculiar molecular scaffolds that distribute proteins into highly organized microdomains, and the integrin family is one of the most prominent classes of adhesion receptors that bind TSPANs. In this study, we screened several integrins, which could interact directly with TSPAN1 protein and identified integrin α6β1. Lastly, we demonstrated that TSPAN1 promoted integrin α6β1 downstream phosphoinositide-3-kinase (PI3K)/AKT/glycogen synthase kinase (GSK)-3β/snail family transcriptional repressor (Snail)/phosphatase and tensin homolog (PTEN) feedback loop, which is responsible for the EMT of CCA.
Methods
Cell lines and culture
The HIBEpiC normal human biliary cell line was obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). The L02 immortalized normal liver cell line was purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Science, China. The HuCCT1 cell line was kindly provided by the Cancer Cell Repository of the Tohoku University in Japan. CCLP1 and KMBC cell lines were purchased from BeNa Culture Collection (Beijing, China). RBE and HCCC9810 cell lines were purchased from Shanghai Bioleaf Biotech Corporation (Shanghai, China). Huh28 was purchased from Accegen Biotechnology Corporation (Fairfield, NJ, USA). Huh28, RBE, HCCC9810 and HuCCT1 were authenticated using Short Tandem Repeat (STR) analysis. All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) at 37 °C and exposed to an atmosphere of 5% CO2.
CCA tissue collection
We collected 60 CCA samples legally from patients who underwent routine surgical procedures between 2010 and 2016 at The First Affiliated Hospital of Harbin Medical University. The histopathological diagnosis was based on the World Health Organization criteria. Clinical tumor typing was assigned based on the sixth edition of the TNM classification system published by the International Union Against Cancer. The histological grade of tumor differentiation was confirmed in accordance with the classification proposed by Edmondson and Steiner. Ethical approval was obtained from The First Affiliated Hospital of Harbin Medical University Research Ethics Committee and written informed consent was obtained from each patient. The detailed clinicopathological characteristics of the 60 CCA specimens used in this study are listed in Table
1.
Table 1
Correlation between TSPAN1 staining and clinicopathologic characteristics in 60 CCA patients
Age (years) |
> 60 | 27(64.29%) | 11(61.11%) | 0.8151 |
≤ 60 | 15(35.71%) | 7(38.89%) | |
Gender |
Male | 23(54.76%) | 9(50.00%) | 0.7347 |
Female | 19(45.24%) | 9(50.00%) | |
Histological differentiation |
Well | 10(23.81%) | 8(44.44%) | 0.2752 |
Moderate | 15(35.71%) | 5(27.78%) | |
Poor | 17(40.48%) | 5(27.78%) | |
TNM stage |
I-II | 16(38.10%) | 14(77.78%) |
0.0048
|
III-IV | 26(61.90%) | 4(22.22%) | |
CA19–9 (U/ml) |
≤ 37 | 13(30.95%) | 5(27.78%) | 0.8058 |
> 37 | 29(69.05%) | 13(72.22%) | |
Lymph node metastasis |
No | 19(45.24%) | 14(77.78%) |
0.0202
|
Yes | 23(54.76%) | 4(22.22%) | |
Distant metastasis |
No | 29(69.05%) | 17(94.44%) |
0.0331
|
Yes | 13(30.95%) | 1(5.56%) | |
Lentivirus, antibodies, and reagents
The lentiviral vectors overexpressing the human
TSPAN1,
miR-194-5p, and
Snail gene (LV-TSPAN1, LV-miR-194-5p, and LV-Snail, respectively) and their corresponding knockdown forms (LV-shTSPAN1, LV-anti-miR-194-5p, and LV-shSnail, respectively) were constructed and synthesized by Shanghai GeneChem Corporation (Shanghai, China). Empty vectors were used as the corresponding controls. The lentiviral vector encoding the human firefly luciferase gene was constructed and purchased from RiboBio Corporation (Guangzhou, China). The target sequences are listed in Additional file
1: Table S1. Information on the primary antibodies for the western blot, immunohistochemistry (IHC), immunofluorescence (IF), and co-immunoprecipitation (co-IP) analyses is provided in Additional file
1: Table S2. Detailed information of the primers and probes used in the quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is listed in Additional file
1: Table S3. Laminin 5 and LY294002 were purchased from Abcam Corporation (Cambridge, Cambridgeshire, UK).
Cell proliferation analysis and colony formation assay
Transfected cells were seeded in 96-well plates (1–2 × 103 cells per well) and incubated overnight at 37 °C with 5% CO2 to allow attachment. Cell viability at various time points was measured using Cell Counting Kit-8 (CCK-8) assays (CK04–01; Dojindo Molecular Technologies, Inc., Japan). The primary medium in the wells was replaced with 100 μL of complete medium supplemented with 10 μL of CCK-8 reagent, and after incubating at 37 °C under 5% CO2 for 2 h, 450-nm absorbance was measured. The experiments were performed in triplicate. For the colony formation assay, transfected cells were seeded in six-well plates at a density of 500–800 cells/well and incubated for 14 days at 37 °C under an atmosphere of 5% CO2. Subsequently, the cells were fixed with 4% (w/v) paraformaldehyde, stained with 0.5% crystal violet, and counted.
Wound healing assay
Cells were seeded in a six-well plate and cultured at 37 °C under an atmosphere of 5% CO2 until the cells had reached confluence. Cells were washed three times with phosphate-buffered saline (PBS), and the bottom of each well was scratched with a 200 μL pipette tip. An Eclipse TS100 microscope (Nikon, Tokyo, Japan) was used to capture the images at 0 and 24 h.
Migration and invasion assays
A BD Falcon 24-well insert system (BD Biosciences, San Jose, CA, USA) was used to perform experiments. For the cell migration assay, 2–4 × 104 cells were suspended in 500 μL serum-free medium, seeded in the upper chambers of the transwell, and then the culture medium containing 10% FBS was added to the lower chambers. For the Matrigel invasion assay, filters were precoated with 30 μL Matrigel (BD Biosciences, San Jose, CA, USA) for 3 h and the follow-up procedures were same as those for the migration assay. After incubation at 37 °C under an atmosphere of 5% CO2 for 24–48 h, the non-migrating or non-invading cells remained on the upper surface of the filters. Cells that penetrated the membrane filters were fixed in methanol, stained with crystal violet, and counted under a light microscope.
Western blot analysis
Whole cell or tissue extracts were prepared using radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. After determining the protein concentration, the samples were denatured, separated using sodium dodecyl-polyacrylamide gel electrophoresis, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA). The membranes were probed with the antibodies listed in Additional file
1: Table S2. Protein bands were visualized using an enhanced chemiluminescence assay kit (Pierce, Rockford, IL, USA).
QRT-PCR
Total RNA was extracted from cultured cells and clinical tissues using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Furthermore, complementary DNA was generated using the High-Capacity RT kit (Applied Biosystems, Foster City, CA, USA) after RNA quantification. The qRT-PCR assays were performed using Power SYBR Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA), using an ABI Prism 7900HT instrument (Applied Biosystems, Carlsbad, CA, USA) with the primers listed in Additional file
1: Table S3 and normalized to the relative glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level. For the miRNA analyses, a TaqMan® MicroRNA RT kit and TaqMan® Universal Master Mix II no UNG (Applied Biosystems, Carlsbad, CA, USA) were used to perform the RT and PCR, respectively. The miRNA expression levels were normalized to that of U6.
IHC assay
The formalin-fixed and paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated with a gradient ethanol series. Then, antigen retrieval was performed using an antigen-unmasking solution (citrate-based). Tissue sections were blocked and incubated with primary antibodies at optimal concentrations overnight at 4 °C. Then, the biotinylated sections were incubated with the secondary antibody (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature. Lastly, the sections were stained with diaminobenzidine (DAB) kit (Vector Laboratories) and counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA). The results were scored based on the intensity and the extent of staining. TSPAN1 staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate) and 3 (strong). The staining extent was scored based on the percentage of positive cells using the following scale: 0 (negative), 1 (0.01–25%), 2 (25.01–50%), 3 (50.01–75%), and 4 (75.01–100%). The histologic score (H score) for each section was calculated with the following formula: histologic score = proportion score × intensity score. Thus, the total score could be 0, 1, 2, 3, 4, 6, 8, 9, or 12, and the staining could be classified as negative/low (0, 1, 2, 3, 4) or positive (6, 8, 9, 12).
IF assay
For the IF assay, transfected cells were fixed with 4% (w/v) paraformaldehyde for 10 min, permeabilized with 0.1% (v/v) Triton X-100 for 20 min at ambient temperature, and blocked in 10% normal goat serum for 30 min. Cells were then incubated with primary antibodies overnight at 4 °C. After thorough washing with PBS, the cells were incubated with fluorescent secondary antibodies (Invitrogen, Eugene, OR, USA) for 1 h at room temperature. Finally, the cells were washed and counterstained with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA) and then images were captured using a DMRA fluorescence microscope.
Co-IP assay
Cells were seeded in six-well plates, incubated with 200–400 μL ice-cold IP lysis buffer per well on ice for 5 min with periodic mixing, and supplemented with 10 μL/mL Halt™ protease and phosphatase inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). Then, we transferred the lysate to a microcentrifuge tube, which was centrifuged at 13000×g for 10 min to pellet the cell debris at 4 °C. A Dynabeads™ protein G IP kit and Magnet Starter Pack (Thermo Scientific) were used to immunoprecipitate the Dynabeads®-Ab-Ag complex following the manufacturer’s protocol, and samples were analyzed using western blotting as previously described.
Luciferase assay
In brief, a luciferase assay kit (Promega, Madison, WI, USA) was used to evaluate the luciferase activity. Cells were seeded in 24-well plates and incubated for 24 h. For the transfection, specific plasmids were cotransfected with 1 ng pRL-TK Renilla luciferase plasmid into the cells. Lastly, the Dual-Luciferase Reporter Assay system (Promega) used to measure the luciferase activity after 48 h according to the manufacturer’s instructions.
Animal studies
Male BALB/c athymic nude mice (4–6-week-old) were obtained from the Experimental Animal Center of Shanghai Institute for Biological Sciences and maintained in a pathogen-free environment according to the institutional guidelines for animal care. To establish a subcutaneous xenograft model, 5 × 106 cells suspended in 150 μL PBS were subcutaneously injected into the flank of mice. The tumors were excised after 6 weeks to calculate their volumes.
For the in vivo metastasis assays, intrahepatic, peritoneal, and pulmonary metastases were established. The intrahepatic metastasis assay was performed by injecting 5 × 106 cells in 200 μL PBS into the mouse livers through the spleen parenchyma. To avoid intrasplenic tumor growth, the mouse spleens were removed. After 4 weeks, the mice were euthanized, and their livers were harvested to count the nodules.
To evaluate the peritoneal metastasis, 5 × 106 CCA cells in 200 μL PBS were inoculated into the intraperitoneal cavity of the mice, which were subsequently euthanized, dissected, and representative images were captured using a camera, 4 weeks after the injections.
Furthermore, 5 × 106 cells were injected into nude mice through the tail vein, and 6 weeks later, tumor formation and metastasis in the lungs were measured using a Berthold NIGHTOWL LB983 imaging machine with D-luciferin (Xenogen, Hopkinton, MA, USA). After imaging, all the mice were euthanized, the lungs were excised, and the metastatic nodules were counted following hematoxylin and eosin staining (H&E) staining.
Statistical analysis
The statistical analysis was performed using the statistical package for the social sciences (SPSS) 16.0 software (SPSS, Chicago, IL, USA) and the GraphPad Prism software package (v. 6.01, San Diego, CA, USA). The results are presented as means ± SD. The variance between the two groups was analyzed using Student’s t-tests, and p < 0.05 was considered statistically significant.
Discussion
The
TSPAN1 gene is located on chromosome 1p34.1 and encodes the 26 kDa protein of the same name (TSPAN1), which is mainly expressed on the plasma membrane, and on some intracellular vesicles in various tissues [
31]. Recent research studies on TSPAN1 have focused on its ability to promote proliferation and migration in cancers. Nevertheless, the mechanism underlying the actions of TSPAN1 are not well understood, and there are limited studies. We first time identify the TSPAN1 function and specific mechanism in human CCA progress.
In the present study, we evaluated TSPAN1 expression in clinical CCA specimens and relevant cell lines, and the results indicated that TSPAN1 was highly expressed at the mRNA and protein levels. The statistical analysis determined that TSPAN1 upregulation was associated with the TNM stage and metastasis. In agreement with the clinicopathological features, our data revealed that TSPAN1 overexpression enhanced tumorigenesis, and as emphasized by research studies, some metastasis assays in vitro and in vivo confirmed that TSPAN1 promoted metastasis in CCA. In the next step, we verified that TSPAN1 induced EMT, and TSPAN1 knockdown resulted in the upregulation of E-cadherin and decrease of N-cadherin and vimentin.
We sought to investigate how miRNA deregulation affects tumors, so we screened deregulated miRNA, which is a contributing element in the high expression of TSPAN1 in CCA. Low expression of miR-194-5p was observed in CCA tissues and miR-194-5p suppressed CCA metastasis in our study. Then, we validated the complementary binding between miR-194-5p and TSPAN1–3′-UTR through the luciferase reporter assay. Most importantly, we proved that a combination of high TSPAN1 expression and low miR-194-5p expression predicts a poor prognosis in CCA patients.
EMT has been shown to significantly accelerate metastasis in epithelium-derived carcinoma including CCA [
32,
33]. EMT is a complex, transient, reversible biological process that makes tumors epithelial cells lose polarity, turns adherent phenotypes into mesenchymal forms, and increases cell invasion and migration ability. Alteration of adhesion molecules and the ECM are two key factors in the EMT process [
34]. For reference, we paid close attention to the functional characteristics of the TSPAN superfamily of proteins and discovered that they are always associated with adhesion receptors of the integrin family in regulating integrin-dependent cell migration [
35]. For example, Herlevsen et al. [
36] found that the association of TSPAN8 with α6β4 integrin supported cell motility and liver metastasis formation, and Ai et al. [
26] discovered that TSPAN24 interacted with Integrin α6β1 to amplify PI3K/AKT signaling to induce EMT in HCC. Therefore, we focused on several integrins that play stimulatory roles in CCA migration: Utispan et al. [
22] reported that integrin α5β1 promoted the invasion of CCA through a PI3K/AKT-dependent pathway. Ding et al. [
23] demonstrated that a high level of Integrin a6 expression was associated with a migratory and invasive phenotype of intrahepatic CCA cells, and Patsenker et al. [
24] found that aVβ6 integrin is a highly specific IHC marker for CCA. We confirmed α6β1 from these integrins using co-IP and IF techniques to demonstrate its interaction with TSPAN1. We examined pivotal downstream molecules after TSPAN1 transfection using western blotting and discovered that TSPAN1 was involved in the PI3K/AKT pathway. A time-response experiment with Laminin 5 in CCA cells further implied that TSPAN1 amplified PI3K/AKT pathway signaling. Then, we determined whether there was a visible difference in p-GSK-3β and Snail expression. Finally, we found that Snail inhibited PTEN expression in CCLP1 and HCCC9810 cells, indicating PI3K/AKT/GSK-3β/Snail/PTEN feedback loop existed in CCA.
Acknowledgements
Not applicable.