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Intrahepatic cholangiocarcinoma (ICC) has been reported as the second most frequently happened primary hepatic carcinoma all around the world with an increasing incidence during the past three decades especially in Eastern Asia. The origination of ICC is the transformation from cholangiocytes into intrahepatic tumors which is usually diagnosed late. The therapies of ICC are very limited including the final surgical resection with a high rate of primary recurrence. Due to the reasons above, the patients with ICC have a low overall survival and the prognosis of ICC is quite poor, which alerts us the emergency need to explore new powerful strategies to diagnose ICC earlier and effective therapeutic alternatives. In this case, molecule therapies have more advantages than traditional ones which requires more understanding about molecular mechanisms and biomarkers in the initiation and progression of ICC .
Receptor tyrosine kinases (RTK)s, a group of receptors containing a transmembrane domin from the family of protein tyrosine kinases, are used to combine several cytokines, growth factors, and hormones in the surface of cells [2, 3]. RTKs have been indicated as the essential effect factors in a variety of cellular processes, and furthermore, the crucial regulators during the origination and developing procedure of many kinds of cancers . Serine Threonine Tyrosine kinase (STYK1), which is also known as Novel oncogene kinase (NOK), is one of the RTK members possessing a special structure lacking the ectodomain compared to other subfamilies, which means it does not have the ability to bind specific ligands [5-7].
Previous studies showed that overexpressed STYK1 in nude mice BaF3 cells could lead to malignant transformation and metastasis, as well as cell proliferation independent of growth factors and colony formation independent of surface sticking. [5, 8-11].
Notably, the tumorigenicity of STYK1 has been identified in many types of cancers compassing breast cancer, prostate cancer, ovarian cancer, lung cancer, and acute leukemia to date [5, 11-14]. Thus we conjectured there is a big chance that STYK1 is likewise a pivotal element in the oncogenesis of ICC in line with the previous reports, and STYK1 could be studied as a promising biomarker for the timely diagnosis and novel therapies of ICC.
In the present study, the role of STYK1 in ICC cells was investigated through a group of experiments. The expression levels of STYK1 mRNA in 15 pairs of ICC tumor and adjacent tissues were firstly measured. Whereafter, STYK1 was knocked down in ICC cell lines HCCC-9810 and RBE via a lentivirus-mediated system using short hairpin RNA (shRNA) as a biological tool. Then the cell proliferation, colony formation, cell cycle progress, formation of tumors in nude mice, migration and invasion, as well as the expressions of cell cycle proteins of Lv-shSTYK1 or Lv-shCon infected cells were subsequently analyzed by CCK-8 assay, colony formation, flow cytometry, tumor formation, wound scratch assay, transwell, and western blotting one by one. The findings coming from this study could provide some clues for the development of new molecular targeted treatment methods eliminating ICC concerning the function of STYK1.
Materials and methods
2.1 Tumor and adjacent tissue samples
The 15 pairs of human tumor and adjacent tissue samples of ICC for qPCR (quantitative real-time polymerase chain reaction) were selected from the tissue bank of XXX hospital randomly with the permission of XXX hospital ethic committee.
2.2 Quantitative real-time PCR
The relative basic expression levels (different tissues) and knocked down expression levels (different ICC cells) of STYK1 mRNA were examined via qRT-PCR (quantitative real-time polymerase chain reaction).The qRT-PCR was carried out as described previously with some minor modifications. The internal control used in this study was β-actin instead of GAPDH. The forward and reverse primers of STYK1 held the sequences of 5’-CAGTGGGAAGGAGGGACTGA-3’ and 5’-TGCAGCCCAGTGAAATTGGA-3’ respectively. Whereas the sequences of β-actin primers were 5’-GTGGACATCCGCAAAGAC-3’ (sense) and 5’-AAAGGGTGTAACGCAACTA-3’ (antisense).
2.3 Cell cultures
ICC cell lines HCCC-9810, RBE and human embryonic kidney cell line 293T were purchased together from the Cell Bank of Chinese Academy of Science (Shanghai, China). All these cells were grown in RPMI1640 (Gibco, Carlsbad, CA) in which 10% foetal bovine serum (Biowest) as well as 1% penicillin/streptomycin (Corning, Lowell, MA) were also added at 37oC in an incubator with the atmosphere of 5% CO2. 293T cells were used to package the lentivirus, while HCCC-9810 and RBE cells were targets of transfection.
2.3 Construction of lentiviral vector system and transfection
The specific sequence of shRNA targeting STYK1 was designed as 5’- XXX-3’, while the gene order 5’-XXX-3’was used to build shCon because of its disability to interfere normal human gene. These shRNAs were ligated into the vector pFH-L (containing GFP (green fluorescent protein) as a visible gene marker, after which the constructed vectors were transducted into 293T cells together with another two packaging vectors termed pVSVG-I and pCMVâ-³R8.92 via the Lipofectamine 2000 (Invitrogen) protocol. After the harvest of packaged letivirus particles, HCCC-9810 and RBE cells were incubated in 6-well plates (XX cells/well) in which the Lv-shSTYK1 or Lv-shCon was also added (a MOI of 10) for infection. After the infection of 12h, the cells were subsequently cultured with the basic medium replacing the medium containing lentivirus for 48 more hours. Successful infection was then detected through observing cells fluorescing green because of GFP under fluorescence microscope.
2.4 Western-blot analysis
After 4 days since infection, to get cell extracts of HCCC-9810 and RBE cells for analyzing the proteins, cell lysis was performed by resuspending and incubating the cells in cell lysis buffer containing 100 mM Tris-HCl (pH 6.8), 4% SDS, 10 mM EDTA, and 10% Glycerol. The mixture of the extracted proteins was separated by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The separated proteins were transferred into nitrocellulose membranes with Transfer butter. Membranes were then probed with 1:XXX diluted XX anti-XX-STYK1 primary antibody(Catalogue number: XXX), 1:XXX diluted XX anti-XX-Vimentin primary antibody(Catalogue number: XXX), 1:XXX diluted XX anti-XX-N-cadherin primary antibody Catalogue number: XXX), 1:XXX diluted XX anti-XX-Ecadherin primary antibody(Catalogue number: XXX), 1:XXX diluted XX anti-XX-Cyclin B1 primary antibody(Catalogue number: XXX), 1:XXX diluted XX anti-XX-Cdc2 primary antibody(Catalogue number: XXX), 1:XXX diluted XX anti-XX-Cdc25A primary antibody(Catalogue number: XXX), and 1:60000 diluted GAPDH (Catalogue number: XXX). Whereafter, the membranes were incubated with 1:5000 diluted goat anti-rabbit horseradish peroxidase secondary antibody conjugated to HRP (Catalugue number: XXX) after being washed with TBST. And the chemiluminescent (ECL) detection reagent was added to help with observation .
2.5 CCK-8 cell proliferation assay
To test whether the depletion of STYK1 could affect the proliferation of ICC cells, HCCC-9810 and RBE cells were added into 96well plates ( XXX cells/well) after Lv-shSTYK1 or Lv-shCon transfection, followed by incubation of 24h, 48h, 72h, or 96h. The cell count kit-8 (CCK-8) (China) was used to detect cell proliferation as described previously . After the addition of CCK-8 solution (XX μl/well), another X hours of incubation was carried out before the measurement of absorbance at 460nm. Four more times of the detection of absorbance were also carried out in the next four days respectively.
2.6 Colony formation
The colony formation abilities of HCCC-9810 and RBE ICC cells were analyzed following the below strategy. Firstly, HCCC-9810 and RBE cells were plated on 6-well plates and incubated, after which they were infected with Lv-shSTYK1 or Lv-shCon and cultured for another 4 days. At the end of the incubation, cells were harvested, counted and seeded in new 6-well plates with a concentration of 600 cells each well. In the following 7 days, colonies developed from both groups (Lv-shSTYK1 and Lv-shCon) of HCCC-9810 and RBE cells naturally. The plates were then fixed by paraformaldehyde before stained using crystal purple. All the colonies containing more than 50 cells were picked, then observed and photographed.
2.7 Analysis of cell cycle progression via flow cytometry
After the infections with Lv-shSTYK1 or Lv-shCon, HCCC-9810 and RBE cells were seeded in 6-cm dishes with the concentration that each dish contained 100000 cells. The following incubation lasted for 40 h and subsequently the cells were washed with cold PBS on ice. Whereafter, 75% cold ethanol was used to fix the cells at 4oC overnight. The cells were then resuspended in the mixture of 50 mg/ml propidium iodide (PI; ) and 100 mg/ml DNase-free RNase A and cultured for half an hour. The well stained cells were then collected by filtering the suspension through a 50-mm nylon mesh and detected using a Cytomics FC 500 flow cytometer.
2.8 In vivo tumor formation of nude mice
The inï¬‚uence of STYK1 silence on the tumor development of ICC in vivo was examined. The two types of ICC cells infected with Lv-shCon or Lv-shSTYK1 generated four groups (HCCC-9810-Lv-shSTYK1; HCCC-9810-Lv-shCon; RBE-Lv-shSTYK1; RBE-Lv-shCon) which were subsequently injected into 24 4-week-old Balb/c nude mice (6 mice each group,) at quantity of 5 × 106 per mouse. The progression of oncogenesis in vivo was determined through measuring the size of the tumors via a vernier caliper in a blinded fashion every week after the first week for the primary tumor to form and the measurement lasted for about 6 weeks. The formula, tumor volume (mm3) = width (mm)2×length (mm)×0.5, was used to estimate tumor volume. All mice were sacriï¬ced when the measurement was finished and their corresponding tumors were weighted by a digital balance. In this part, all experiments about animals were approved by the Animal Care Committee of the.
2.9 Wound scratch assay
This assay was conducted as described previously with some minor modifications as following. The ICC cells transfected with Lv-shCon or Lv-shSTYK1 were seeded in a 24-well plate at a density of X cells per well respectively and incubated to form the monolayer cells before scratching its center with a germfree toothpick to develop a damaged area with an equal width. PBS was then used to wash the scratched cell monolayer for three times and the cells were incubated for 24 hours accompanied with the observations and records at the very beginning and the end. Lines showing the edges of the cells were added at a later stage to help compare the motility of infected and non-infected ICC cells.
2.10 The effects of shSTKY1 on the migration and invasion abilities of ICC cells
Transwell assay was used to investigate the migration abilities of ICC cells under the influence of shSTKY1. Transwell chambers with polycarbonate membranes of 8.0μm pore size were obtained firstly. Whereafter, two ICC cell lines were suspended in upper transwell chambers containing F-12 solution with 0.2% BSA (serum free) as culture medium. On the other hand, all the upper tanswell chambers were put in corresponding lower chambers filled with F-12 supplemented with 10% FBS and cultured at the atmosphere of 37°C and 5% CO2, overnight. At the end of the incubation, the cells remained in upper chambers were discarded. The crawl over cells on the lower surfaces of the membranes then underwent 10-minute fixation by 4% paraformaldehyde, 30-minute staining by 0.1% crystal violet, and the rinse in PBS before being counted and photographed under a microscope.
The invasion assay was carried out almost the same as the migration assay except that a layer of matrigel was added on the upper side of the polycarbonate membrane to mimic the extracellular matrix in vivo. Cells attached to the downside of the membrane were also dealt with and counted in the same way as the migration assay.
3.1 ICC tumor expresses much more STKY1 than the corresponding adjacent tissues.
The relative mRNA levels of STKY1 in several randomly selected ICC tumor and adjacent samples were measured via qRT-PCR and compared with each other. As is shown in Figure 1, there is a big difference between the STKY1 mRNA in tumors and the adjacent parts which means the overexpression of STKY1 could be a mark of tumorigenesis of ICC.
3.2 Lentivirus-mediated depletion of STYK1 in ICC cells.
Because of their stable expressions of STKY1, HCCC-9810 and RBE cells were selected to explore the function of STYK1 in ICC cells. HCCC-9810 and RBE cells were firstly transfected with GFP-tagged-Lv-shCon and GFP-tagged-Lv-shSTYK1 respectively. The transfection results were observed through the expression of GFP under a fluorescent microscope (Figure 2A). The relative mRNA levels of STYK1 were decreased dramatically by more than 80% according to the qRT-PCR results in both ICC cell lines (Figure 2B). Meanwhile, the results of western blot confirmed the high knockdown efficiencies of Lv-shSTYK1 in HCCC-9810 and RBE cells on protein levels(Fig 2C).
3.3 Proliferation abilities of ICC cells were weaken by shSTYK1
Given the high knockdown efficiency of shSTYK1, the prolificacies of infected HCCC-9810 and RBE cells were further analyzed via CCK-8 assay. In accordance with Figure 3A and B, it was indicated that ICC cells infected with Lv-shSTYK1 showed much slower proliferation rates comparing with the cells infected with Lv-shCon. The conclusion was then confirmed by colony formation assay which demonstrated that HCCC-9810 and RBE cells transducted with Lv-shSTYK1 could only form much smaller colonies than those developed by the ICC cells transducted with Lv-shCon(Figure 3C and D). Combination of CCK-8 assay results and colony formation results suggested that proliferation abilities of ICC cells were significantly weaken by shSTYK1 transductions, reflecting the essential role of STYK1 in ICC cell growth.