Effect Of Wnt Signaling On Epithelial-mesenchymal Transition Of Colon Cancer

BackgroundWnt genes are widely expressed in invertebrates and vertebrates, and they are highly conserved among different species. The functions of Wnt signaling pathway are relevant to series of complex and various regulation of physiological and pathological processes such as cell proliferation, differentiation, polarity changes, migration and apoptosis. Especially in the proliferating cells(such as stem cells, tumor cells, etc.), canonical Wnt pathway is found highly activated. To date, researches showed that thedisrugulation of Wnt pathway was closely related to a variety of cancers, such as breast cancer, stomach cancer, esophageal cancer, colon cancer, liver cancer, lung cancer, melanoma, etc. Wnt played a key role in the mechanism of tumorigenesis, advancement, and metastasis.Epithelial-mesenchymal transition(EMT) refers to the phenomenon of epithelial cells transforming into mesenchymal-like cells in specific physiological or pathological conditions; the reversed process is called mesenchymal-epithelial transition(MET). Physiological EMT/MET occurs during embryo development and wound healing, while pathological one is mainly envolved in tissue fiberosis and maligancy. It is a progress that the cells lose its epithelial cell phenotype and gradually obtained mesenchymal-like cell phenotype, which is a complex biological process, involving multiple genes. The tight connection between epithelial cells is lost, cell polarity disappeared, and adhesion capability is reduced; meanwhile, the ability of cell migration is enhanced, so that the cells yield invasion and metastasis potential. The specific changes of molecular markers are the downregulated expression of epithelial cell membrane proteins such as E-cadherin(epithelium-cadherin, E-cad), while upregulation of mesenchymal cell ones such as N-cadherin(N-cad), Vimentin(Vim), etc. And more than one transcription factor shows conresponding changes as well.Molecular signaling pathways such as canonical Wnt pathways are underlying the EMT mechanism. play role in both physiological and pathological EMT/MET. For instance, knockout of Wnt genes abolished the mesenchymal cell immigration and subsequently the disruption of development of kidney in mouse. Molecular signaling pathways are underlying the EMT mechanism. Canonical Wnt signaling is called also Wnt/β-catenin pathway.The present studies showed that Wnt/β-catenin signaling pathway was not only involved in tumor proliferation and differentiation, but also involved in the regulating progress of physiological and pathological EMT/MET.In the development of renal tubules, knocking out Wnt gene effectively blocked the migration of mesenchymal stem cell and the formation of localized renal tubular epithelial cells. The method of gene knockout interfering Wnt/β-catenin signaling pathway effectively blocked or even reversed the epithelial-mesenchymal transition for EMT-mediated tumorigenesis. Though it is well accepted that Wnt signaling disrugulation is the dominant cause of colorectal cancer, it is no hint whether this pathway enforce the infiltration and distant metastasis mediated mainly by EMT.ObjectiveIn order to study the effect of the canonical Wnt signaling pathway on epithelial-mesenchymal transition, that is, mechanism on colorectal cancer invasion, we choosed colon cancer cell line HCT116 cells as experimental material, applied agonist(Wnt3a) and antagonist(Dkk1) to regulate the activity of the Wnt signaling pathway, and observed the effect of Wnt signaling on cell migration, and gene expression of EMT featured membrane protein and transcription factors, through scratch assay and real-time quantitative PCR. Methods1.HCT116 cell culture. The colorectal cancer cell line HCT116 was purchased from cell bank of Shanghai CAS. Cells were suspended in My Coy’s 5a medium supplemented with 10% FBS, and seeded into culture flask in a density of 1×105cells/ml(5ml/flask). Then the cells were kept in incubator with 37?C,5%CO2 and saturated humidity. Cells were trypsinized and passaged at the ratio of 1:5 when reaching 90% confluence.2.The experimental groups. HCT116 cells were divided into three groups:(1) control group: HCT116 cells without treatment;(2) antagonist group: HCT116 cells +Dkk1-100ng/ml(3) agonist group: HCT116 +Wnt3a-40ng/ml.HCT116 cells were suspended in the conditional medium as mentioned above, and seaded into 6-well plates in a density of 1×106/well. Three parallel wells were set for each group. Antagonist and agonist administration was started 24 h after the initial seading when the cells adhered to the surface.3.Cell scratch assay. HCT116 cells were seeded in 6-well plate in a density of 1×106 cells /well, and cultured for 24 h in the conditional medium. Whencells covered about 80% or more to the well surface, scratch along the diameter using the tip of a 200μl pipette to draw a straight line in each well. Then scratch off the cells completely in one side of the line and keep the other side intact. Wells were washed three times with PBS to remove floating cells, and replaced with conditional medium containing 2% FBS, added 100ng/ml Dkk1(antagonist group), 40ng/ml Wnt3a(agonist group), or no extra treatment(control group). This time point was recorded as 0h. Under the inverted phase contrast microscope, the each well along the scratch line was divided into 8 continuous view-field in an up-to-down way. All view-fields were observed in 12 h time intervals(0h, 12 h, 24 h and 48h) for 48 h. Images were collected.4.Cell cycle detection. HCT116 cells were adherently cultured for 24 h and treated according to that mentioned in Method 2 for 48 h. Cells from 3 groups were collected and washed with PBS for 3 times. Then cells were fixed with cold ethanol overnight. After centrifugation, cells were resuspended in Propidium Iodide(PI)/RNase Staining Buffer 200μl, and incubated in dark at room temperature for 30 min. After 3 times of PBS washing, cell cycle distribution was detected and recorded with flow cytometry. Data were ananlyzed with software Mod Fit.5.Expression change of Wnt pathway genes, EMT featured protein markers, and transcription factors. Total RNA was extracted from the 3 individual groups using total RNA extraction kit according to the manufacturer’s instruction. the RNA concentration and purity were accurately determined by Nanodrop, and RNA was reversely transcribed into c DNA with the random primers from the same amount of RNA. Elative Real-time fluorescently quantitative PCR was adopted to detect the expression of Wnt signaling pathway-related genes β-catenin,Axin2,C-myc,Wntless; EMT-featured genes E-cad, N-cad, Vimentin; and EMT-featured transcription regulatory factor genes Slug,Snail,ZEB1,ZEB2, Twist, MMP2, MMP3, etc. The expression change was presented as the relative ratio of gene expression, whichwas calculated by –σTT among the antagonist to control group, and agonist group to control group. β-actin acted as a reference gene, while ROX as sampling reference.6. Statistics analysis. All tests were repeated at least twice. Using software SPSS10.1, groups were compared with t-test(α= 0.05). Results1.HCT116 cells were passage at 1: 5. After 24 h all cells form adherent monolayer.Cell morphology was epithelial like, and appeared polygonal or oval in shape. Cells were of highproliferation. Continuous passaging caused no detactable morphology and growth mode change.2.After administration of antagonist and agonist to the cells, cell scratch assay showed that the control group did not occur significant cell migration until 24h; in the agonist group(add Wnt3 a, 40ng/ml), the migration of cells can be observed at 12h; in the antagonist group(add Dkk1,100ng/ml),there was no significantly cell movement in the cells during 48 h.3.After activation and depression of canonical Wnt signaling pathway, cell cycle indicated that cells of S phase accounted for 10.2%±0.45%, 9.4% ±0.2% and 11.1% ±0.3% in control group, antagonist group and agonist group respectively. Statistics analysis showed no significance(α= 0.05).4.The real-time PCR test results showed that when activated, the canonical Wnt signaling-related genes β-catenin, C-myc, Wntless were highly expressed,; epithelial marker E-cad was downregulated, whereas mesenchymal cell marker N-cad, Vimentin expression were significantly upregulated. When inhibited, the gene expression change was opposite. Statistics analysis showed significant change of the ratio of the gene expression(p<0.05).5.As was also showed in real-time PCR, EMT-featured transcription regulatory factors were also changed significantly. When Wnt signaling pathway was activated, the expression of the relevant transcription factor Snail, Slug, ZEB1, Twist, MMP3 was significantly upregulated(p<0.05). However, when Wnt signaling pathway was inhibited, the expression of above transcription factor was abolished significantly(p<0.05).Conclusion1. In vitro the canonical Wnt signaling pathway involves in epithelialmesenchymal transition of colorectal cancer cells. Activation of the signaling pathway can significantly induce the cell immigration and mesenchymal phenotypes; depression of the pathway effectively quenches cell immigration and EMT change.2.Canonical Wnt signaling pathway acts in EMT by regulating the expression levels of the transcription factor Snail, Slug, ZEB1, Twist, MMP3.