Growth Factor-induced Epithelial-mesenchymal Transition In Human Colorectal Carcinoma Cells

[Background and objective]Colorectal cancer is one of the most common malignant tumor in China, which ranks fourth in mortality of cancer and with short survival and very high mortality.And the cancer deaths mainly due to the tumor invasion and metastasis.Therefore, looking for an important and effective factor during process of the invasion and metastasis of colorectal cancer or some molecules associated with metastasis, is very critical for the development of a new concept of diagnostic assessment, clinical diagnosis and prognosis of the Colorectal cancer patients.Epithelial-mesenchymal transition (EMT) is a process of epithelial cells transform into another phenotype with mesenchymal cell properties,through which cells acquire the ability to obtain differentiation or migration.EMT is an important pathophysiological process,which played an important role in the embryonic development, wound healing, tissue regeneration, stem cell differentiation and organ fibrosis.According to the recent studies,the EMT process plays a vital role in the tumor invasion and metastasis during the cancer development, and the study of EMT in tumor has quickly become a new hot spot. EMT of tumor cells is considered a dynamic process with multi-step.The mesenchymal-like phenotype transformation and reconstruction of cytoskeleton, making the loss of adhesion among tumor cells, which enhance the cell mobility and cause the degradation of basement membrane and ECM.All these features Accelerate the invasion and metastasis of cancer cells.The present studies show that the phenotype during EMT changes mainly regulated the by cell signal transduction,which mainly affected by the Stimulation of external environment of cancer cells,such as ischemia, hypoxia and a variety of cell growth factors. The growth factors play an important role in EMT, and the Transforming Growth Factor-β1(TGF-β1) is a classical molecular which induced cells EMT.TGF-β1 are considered high expression in cancer patients'serum,and the express level of which increases the while cancer stage advances.The EMT induction mechanism of TGF-β1 has been partially clarified.However, the EMT is a complex process,which Regulated by multiple factors simultaneously while those factors maybe have some interaction among themselves at the same time.The microenvironment of the tumor cells is also complicated,all those factors associated with the EMT should also be taken into account.Therefore, the current studies of EMT in vitro which use one stimulating factor have thier limitations.Epidermal growth factor (EGF) is closely related with the tumor. Many studies show that patients with colorectal cancer serum EGF expression was significantly higher in patients than normal, the expression of its receptor EGFR have also increased.The expression level of EGF and the degree of cancer stage are positively correlated.We hypothesized that EMT-related tumor invasion and metastasis is associated with the EGF levels.There is no related literature regarding the EGF induced EMT in colorectal cancer cells and then enhance the ability of cell invasion and metastasis domestic and abroad.This research was to study the EMT features of colorectal cancer cells by the stimulation of EGF,and to compare the different characteristics of EMT with TGF-β1 treatment and TGF-β1/EGF co-treatment of colorectal cancer cells.microRNA (miRNA) is a class of newly discovered small molecule non-coding single-stranded RNA which closely related with the gene regulation, and it is a hot spot in current studies. Multiple signaling pathways, cytokines, transcription factors, oncogenes, etc. involved in the EMT process,so its surely associated with the changes in the genetic level.Therefore,miRNAs have an important regulatory role in EMT process.There is no related literature regarding the changes in microRNA expression profile in EMT-induced colorectal cancer cells.This research was to study the miRNAs expression profiling in EMT-induced colorectal cancer cells by miroRNA gene chips.[Methods](1) To use exogenous recombinant human transforming growth factorβ1 (rhTGF-β1) and recombinant human epidermal growth factor (rhEGF), respectively or co-induce colon cancer cell line SW480, then to detect the EMT-related gene expressions in these different induced SW480 cell groups, which include morphological changes observed by inverted phase contrast microscope.To use Transwell invasion model in vitro and scratch assay to compare cell movement or migration ability in different groups.To use Western Blot to detect the epithelial markers (E-cadherin) expression in different groups.To use the quantitative real-time PCR to detect EMT-related gene such as epithelial marker (E-cadherin), mesenchymal markers (Vimentin, Fibronectin), and related transcription factors (ZEB1, Snail, Slug) expression.(2) We took several different time points of EMT-induced SW480 cells, then used high-throughout microRNA array chip to discuss microRNA expression profile of colon carcinoma cell line SW480 after the EMT induction.[Results](1) TGF-β1 can induce the occurrence of typical EMT phenomenon in colorectal cancer cell line SW480, including the morphology change and significant difference expression of the EMT-related markers, also enhanced cell migration and invasion ability.After the EGF treatment, the morphological changes of SW480 cells meet the standerd EMT characteristic changes.And the EMT-related molecular markers (E-cadherin, Vimentin, Fibronectin, Snail, Slug , ZEB1) also consistent with the characteristic changes of EMT.EGF treatment also enhanced the cell migration and invasion ability.Compared with the EGF treatment group and TGF-β1 treatment group, the TGF-β1/EGF co-treatment group induced by treatment group can cause more typical EMT-related changes, including morphological and molecular markers changes.Some of the markers(E-cadherin,Vimentin,Fibronectin,Slug) show significant difference compared with the EGF treatment group and TGF-β1 treatment group, respectively. We can observe more invasive, movement and maintenance of the phenotype abilities in co-treatment group.(2) Some of the microRNAs changed after EMT-induced in SW480 cells according to the detections of microRNA expression profiling microarray chips. The expression levels of miR-1308, miR-138, miR-139-5p and miR-1180 changed in EMT induced SW480 cells compared to EMT uninduced cells. miR-1180 and miR-138 expression levels downregulated; miR-1308 and miR-139-5p expression levels upregulated.【Conclusion】(1) Typically EMT phenomenon can be induced by both EGF and TGF-β1 in SW480 cells. More typical EMT phenomenon and related phenotype were observed after the EGF/TGF-β1 co-treatment when compared to the EGF treatment group and TGF-β1 treatment group, respectively. The EGF/TGF-β1 co-treatment is more suitable for EMT-ralated research of colon cancer cells in vitro.(2) Some new microRNA molecules have been found in growth factor-induced EMT of colon cancer SW480 cells by the microRNA expression profile array chips, and provide a new direction for future research of the EMT of colon cancer in the level of micoRNAs.