| Part one Building epithelial-mesenchymal transition (EMT) uroepithelium carcinoma cell line induced by TGF-β1, and verify it experimentally.Objective To explore the best induced concentration and time of TGF-β1in EMT of human uroepithelium cell carcinoma (T24), and to verify its gene expression and biological behavior.Methods Human bladder urothelial carcinoma cell line (T24) was cultivated. Different concentrations of TGF-β1factor was used to induce EMT in T24cell line, then the cell morphological changes were observed at different time points. The real-time quantitative PCR was used to determine the expression level of EMT marker (such as Vimentin, MMP2, E-cadherin, slug, ZEB1, and so on). Meanwhile, the cell scratch test, Transwell migration test was used to detect the changes of biological behavior in T24cells induced by TGF-β1.Results The10ng/ml TGF-β1induce T24cells about36hours were the best induced concentration and time. The cell morphological changes suggested the epithelial-mesenchymal transition of T24cells. At the same time, the expression level of EMT marker gene also indicated a corresponding change, such as:the expression levels of mesenchymal cells markers (Vimentin and MMP2) were increased, the expression level of epithelial marker (E-cadherin) was decreased, the expression level of the EMT factor (slug, ZEB1)were increased. In addition, the cell scratch test showed that, there was a significant difference in the average healing area (percentage)(0.81vs1, P<0.05) between the two groups (before and after the10ng/ml TGF-β1induced); Similarly, Transwell migration test also indicated the cell invasion and migration ability were obviously enhanced when the T24cell line were induced by10ng/ml TGF-β1(54.6/HP vs97.65/HP, P<0.05).Conclusions The cell morphology changes indicated that the10ng/ml TGF-β1induce T24cells about36hours were the best induced concentration and time for T24cell line EMT. Further, both the EMT related gene expression and its biological behavior shows that the cell invasion and migration ability were significantly enhanced when the T24cell line were induced by TGF-β1. Part two The mRNA and MicroRNA expression profiles of human uroepithelium cell carcinoma induced by TGF-β1Objective To obtain the mRNA and MicroRNA expression profiles of T24cell line induced by TGF-β1factor.Methods The total RNA of the two groups (before and after the10ng/ml TGF-β1induced) were extracted and tested. After that, the qualified total RNA were purified, incubated and tailed, fluorescence marked, chip hybridized, cleaned and dyed, chip scanned, data extracted and standardized. The chips used in this study were GeneChip(?) Prime ViewTM Human Gene Expression Array chip and Multispecies micrornas2.0Array chip. Then we registered and used the MAS3.0System (Capital Bio(?) Molecule Annotation System V3.0) to analyze the results of the gene chip and microRNA chip (such as GO, KEGG pathway, Target gene prediction, Contingency analysis, etc.).Results When the T24cell line were induced by10ng/ml, there were1062genes, including SPC25etc, their expression levels were decreased, meanwhile, about572genes, including genes ASNS, their expression levels were increased. The KEGG pathway analysis found that, including Cell cycle, there were about40signaling pathways are closely related with this process. According to the GO database, about1250related GO information were detected, most of them were closely related with cell cycle, cell division, adhesion, metabolic regulation, immune responses, cell sports and so on. The microRNA chip scan results showed that, there were6microRNA, including miR-1184etc, their expression levels were decreased, meanwhile, about8microRNA, including miR-21, their expression levels were increased. The contingency analysis of microRNA and mRNA chip found that three microRNAs, including microRNA-21, microRNA-146a, and microRNA-638, were significantly related with some differentially expressed genes expression trends, including ACVR1C, NOG, THBS1, TUBB2A, which were closely related with EMT process.Conclusions We obtained the mRNA and MicroRNA expression profiles of T24cell line induced by TGF-β1factor; Some molecular signaling pathways, including Cell cycle, were involved with the process of EMT of T24Cell line, further, they are closely related with cell growth, connection, biological behaviors (such as adhesion, and migration); miRNA-21, miRNA-146a, and miRNA-638very likely play a role in the process of EMT in T24cells by means of targeted ACVR1C etc genes. Part three Verify the results of microarray which is focus on TGF-β1induced bladder cancer T24cellsObjective The purpose of part is to verify the results of microRNA gene chip analysis and bioinformatics analysis in vitro, and to improve the credibility of the results.Methods Overexpress and inhibit the expression of microRNA by use of microRNA mimics and microRNA inhibitor. Verity whether the expression of three microRNA are same as the microassay results, the effect of mimics and inhibitor, as well as the regulation effort to target gene expression by Real-time PCR. Observe the cell invasion, migration, and metastasis by scratch test and transwell chamber experiments.Results The results from the PCR showed that the expression trends of microRNAs such as microRNA-21, microRNA-146and microRNA-638, are the same as the the gene chip results. MicroRNA mimics and inhibitor act well. Furthemore, the expresstion of target genes of microRNAs were the same as bioinformatics analysis. Scratch tests showed that the invasion capability enhanced in all the mimics groups except normal control groups, while the invasion capability weakened in all inhibitor groups. Transwell chamber showed that migration and metastasis capability enhanced in all the mimics groups except normal control groups, while the migration and metastasis capability weakened in all inhibitor groups.Conclusions MiRNA-21, miRNA-146a and miRNA-638and other microRNAs come into play in the T24bladder cancer cells EMT process by regulating target genes such as ACVR1C. Inhibit the expression of these microRNAs can inhibit urothelial carcinoma epithelial-mesenchymal transition, thereby change the cell invasion and migration capabilities. |
PDF Full Text Request