The Anti-tumor Effect Of BTG2 Gene Overexpression On Cytobiology Characteristics Of EJ Bladder Cancer Cells

Objective:To investigate the role of antiproliferative protein BTG2 (B cell translocation gene 2) in development of bladder cancer. In this study, we used real-time PCR to determine the expression levels of BTG2 in several bladder cancer cell lines (EJ, BIU-87,5637 and T24) and a normal human bladder epithelial immortalized cell line SV-HUC-1. We analyzed the differential expression of BTG2 between malignant bladder cancer cell lines and SV-HUC-1. To further clarify the influence of BTG2 on the biological characteristics of bladder cancer line EJ, BTG2 was subcloned into pcDNA3.1 (+) vector. The plasmid was then transfected into bladder cancer EJ cells, and stably-transfected cell lines were screened and established.We next investigated the influence of BTG2 on survival, proliferation and migration of bladder cancer line EJ, explored the role of BTG2 in the development of bladder cancer, and these data will provide theoretical basis for the future gene therapy of bladder cancer.Methods:Compare BTG2 expression in different bladder cell lines:To clarify the role of BTG2 in development of bladder cancer, we analyzed the expression level of BTG2 in several bladder cancer cells and human bladder epithelial immortalized cell line SV-HUC-1, and determined the relationship between BTG2 expression and bladder cancer. Bladder cancer cells EJ, BIU-87, T24 and 5637 were cultured in RPMI-1640 medium containing 10% fetal bovine serum. Human normal bladder epithelial immortalized cell line SV-HUC-1 was cultured in F12K medium containing 10% fetal bovine serum. Total cellular RNA Cell was extracted with Trizol reagent and BTG2 expression was measured in by real-time PCR. Construct BTG2 eukaryotic expression vector, and establish stably-transfected cell lines:First, primers for BTG2 were designed according to human its coding sequence. BTG2 fragments were obtained by PCR and inserted into pcDNA3.1 (+) vector, which carries a Neomyein resistance gene, we used restriction endonuclease digestion and DNA sequencing to confirm the sequence of recombinant vector. The plasmid was then transfected into human bladder cancer EJ cells and monoclonal stably-transfected cells were subsequently established by G418 (400 mg/L) selection. Elevated expression of BTG2 in stably-transfected EJ cells were confirmed by both quantitative PCR and semi-quantitative PCR, supporting the successful establishment of BTG2-overexpressing stablely-transfected cell lines. The impact of BTG2 on the biological characteristics of bladder cancer EJ: We used inverted microscope to observe the effect of BTG2 on shape and number of EJ cells. The effect of BTG2 on EJ cell growth was estimated by growth curve method, colony formation assay was performed to observe the proliferation capacity of EJ cells and to evaluate the colony formation ability in vitro. The cell migration ability was detected by wound-healing assay. Results:Using real-time quantitative PCR assay BTG2 mRNA of bladder cancer cell lines and SV-HUC-lcell,the data displayed:BTG2 expression of SV-HUC-1 was significantly higher than other bladder cancer EJ,BIU-87, T24,5637.In the rest of the bladder cancer cells,we observed the lowest expression of BTG2 is T24 cells,that is highest malignancy in EJ,BIU-87, T24.5637. Similar BTG2 level is detect in EJ and BIU-87,and the highest expression of BTG2 is 5637cells. Accordingly we found higher BTG2 was detect in lower malignant bladder cancer, so we can speculate that BTG2 play a suppressor role in the development of bladder cancer. We transfected pcDNA3.1 (+)-BTG2 expression vector with bladder cancer EJ, construct stably transfected cell lines.we observed that stably transfected cells B2C6 group is slower than transfected with empty vector E2C5, and cell becomes longer,irregular. The effect of BTG2 on EJ cell growth curve was estimated by growth curve method, Observed that transfected pcDNA3.1 (+)-BTG2 group cell growth was more robust than transfected with empty vector pc-DNA3.1 (+) group and untreated cells EJ, and cell proliferation was significantly inhibited,after transfected BTG2 expression plasmid.In cloning experiments,we observed that transfected eukaryotic expression vector cell clones are smaller than transfected with empty vector and untreated group. Moreover, in order to understand the impact of BTG2 in invasion of EJ cell,the result showed that invasion of EJ cell is lower,after transfect plasmid vector group than the non-transfected group and transfected with empty vector group. So BTG2 gene can inhibit the migration of EJ, and significantly reduced the rate of cell migration.Conclusion:Our data proved that BTG2 expression in bladder cancer cell lines is much lower than normal human bladder epithelial cell line SV-HUC-1. Overexpression of BTG2 inhibited the growth of EJ cells and weakened the migration ability of EJ. Our findings implies that BTG2 plays the role of tumor suppressor gene in bladder cancer, impaired expression of this gene maybe involved in the initiation and development of bladder cancer. These results may provide theoretical basis for the diagnosis and treatment of bladder cancer in the future.