The Experimental Study On Molecular Biology Behavior Of Transforming Growth Factor-β1 In Colon Cancer Cells

Objective: To construct the recombinant human transforming growth factor-β1 eukaryotic expression vector and The expressing plasmids targeting on TGF-β1 mRNA. They were transfected into the SW48O cells respectively by Lipofeetamine 2000 . and to detect the different effects in the molecular biology behavior of colon cancer cell line . In order to clarify the role of TGF-β1 in colon cancer initiation , progression and metastasis ,and lay the foundation for further study the applications of TGF-β1 on gene therapy in colon cancer.Methods: (1) Total mRNA was extracted from human hepatic tissue and the TGF-β1 cDNA was obtained by RT-PCR and cloned into pEGFP-N1 vector, and then the pEGFP-N1-TGF-β1 vector was constructed, identificated and transfected into COS-7 cells. its expression was detected by fluorescent microscopy, RT-PCR and Western blot.(2) Two expressing plasmids targeting on TGF-β1 mRNA and the corresponding negative control vector were successfullv constructed, and transfected into the SW480 cells by Lipofeetamine 2000 after the recombinant expressing plasmids were identified by using enzyme digestion and sequencing,then The expression of TGF-β1mRNA was detected by RT-PCR and the expression of TGF-β1 protein by Westen blot. (3) The recombinant expressing plasmids( pEGFP-N1-TGF-β1) and the expressing plasmids targeting on TGF-β1 mRNA was transfected into SW480 cells respectively by Lipofeetamine 2000. Its expression was detected by fluorescent microscopy,The expression of TGF-β1mRNA was detected by RT-PCR and the expression of TGF-β1 protein by Westen blot. the growth of SW480 cells was tested by MTT assay,SW480 cell cycle was analyzed by flow cytometry,and the invasion ability of SW480 cells were investigated by transwell-matrigel invasion chambers.Results: (1)Enzyme digestion analysis and sequencing showed that the target genes was found to be 1173bp, and had been inserted into recombinant vector, as compared with gene reported by GenBank, 0.08% of the gene mutation (531th: T→C) and no amino acid residues change in hTGF-β1. RT-PCR and Western blot demonstrated that the recombinant vector pEGFP-N1-TGF-β1 was expressed in COS-7 cells, and the relatived molecular mass of the GFP-TGF-β1 fusion protein was about 52×103, while the relatived molecular mass of the GFP protein expressed was about 27×103. The western blot analysis indicated that the GFP-TGF-β1 fusion protein could be recognized by anti-GFP antibody, suggesting that this fusion protein had good biological activity.(2) Enzyme digestion analysis and sequencing showed the expressing plasmids targeting on TGF-β1 mRNA were constructed successfully.after transfected into SW480 cells , both mRNA and protein levels of TGF-β1 were effectively down regulated. (3) After pEGFP-N1-TGF-β1were transfected into SW480 cells, The mRNA and protein levels of TGF-β1 were effectively increased. the ability of cell proliferation transfected with pEGFP-N1-TGF-β1 was decreased significantly (P<0.05), the cells cycle were arrested at G0-G1 stage, and the invasive ability were evidently increased. In the cells transfected with the expressing plasmids targeting on TGF-β1 mRNA, The levels of TGF-β1 were effectively down regulated. the invasive ability were evidently decreased,but there were no significant changes in the proliferation and cycle of SW480 cells .Conclusion: After pEGFP-N1-TGF-β1were transfected into SW480 cells, the ability of cell proliferation transfected was decreased significantly, the cells cycle were arrested at G0-G1 stage, and the invasive ability were evidently increased. Inconsistent effect SW480 cell proliferation and invasiveness may be achieved through different signal paths.In the cells transfected with pshRNA-TGF-β1, The levels of TGF-β1 were effectively down regulated. the invasive ability were evidently decreased,but there were no significant changes in the proliferation and cycle of SW480 cells .From this,TGF-β1 plays a vital role in colon cancer initiation,progression and metastasis,Treatment with TGF-β1 gene as the target , would be expected to become one of the important strategy for the treatment of colon cancer.