| Objective: Twist, a Basic helix loop helix (bHLH) transcription factor existed in Placenta, mouse and human, was expressed mainly in Placenta, mesoderm of embryo and some undifferentiated tissues from adult mesodermal tissues . Twist protein was consisted of 202 amino acids (human), 206 amino acids (mouse), 166 amino acids (toad), and 490 amino acids (drosophila) in different genus, there are 96% homology between mice and human in amino acid sequence , and 100% conserved sequence in DNA-binding domain between mouse and human,That means they can bind the same DNA sequence"E-box": CANNTG, Twist regulates expression of lots of genes in transcriptional level,its original function was found to regulate the development of Embryo by EMT. It is recently confirmed that twist was engaged in epithelial mesenchymal transition(EMT)in some epithelium-original tumors and promote its invasion and metastasis. Meanwhile, twist can induce a set of mesenchymal arkers in the progress of tumors separately. It was shown that twist was mover-expressed in various kinds of solid tumors, like breast cancers, prostate cancers, melanoma and osteosarcoma; it can promote apoptosis of human breast cancers and melanoma. The expression of twist was also confirmed in human breast cancers, colon cancers, kidney cancers, gastric cancers, hepatic carcinoma and normal hepatic cells, its expression was highest in colon cancer cell line, while the lowest in normal hepatic cells. It is shown that Twist gene was high-expressed in HCT116cell lines of colon cancer, but low-expressed in the SW480 cell stub of colon cancers. In this experiment, we transfered SW480 cellses with the plamid of high expression Twist gene and observe the effect of twist restoration in SW480 cells on cell proliferation, apoptosis migration and invasiveness in vito.Methods: SW480 cell which express low level of Twist were transfected with high-expressed twist plasmid or empty vector plasmid(control).To obtain cell line transferred stably with empty vector or Twist plasmid ,selection was initiated using G418. Cell proliferation was determined using MTT methods ,cell motility rate was observed with using Wound Healing assay; cell invasiveness was evaluated by Matrigel invasion assay and expression of Twist protein were detected with western blot after transfected stably with either empty vector or Twist plasmid.Results:1 The Twist gene mRNA and protein expression in SW480 cells tranfected stably with Twist plasmid incresed significantly compared with those in control group(P<0.05)2 MTT method result show :compared with control group, the growth rate of stably transfectec group was significantly higher than control group(P<0.05).3 Wound Healing assay results: The transfected group cells migrated rapidly and closed the wound by (40.06±5.56)after 24 h and (25.24±2.65)after 48h.In contrast ,control group cells only closed the wound by (75.77±8.06)and (35.37±6.79) in the same time interval. The wound closed rate in transfected group cells was significantly higher than in control groups (P<0.05).4 The number of invasive cells in transfected groups were(154±12)and were(73±14) in control group. The invasive cells number in transfected groups was significantly higher than in control group(P<0.05).Conclusion:1 In this exprement Twist gene was transfected successfully in the SW480 cells and can be stably experssed.2 After up-regulated the Twist gene it can be improve the vito proliferation of the SW480 cell .It is show that Twist gene can improve the vito proliferation of the SW480 cells. 3 After up-regulated the Twist gene it can be improve the vito migration of the SW480 cell .It is show that Twist gene can improve the migration of the SW-480 cells.4 After up-regulated the Twist gene it can be improve the vito invasion of the SW480 cell .It is show that Twist gene can improve the vito invasion of the SW480 cells.
|
PDF Full Text Request