| ObjectiveThe treatment of tumors is a worldwide problem, the fight against cancer recurrence and metastasis after surgery is important and difficult of today's medical technology research.CD151is the member of the tetraspanin family to be associated as a promoter of tumor metastasis of malignant cells. Tetraspanins form membrane complexes with integrins. Here we constructed CD151-AAA mutant to assess the roles of Rac, Cdc42and P-Rac/Cdc42, and the effects of CD151-integrin complexes on proliferation, migration and invasion of HepG2cells. pAAV-CD151and CD151-AAA mutant were constructed and used to transiently transfect HepG2mediated with Qiagen Attractene Transfection Reagent. After transfection, the expression of CD151was measured by Western blotting. Cell proliferation assay was evaluated using the Cell Counting Kit-8(CCK-8) method, Cell migration assay was assessed by cell-wounding healing assay and cell invasion assay was evaluated in Microchemotaxis Chambers using FBS as the chemotactic stimulus. The potential involvement of various signaling path-ways was explored using relevant antibodies. The association between CD151and integrins was evaluated by immunoblotting analysis. We found that CD151promotes cell proliferation migration and chemotaxis. In addition, CD151increases phospho-Rac/cdc42activity. Further, CD151-AAA mutant decreased cellular proliferation, migration, and invasion. Moreover, CD151-AAA mutant abrogated the association between CD151and integrins. These data suggest that CD151form complexes by interacting with integrins, particularly α3β1and α6β1, to exert its function on HepG2cells. The mechanism is possibly related to the Rac, Cdc42and P-Rac/Cdc42signalling pathway. Methods1. extract plasmid pAAV-CD151, pAAV-CD151-AAA194-196,pAAV-GFP.2. Using Attractene Transfection Reagent liposomal to transfect HepG2cells, divided into four groups, pAAV-CD151group, pAAV-CD151-AAA194-196group, pAAV-GFP group and blank control group.Extracting each groups cell protein to detect the expression of CD151by Western blot,the proliferation of the cells was observed by CCK-8assay, Cell migration assay was assessed by cell-wounding healing assay and cell invasion assay was evaluated in Transwell plate (pore size8μm). observe the expression of integrin α3,α6and (31by co-immunoprecipitation assay, detect the expression of Cdc42,Rac1/2/3and P-Rac/Cdc42by Western blot.Results1. Successfully extract pAAV-CD151-AAA194"196mutant.2. The expression of CD151in the cells transfected with pAAV-CD151significantly increased as compared with the control and pAAV-GFP groups48h after transfection. In addition, pAAV-CD151-AAA transfection showed no significant difference of protein expression compared with CD151group.3. HepG2transfected with pAAV-CD151significantly enhanced HepG2proliferation as compared with the groups transfected with pAAV-GFP and the control. pAAV-CD151-AAA transfection significantly inhibited the proliferative effects as compared with the groups transfected with pAAV-GFP and the control group.4. In the wound healing assay cells transfected with pAAV-CD151significantly enhanced wound healing, The ability of pAAV-CD151-AAA mutant cells to wound healing was markedly delayed compared with that of pAAV-CD151transfectant cells.5. pAAV-CD151significantly promoted HepG2invasion compared with the control and pAAV-GFP groups. pAAV-CD151-AAA could not promote cell invasion.6. transfection with pAAV-CD151increased expression of phosphorylated phospho-Rac/cdc42compared with the control group and the GFP group, whereas transfection with pAAV-CD151-AAA had no change on the expression of phospho-Rac/cdc42.7. pAAV-CD151group could detect the expression of a3, a6and (31by Immunoblotting analysis,while pAAV-CD151-AAA group could not.Conclusion1. Successfully extract pAAV-CD151-AAA194-196mutant..2. Studies show that the pAAV-CD151promote HepG2cells proliferation, migration and invasion, while the function of mutant group is weakened, the mechanism may be through the increase the expression of P-Rac/Cdc42.3. PAAV-CD151promoting HepG2cells proliferation migration and invasion in vitro depends on the formation of CD151-integrin α3β1/α6β1complex.
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