| Objective:By observing the expression level of Ras homology proto-oncogene C and myosin light chain 2,which are the target molecules of Rho-associated coiledcoil-forming protein kinase signaling pathway, to expore the quality of adrenomedullin and the signaling pathway inhibitor RKI-1447 for the mechanisms of the growth and the migration ability of ovarian cancer CAOV-3 cell.Methods: 1.As the experimental group,CAOV-3 cells were cultured in vitro and treated by different concentrations of RKI-1447(0,1,10,20,30,40,50μmol/L) in different time period(12h,24 h,48h).The inhibition effection of cell proliferation was detected by CCK8 method and count the half inhibition concentration.The cell morphology was observed under light microscope.Cell apoptosis was detected by FCM. 2. The migration of CAOV-3 was examined by wound healing test and transwells test.The expression levels of Rho C m RNA was examined by RT-PCR. The expression levels of Rho C and phospho-MLC-2 protein were examined by Western blotting. 3. Using transwell migration assay and wound-healing test to detecte the inhibitors RKI-1447 by influencing the Rho C m RNA expression and protein Rho C and phospho-MLC-2 protein to hinder the influence of AM in CAOV-3 cell.Results: 1. The half inhibition concentration of CAOV-3 cell was 20 μmol/L of RKI-1447 with a dose-dependent manner, because of the direction of RKI-1447(the concentration of RKI-1447≤0.30%), so we had to choose 1μmol/L and 10μmol/L. With this concentration and working time, apoptosis of CAOV-3 cells was induced(P<0.05). 2.RKI-1447 can inhibit the migration of CAOV-3 from wound healing test and transwell test, while it can reduce the expression of Rho C and phospho-MLC-2 protein, as well as the expression of Rho C m RNA.3. We investigated AM promote CAOV-3 cell migration by transwell migration assay and wound-healing test, while the western blot and real-time PCR were used to detect Rho C m RNA and its protein production, as well as phospho-MLC-2 protein.The results showed exogenous AM could promote the proliferation of CAOV-3 cell, raise the level of Rho C m RNA, increase the protein expression of Rho C and phospho-MLC-2 protein. However, all of these effects were inhibited by RKI-1447. Conclusion: RKI-1447 may inhibite of the key molecular Rho C and tphospho-MLC-2 protein, thus antagonism the influence of AM to the migration of CAOV-3 cell. |
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