Construction Of Tumor Suppressor OVCA1 Mutants And The Preliminary Research On Their Degradation Mechanism

Objective:Breast cancer and ovarian cancer are common female malignant tumors,their pathogenesises are closely related to the mutation and abnormities of a variety of tumor suppressor genes and oncogenes.Tumor suppressor gene OVCA1(ovarian cancer gene1)discovered in 1996 is an breast cancer,ovarian cancer related genes.Early study found ubiquitin-proteasome pathway is one of the wild OVCA1 protein degradation mechanism.And a study showed that OVCA1 gene Ala34 mutates into Asp34,Ser389 mutates into Arg389.To provide exprimental basis for revealing the degradation pathway of OVCA1 mutants 34M/389 M preliminarily and the relation between OVCA1 mutants 34M/389 M and tumors,In this paper we mutate the wild OVCA1 34 and 389 loci point,and explore the influence of MG132 to their expression.Methods:1.Based on the OVCA1 recombinant plasmid for eukaryotic expression prepared and saved in our laboratory,recombinant PCR technology was used to construct the mutative OVCA1(OVCA1-34 M,OVCA1-389M)plasmids for eukaryotic expression.2.MTT assay was used to determine the MG132 maximum concentration that don’t affect the activity of breast cancer cell MCF-7.3.Liposome method was used to transfect wild OVCA1 and mutative OVCA1(OVCA1-34 M,OVCA1-389M)plasmids for eukaryotic expression into HEK293 and MCF-7 cells,and set untransfected cells and transfected p EGFP-Vector cells as transfection control groups.24 hours after transfection put in MG132 effecting 24 hours,.and set the groups without MG132 treatment as control groups.After photograph under fluorescent microscopy,Western blot were used to detect the protein expression of exogenous wild and mutative OVCA1.Results:1.It was confirmed that we acquired OVCA1 mutants(OVCA1-34 M,OVCA1-389M)after sequencing.2.When the concentration of MG132 is 0μM,5μM,10μM and 15μM,the activity of MCF-7 cell does not have a significant impact(p>0.05).When the concentration of MG132 is 20μM,40μM,60μM,80μM and 100μM,the activity of MCF-7 cell has a significant impact(p<0.05).So the MG132 maximum concentration that don’t affect the activity of breast cancer cell MCF-7 is 15μM.3.In the HEK293 cell,Respectively for 389 M and 34 M OVCA1,the protein expression of OVCA1 in the groups without MG132 treatment are 0.066± 0.013、0.576±0.028;the protein expression of OVCA1 in the groups with MG132 treatment are1.006±0.064、1.170±0.132.The protein expression of OVCA1 in the groups with MG132 treatment was significantly higher than that in the groups without MG132treatment(p<0.05).In the MCF-7 cell,Respectively for 389 M and 34 M OVCA1,the protein expression of OVCA1 in the groups without MG132 treatment are 0.044±0.008、0.357±0.043;the protein expression of OVCA1 in the groups with MG132 treatment are 0.712±0.029、0.732±0.041.The protein expression of OVCA1 in the groups with MG132 treatment was significantly higher than that in the groups without MG132 treatment(p<0.05).Conclusion:1.Construct OVCA1 mutants(OVCA1-34 M,OVCA1-389M)plasmids for eukaryotic expression successfully.2.MG132 inhibits the protein degradation of OVCA1 mutants 34 M and 389 M.And it states that Ubiquitin-proteasome system may be involved in the protein degradation of OVCA1 mutants 34 M and 389 M.