The Effect On MCF-7 Cell Line In Response To Functional Inhibition Of UBE2C Through SiRNA.

Background: The tumorigenesis of breast cancer is a process in which involves numerous genes and is associated to sophisticated regulation of gene network.The process is closely related to the dysfunction of Ubiquitin-Proteasome Pathway(UPP).Increasing evidences reveal that protein degradation regulated by UPP plays an important role in tumorigenesis of breast cancer.With the further research on UPP,people were gradually attracted by one important component of UPP,which is UBE2 C.The abnormal expression of UBE2 C gene induces tumorigenesis in several ways such as terminates cell mitosis through a disordered mode,degenerates cell proliferation related protein aberrant,and so on.Hence targeting UBE2 C gene might be an new available strategy for the treatment of breast cancer.However,the precise molecular mechanism of UBE2 C involves in the tumorigenesis of breast cancer is still no clear.Objective: The aim of this study was to determine the effect on proliferation,apoptosis and invasive ability of human breast carcinoma cell line mcf-7 in response to functional inhibition of UBE2 C.Furthermore,to investigate the molecular mechanism of biological behavior above mentioned via small interfering RNA mediated UBE2 C gene and p53 gene silence,in order to provide theoretical basis for targeting UBE2 C gene to treat breast cancer.Methods: Chemically synthesized Si RNA-UBE2 C sequence targeting UBE2 C gene and Si RNA-P53 sequence targeting P53 gene.Transfected sequences into mcf-7 cells via lipofectin.24 hour after transfection,Real-Time PCR and Western blotting was performed to detect the expression of m RNA and protein of UBE2 C and P53.Untreated cells served as blank control group,and cells transfected with invalid sequence served as negative control group.Transfected MCF-7 cells with si RNA-UBE2 C and si RNA-UBE2 C combine with si RNAP53 respectively,untreated cells served as blank control group.Cell proliferation was measured by CCK8 assay,cell apoptosis was detected via flow cytometry,cell migration was determined through wound healing test.Results: 1.Real-Time PCR and Western blotting revealed that the expression of UBE2 C m RNA and protein in si RNA transfection group was significantly lower than that in blank control group and negative control group,while the expression of p53 m RNA was increased than control groups.2.Western blotting revealed that the expression of UBE2 C protein in si RNA transfection group was significantly lower than that in blank control group and negative control group,while the expression of p53 protein was increased than control groups.3.Western blotting indicated that the expression of P53 protein in si RNA-P53 transfection group was significantly lower than that in blank control group and negative control group,while the expression of UBE2 C protein showed no significance than control groups.4.CCK8 assay indicated that the cell proliferation rate in si RNA-UBE2 C transfected group was notable decreased than blank control group,while the proliferation rate in combined transfected group(si-UBE2C+si-P53)was higher than si RNA-UBE2 C transfected group,but still under blank control group.4 flow cytometry revealed that the cell apoptosis rate in si RNA-UBE2 C transfected group was notable increased than blank control group,while the rate in combined transfected group was under si RNA-UBE2 C transfected group,but still higher than blank control group.5.wound healing test suggested that compare with the blank control group,the migration ability of si RNA-UBE2 C transfected group was clearly weakened;the combined transfected group showed stronger ability of migration than that of si RNAUBE2 C transfected,but was still inferior to that of blank control group.Conclusions:1.Silent UBE2 C gene upregulated the expression of P53 gene in mcf-7 cell line,while silent P53 gene made no difference to the expression of UBE2 C gene.This reveals that UBE2 C gene might regulates the transcription and translation of P53 via certain pathway.2.Silent UBE2 C gene increased the apoptosis rate and decreased the ability of migration and proliferation of mcf-7 cell line.This reveals that UBE2 C gene might regulates the apoptosis,migration and proliferation via certain mechanism.3.The change of biological behavior mentioned above might be attributed to the regulation of P53 gene from UBE2 C.