The Interaction Between Pinin And CtBPs And Functional Role Of Pinin In Ovarian Cancer

Objective:Ovarian cancer is the leading cause of mortality from gynecological malignancy despite advancements in novel therapeutics.Pinin is a multifunctional protein with dual localization at desmosome-intermidiate filament complex and in the mucleus.The study aimed to investigate the interaction between Pinin and CtBP(C-terminal binding protein),which is a transcriptional corepressor,and the functional role of Pinin in ovarian cancer.Methods:Immunohistochemistry was performed to show the expression of pinin in different ovarian tissues.Western blot was done to investigate over-expression of pinin and CtBPs in different ovarian tissues and ovarian cancer cell lines.By shRNA,different gene-knowckdown ovarian cancer cell lines(shControl,shPinin-1,shPinin-2,shPinin-3,shCtBP1,and shCtBP2)were established.Co-immunoprecipitation experiment,western blot and immunofluorescence experiment were performed to describe that pinin interacted with CtBP2.By means of MTT,the relative cell growth,survival after confluence and reaction of different Pinin-knockdown ovarian cancer cell lines to Taxol were determined.And soft agar experiment was done to investigate the transformation capacity of ovarian cancer cells with Pinin-knockdown.Results:The result of immunohistochemistry showed that pinin is highly expressed in borderline and invasive tumors compared with healthy and benign ovarian tissues.In terms of histology,mucinous tumors showed a reduction of expression compared with other histological subtypes.Western blot showed over-expression of pinin in different ovarian cancer cell lines.The result was similar to CtBP2.For the co-immunoprecipitation,CtBP2 antibody was used to pull down CtBP2 complex.In the western blot,pinin antibody was used to detect any pinin in the CtBP2 complex.The result showed that pinin was pulled down by the CtBP2 antibody(in the CtBP2 lane),but not in the control antibody lane.Immunofluorescence experiment found Pinin in green(dyed by Alexa Fluor 546),co-localized with CtBP2 in red(dyed by Alexa Fluor 647)in the nuclei of the cancer cells.Western blot showed the reduction of Pinin expression in both CtBPl and CtBP2 knockdown cancer cell lines and the reduction of CtBP proteins in the Pinin-knockdown cancer cell lines.Comparing the growth rate of the Pinin-knockdown cell lines with control cell line by means of MTT described Pinin-knockdown cell lines grew at least as fast as the control cancer cell line.After the cells reach confluence,there was a significant cell death for the Pinin-knockdown cell lines when compared with control cell line.We also performed soft agar to measure clonogenecity of the cancer cell lines.Three Pinin-knockdown ovarian cancer cell lines showed significant reduction of colonies compared with the control cell line:Adhesion assay by growing cells on different extracellular matrices was performed.The result demonstrated the loss of adhesion of the pinin-knockdown cancer cells to different extracellular matrices,compared with the control cells.The reaction of pinin-knockdown cancer cells to Taxol increased.Conclusions:Pinin is highly expressed in ovarian tumors.Pinin and CtBPs could affect each other's expression in the ovarian cancer cells and Pinin knockdown reduces the expression of CtBP proteins.Pinin seems not to regulate cell proliferation,but may affect cell survival.Pinin also regulates cell adhesion and transformation capacity.