HepaCAM Inhibits Renal Cancer786-O Cell Proliferation And Migration Via Blocking PKCε Translocation From Cytoplasm To Plasma Membrane

Part I Protein expression of HepaCAM and PKCε in renal clearcell cancer tissuesObjective：To investigate the protein expression of HepaCAM andPKCε in renal clear cell cancer tissues.Method：The HepaCAM and PKCε protein in thirty-six human renalclear cancer tissues and adjacent non-cancerous tissues were determined byimmunohistochemistry. Inverted microscope was used to capture images,and the IPP6.0software was used to analyze mean density.Results：HepaCAM expression decreased significantly while PKCεincreased in renal clear cell carcinoma; HepaCAM expression increasedwhile PKCε decreased in adjacent non-cancerous tissues. There is a inverserelationship in PKCε and HepaCAM. And PKCε was related to tumorgrade and stage. The differences were statistic significant (p<0.05).Conclusion： The PKCε and HepaCAM protein were verified intissues and analysized their relationship. Part II HepaCAM re-expression affected PKCε located in cytoplasm orcell membraneObjective：To detect PKCε protein expression and location in786-Ocell line after infected with Ad-GFP-HepaCAM.Methods： HepaCAM, PKCε membrane protein, PKCε cytoplasmprotein, PKCε total protein were determined by western blot after infectedwith Ad-GFP-HepaCAM. PKCε location was further detected byimmunocytofluorescent.Results：After HepaCAM expressed successfully in experiment group,PKCε membrane protein level decreased, PKCε cytoplasm proteinincreased, while total protein not changed. Immunocytofluorescentuncovered PKCε cytoplasm protein increased in experiment group whencompare with blank and Ad-GFP group (p＜0.05).Conclusion: HepaCAM expression affected PKCε distribution betweencytoplasm and cell membrane, but did not affected total protein expression. PartⅢ The effect of PKCε specific translocation inhibitor εV1-2on786-O cell proliferation and migrationObjective：To verify the effect of εV1-2on786-O cell proliferation and migration ability; comparing the inhibition of clone formation and cellmigration ability between HepaCAM and εV1-2.Methods： To make clear the best concentration and time point ofPKCε specific translocation inhibitor εV1-2on786-O cell by CCK-8assay.Western blot was used to demonstrate the protein expression of MMP-9,cyclinD1and p-AKT. Colony formation assay and wound healing assaywere used to illuminate the effect of HepaCAM and εV1-2on proliferationand migration ability.Results：The best concentration and time point of εV1-2on786-O cellwere10μmol/L and24h. MMP-9, cyclinD1and p-AKT decreased afterHepaCAM re-expression and exposed to εV1-2by western blot. Colonyformation assay and wound healing assay uncovered that HepaCAMre-expression have a greater ability of inhibiting colony formation and cellmigration than exposed to εV1-2. And the joint use of them have a soundeffect (p<0.05).Conclusion: Both of exogenous expression of HepaCAM and exposedto εV1-2can inhibited key molecules expression of cell proliferation andmigration. HepaCAM expression can effectively inhibit the cell quantity ofclone and cell migration when compare to εV1-2group. HepaCAM mayact as an upstream molecule to modulate786-O cell proliferation andmigration.