| BackgroundOvarian cancer is one of the most common gynecological malignancies,and the incidence is rapidly increasing in our country.Ovarian cancer is difficult to treat and characterized by poor prognosis and high mortality.The metastasis and invasion of advanced ovarian cancer are main causes for its high mortality.Epithelialmesenchymal transition(EMT)is a process in which epithelial cells gradually lose the characteristics of epithelial cells and acquire the characteristics of mesenchymal cells due to external stimulation and internal factors.Studies have shown that EMT is the initial step of cancer metastasis and invasion,and is an important characteristic event of cell biology that often occurs in the process of invasion.EMT has shown important values in the studies of occurrence,development,diagnosis,and treatment of cancers.Studies have shown that EMT plays an important role in the metastasis and invasion of ovarian cancer.After EMT,the epithelial properties of epithelial ovarian cancer(EOC)cells are weakened,and many cells acquire the mesenchymal properties.Single or multiple clumps of cells detached from the primary tumors flow freely with the peritoneal fluid,and then they adhere to the surfaces of other tissues;thus,they metastasize to distant sites and invade deeply into other tissues,forming local or large-scale metastases.Research and development of drugs targeting EMT has become a hot area in development of anti-cancer drugs.Recent studies have shown that proteins of PKC family play an important role in the occurrence and progression of cancers,and are potential targets for anti-cancer drug development.PKCε,encoded by the PRKCE gene,is a member of the novel PKCs.PKCε has been demonstrated to be highly expressed in various cancers,including lung cancer,breast cancer,prostate cancer,and lymphomas,and associated with poor prognosis of these cancers.These studies mainly focus on the roles of PKCε in promoting tumorigenesis and proliferation of cancer cells.So far,sporadic studies have shown that PKCε may be associated with EMT.It may activate some intracellular signaling pathways involved in EMT,and may participate in the EMT process of cancers.Given the significance of EMT in migration,invasion and drug resistance of ovarian cancer,we decided to study the roles of PKCε in epithelial ovarian cancer,especially its role and molecular mechanisms in EMT of ovarian cancer.Hopefully this study can provide novel,reliable,and specific therapeutic targets for ovarian cancer and novel strategies for ovarian cancer treatment.Methods(1)Data analysis platforms of bioinformatics were used to search and analyze the expression of PRKCE gene in ovarian cancer tissues,and the clinical correlation between its expression and clinicopathological characteristics of ovarian cancer patients was analyzed.(2)The cancer tissue and adjacent normal tissue sections of local ovarian cancer patients were collected,and the protein levels of PKCε were detected by immunohistochemical(IHC)experiments.The IHC H-scores and clinicopathological information of ovarian cancer patients were statistically analyzed to find out the correlation between the protein levels of PKCε and local ovarian cancer patients.(3)The protein levels of PKCεin different ovarian cancer cell lines SKOV3,ES-2,3AO,and A2780 were detected by western blot(WB).(4)Appropriate cell lines with different PRKCE expression were selected to be knocked down by si RNAs,overexpressed with lentivirus infections,or inhibited by the specific peptide inhibitor.The effects of PRKCE expression on cellular functions of ovarian cancer,such as cell proliferation(via MTT),and cell EMT phenotypes(via cell wound healing,transwell migration and invasion experiments),were investigated.(5)An EMT biomarker library was constructed for screening.Knockdown or overexpression of PRKCE were perfoemed in different ovarian cancer cells,and screening was performed by q RT-PCR experiments.The EMT biomarkers regulated by PRKCE were screened out.The validation of EMT biomarkers with significant changes was also carried out via WB experiments.(6)Cell adhesion assays were used to investigate the regulation of PRKCE on adhesion of ovarian cancer cells.(7)Regulation of downstream signaling pathways of PRKCE on EMT of ovarian cancer was studied by WB,sh RNA lentivirus infection,transwell migration and invasion,and other assays.(8)Mouse orthotopic tumor models of ovarian cancer cells were constructed to investigate the effects of PRKCE expression on ovarian cancer tumorigenesis and metastasis.(9)Regulation of PRKCE expression on the signaling pathways in the cancer tissues of the mouse lung metastasis models and the orthotopic tumor models of ovarian cancer cells were verified via IHC experiments.(10)Expression of core molecules of downstream signaling pathways of PRKCE abd the correlation with ovarian cancer were evaluated through the bioinformatical data analysis.(11)Expression of core molecules of downstream signaling pathways of PRKCE was detected by IHC experiments;IHC H-scores and clinicopathological information of ovarian cancer patients were analyzed to find out the correlation between their expression levels and clinicopathological characteristics of local ovarian cancer patients.Results(1)Expression of PRKCE gene in internationally recognized databases such as TCGA and GEPIA is significantly higher in epithelial ovarian cancer tissues than normal tissues.Expression of PRKCE gene in epithelial ovarian cancer tissues was associated with the prognosis of these patients as shown by the results of the Kaplan-Meier data analysis and it was negatively correlated with overall survival(OS)and progressionfree survival(PFS).(2)PKCε protein is highly expressed in local ovarian cancer patients,and its levels are closely related to the stages and differentiation status of cancer tissues.But its levels are not significantly correlated with ages or histological subtypes of cancer tissues.(3)PKCε protein is abnormally and highly expressed in multiple ovarian cancer cell lines when compared to normal ovarian epithelial cells,.(4)Overexpression of PRKCE in A2780 and 3AO cells significantly promoted the EMT phenotypes of ovarian cancer cells,such as migration and invasion,and had no significant effects on cell proliferation.And knockdown of PRKCE in SKOV3 and ES-2 cells significantly reduced the EMT phenotypes of these cells,inhibiting migration and invasion of ovarian cancer cells,and have no significant effects on cell proliferation.PKCε peptide inhibitors also significantly reduced the EMT phenotypes of ovarian cancer cells SKOV3 and ES-2 by inhibiting their migratory and invasive abilities.(5)A library of EMT biomarkers of 16 genes closely related to the EMT process of cancer cells for screening was constructed based on the published literatures.m RNA expression of EMT biomarker molecules SNAIL1,E-cadherin and Vimentin in ovarian cancer cells was demonstrated to be regulated by knockdown or overexpression of PRKCE gene in SKOV3,ES-2,A2780,and 3AO ovarian cancer cells via screening with q RT-PCR experiments.Protein levels of EMT biomarkers SNAIL1,E-cadherin,and Vimentin were also demonstrated to be regulated by PRKCE expression in ovarian cancer cells by WB experiments.(6)Knockdown of PRKCE in SKOV3 and ES-2 cells significantly inhibited cell adhesion.Overexpression of PRKCE gene in A2780 and 3AO cells significantly enhanced cell adhesion.(7)Protein levels of Integrin β1 and ILK were regulated by PRKCE in ovarian cancer cells as demonstrated by WB results.Integrin β1 is expressed in multiple ovarian cancer cell lines.Knockdown of Integrin β1 gene significantly suppressed EMT phenotypes of ovarian cancer cells SKOV3 and ES-2.These results suggest that PRKCE regulate the EMT process of ovarian cancer cells through the Integrin β1-ILK-SNAIL1 pathway.(8)Overexpression of PRKCE gene promotes metastasis of ovarian cancer cells of orthotopic mouse models.And the protein levels of Integrin β1,SNAIL1,Vimentin,and E-cadherin were regulated by overexpression of PRKCE in tumor tissues of orthotopic mouse models.In addition,the protein levels of Integrin β1,SNAIL1,Vimentin,and E-cadherin were consistantly regulated by knockdown of PRKCE in lung metastasis models.(9)Expression of Integrin β1 gene was positively correlated with expression of PRKCE and ILK in the TCGA database of ovarian cancers.And its expression was correlated with the prognosis of ovarian cancer patients.The overall survival and progression-free survival are lower in tissues with higher expression of Integrin β1gene.Protein levels of Integrin β1 were higher in local ovarian cancer tissues than in normal ovarian epithelial tissues.And its levels are closely correlated with the stages and differentiation staus of local ovarian cancer patients,but its levels are not significantly correlated with ages or histological subtypes of cancer tissues.ConclusionPRKCE gene is significantly correlated with epithelial ovarian cancer clinically,and its expression is closely related to the prognosis of patients with epithelial ovarian cancers.PRKCE gene can regulate multiple EMT phenotypes and cell adhesion of various epithelial ovarian cancer cell lines.PRKCE gene regulates the EMT process of ovarian cancer through the Integrin β1-ILK-SNAIL1 pathway,and finally implemented by E-cadherin and Vimentin.Our findings may provide new,reliable and specific therapeutic targets for ovarian cancer,and provide novel,reliable,and specific therapeutic targets for ovarian cancer and novel strategies for ovarian cancer treatment. |
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