Effect Of RNAi Targeting PKCε Expression On Glioma Cell Proliferation, Apoptosis, Invasion And Related Molecular Mechanism Research

Gliomas are highly invasive tumors associated with high levels of mortality incentral nervous system. Despite advances in the understanding of gliomas, the mediansurvival is only12to15months for patients with glioblastomas（WHO grade IV）.Hence, the treatment of gliomas remains a major neurosurgical challenge. PKCε, as amember of protein kinase C family, is an important intracellular signaling moleculeand modulates diverse cellular functions. It is reported that PKCε is overexpressed ina variety of tumors and has been considered as an oncogene. Stat3, as a member of thesignal transducer and activator of transcription family, is a point of convergence formultiple signaling pathways and plays an important role in regulating cell growth,survival and differentiation. Previous studies have shown that aberrant activation ofStat3has been detected in a wide variety of tumors, and Stat3plays a critical role indevelopment and progression of various tumors.We employed RNAi, immunochemistry staining, co-immunoprecipitation,Western blot, nude mouse xenograft model and so on to investigate the effect ofPKCε on development and progression of glioma, as well as the molecularinteraction mechanism of PKCε and Stat3in glioma.PKCε proteins are overexpressed in human malignant glioma tissues comparedwith human normal brain tissues by immunohistochemical staining, suggestingPKCε has important roles in development and progression of glioma. PKCε proteinsare expressed in human glioma cell lines U251and U87, as well as mainly appear inthe nuclear part of the two cells by immunofluorescence staining.To reveal the biological function of PKCε in glioma, a double-strandedoligonucleotide against PKCε was designed. Western blot analysis showed that theexpression of PKCε in human glioma cell lines U251and U87was significantlyreduced by PKCε RNAi plasmid. Cell proliferation assay showed that down-regulation of PKCε expression can inhibit proliferation in human glioma cellline U251or U87. Cell mitochondrial transmembrane potential assay showed thatdown-regulation of PKCε expression can reduce the mitochondrial transmembranepotential in human glioma cell line U251or U87. Cell apoptosis assay showed thatdown-regulation of PKCε expression can induce apoptosis in human glioma cell lineU251or U87. Cell invasion assay showed that down-regulation of PKCε expressioncan decrease invasion activity in human glioma cell line U251or U87. In addition,U87cells were successfully implanted into the nude mice and formed tumors in vivo.The data showed that intratumoral transfection of PKCε RNAi plasmid achieved byin vivo electroporation can inhibit growth of tumors implanted in nude mice.The molecular interaction mechanism of PKCε and Stat3was further explored.The co-localization of the endogenous PKCε with Stat3was detected in humanmalignant glioma tissues, human glioma cell lines U251and U87byimmunofluorescence staining. The interaction of the endogenous PKCε with Stat3was detected in human glioma cell lines U251and U87by co-immunoprecipitation.Western blot analysis showed that down-regulation of PKCε expression can suppressStat3Ser727phosphorylation and Bcl-xL expression but not total Stat3level,Stat3Tyr705phosphorylation and Bcl-2expression in human glioma cell line U251or U87.In summary, these results indicate that PKCε plays an important role indevelopment and progression of glioma, and PKCε-mediated cellular signaling mightbe partially through Stat3. PKCε is a promising molecular target for gene therapy ofgliomas, which offers new insight on the treatment of gliomas.