Role Of PRKCD In HCC Progression And Its Regulation Of Autophagy Research

Objective: Hepatocellular carcinoma(HCC)is an extremely harmful primary malignant tumor and an important cause of cancer-related death.HCC has a high morbidity and mortality rate and is recognized as one of the global health problems.At present,there is no cure treatment for HCC completely.Protein kinase Cδ(PRKCD)is one of the novel isoenzymes in the protein kinase C family,and expressed in most tissues and organs of the body,and acted as a crucial role in the pathogenesis of inflammation,tumor and other diseases.This study aims to explore the role of PRKCD in the development and progression of HCC and its possible mechanisms from three aspects of bioinformatics,cell and tissue.Methods: The RNA-seq data and clinical data related to HCC were downloaded from the TCGA database,including complete RNA-seq data from 374 HCC samples and 50para-carcinoma tissues.370 patients had RNA-seq and survival data,and 311 patients had RNA-seq and selected clinical features data.Single-factor T-test was used to compare the differential expression of PRKCD in HCC tissues and paracancer tissues,and qRT-PCR,western blotting and immunohistochemical staining were used to detect the differential expression of PRKCD in HCC tissues and paracancer tissues.qRT-PCR and western blotting were applied to detect the expression changes of PRKCD between hepatocellular carcinoma cells and normal cells.At the same time,the correlation between PRKCD and clinicopathological features was analyzed by multivariate variance.The survival and prognosis analysis of PRKCD in HCC was analyzed in R software package and the survival curve and ROC curve of PRKCD in HCC were drawn.western blotting and qRT-PCR were used to detect the silent expression of PRKCD in hepatoma Huh-7 cells.CCK-8 assay and plate clonal formation assay were used to detect the effect of Huh-7 on cell proliferation after silencing PRKCD expression in hepatoma cells.Cell scratch assay and Transwell invasion and migration assay were used to detect the effect of Huh-7 hepatoma cells silenced by PRKCD on cell invasion and migration.The effects of Huh-7 cells on apoptosis and cell cycle after silencing PRKCD were detected by flow cytometry and Western blotting.The effects of Huh-7 cells silencing PRKCD on epithelial-mesenchymal transformation and autophagy were detected by Western blotting.Pearson correlation analysis was used to study the correlation between PRKCD protein and autophagy associated proteins LC3 and p62.The effect of PRKCD on autophagy of HCC was verified by western blotting,immunohistochemical staining and.immunofluorescence assay.Results: PRKCD was highly expressed in HCC tissues and hepatoma cell lines(Huh-7,SMCC-7721,BEL-7404 and HLE)(P < 0.05),and was significantly correlated with clinicopathologic features of HCC(P < 0.05),and survival time of patients with high expression of PRKCD was significantly shortened(P < 0.05).ROC curve showed that the AUC of PRKCD in predicting 1-year,2-year and 3-year survival of HCC patients were 0.704,0.633 and 0.651,respectively.After silence of PRKCD,the proliferation,migration and invasion ability of hepatocellular carcinoma Huh-7 cells were significantly decreased(P < 0.05).After PRKCD silencing,the apoptosis rate of hepatoma Huh-7 cells was significantly increased,the cell cycle was stagnated in G1 and S phases,the expressions of apoptosis proteins Bax-1 and Caspase3 were significantly up-regulated,and the expression of Bcl-2 was decreased.The expression levels of CDK4,CDK6 and Cyclin D1 were significantly decreased(P < 0.05).After the silencing of PRKCD,the expression of N-cadherin,Slug and Vimentin,which are related to epithelial-mesenchymal transition,were decreased in Huh-7 cells,while E-cadherin was up-regulated.The expression levels of LC3 and p62 in HCC tissues were significantly up-regulated.PRKCD protein was positively correlated with LC3 B and p62 protein(r=0.21 and 0.23,P < 0.001).The expression levels of autophagy marker proteins LC3 A,LC3B and p62 were significantly down-regulated after PRKCD silencing(P<0.05).Western blotting assay,immunohistochemical assay and immunofluorescence assay confirmed that LC3 and p62 were up-regulated in HCC,and both of them were binding to PRKCD in the nucleus to affect the autophagy function of HCC.Conclusion:(1)PRKCD is highly expressed in both HCC tissues and cells,and is associated with poor prognosis and clinicopathological features of HCC patients(2)The silencing of PRKCD can inhibit the proliferation,migration and invasion of HCC cells,promote cell apoptosis and block cell cycle(3)The effect of PRKCD on the biological function of hepatocellular carcinoma cells may be related to autophagy(4)PRKCD has a crucial role in the pathogenesis of HCC and is expected to become a new tumor marker,providing theoretical support for clinical diagnosis,treatment and prognosis evaluation of HCC.