Inhibition Of Hepatocellular Carcinoma Cells By Knockdown NET-1 Gene And Potential Mechanism

ObjectiveThe purpose of this study was to demonstrate the effects of sh RNA(short hairpin RNA)interference against NET-1(tetraspanin1)on hepatocellular carcinoma(HCC)cells and the possible mechanism.Methods1.For screening experiment HCC cell lines,Real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blots(WB)were used to detect mRNA and protein expression of NET-1 in different HCC cells(MHCC97H,MHCC97 L,HepG2 and SMMC7721).2.NET-1 RNAi library was constructed for screening the most effective siRNA against NET-1 and NET-1 shRNA was following constructed for transfection into experiment HCC cells.Subsequently we detected influence of NET-1 shRNA on proliferation and apoptosis of HCC cells using MTT assay,flow cytometry,Hoechst staining and Annexin V/PI double labeling.3.We obtained differently expressed genes by microarray assay with the HCC cells treated with NET-1 shRNA and explored biological function and related signaling pathways about the differently expressed genes through GO and KEGG analysis.To explore the possible mechanism that wheather NET-1 shRNA inhibits HCC cells through activating P53,our team detected the expression and interactions of NET-1,MDM4,CHEK2 and P53 in HCC cells using RT-qPCR,WB,Co-IP and IF assays respectively and constructed the overexpression vector of NET-1,MDM4 and P53 which were transfected into HEK293 T cells for conforming the interactions of these proteins.Results1.The mRNA and protein expression of NET-1 in different HCC cells(MHCC97H,MHCC97 L,HepG2 and SMMC7721)were significantly higher than that in normal liver cells(LO2)(P<0.05).In HCC cells,MHCC97 H and MHCC97 L were chosen as experiment cell lines that which contains much more NET-1 than HepG2 and SMMC7721(P<0.05).2.143 NET-1 siRNA locations were obtained using RNAi libriary and the best one was gotten by RT-qPCR for constructing NET-1 shRNA.3.NET-1 protein expressed in cell cytoplasm and cell membrane in the HCC cells.Compared with shRNA NC group in HCC cells,NET-1 sh RNA could knockdown the mRNA and protein expression of NET-1,blocked cells during G1 phase with decreasing proliferative ability and induced apoptosis rate increasing(P<0.05).4.Affymetrix gene microarray screened different expressed genes which showed 2-folds changes in HCC cells with NET-1 shRNA treated.There were 7393 different expressed genes with 3389 up-regulated and 4004 down-regulated in MHCC97 H while 115 in MHCC97 L with 55 up-regulated and 60 down-regulated.5.GO and KEGG analysis showed that:(1)In MHCC97 H,the remarkably gene biological function concludes: signal transduction,cell adhesion,apoptosis,cell cycle,negative regulation of cell proliferation and angiogenesis;and the significantly signaling pathway concludes: Metabolic pathways,Pathways in cancer,p53 signaling pathway,MAPK signaling pathway,TGF-beta signaling pathway,VEGF signaling pathway and mTOR signaling pathway.(2)In MHCC97 L,the remarkably gene biological function concludes: DNA-dependent regulation of transcription,cell proliferation,regulation of growth,positive regulation of protein phosphorylation,positive regulation of protein import into nucleus translocation,negative regulation of cell cycle arrest;and the significantly signaling pathway concludes: Pathways in cancer,Adherens junction,Jak-STAT signaling pathway,Notch signaling pathway and RIG-I-like receptor signaling pathway.6.The different expressed genes were verified at mRNA and protein level using RT-qPCR,WB and IF.Compared with shRNA NC group,the mRNA and protein of CHEK2 were increased in MHCC97 H cells with NET-1 shRNA treated.Although the relationship between NET-1 and CHEK2 was still unclear,CHEK2 and P53 located in nucleus and interacted with each other in MHCC97 H cells.The mRNA and protein of MDM4 were decreased in MHCC97 L cells that treated by NET-1 shRNA.MDM4 expressed in cytoplasm and nucleus while located in cytoplasm with NET-1 at the same time.The interactions of NET-1/MDM4,MDM4/P53 were detected by Co-IP in both MHCC97 L and HEK293 T cells.Conclusions1.The most effective siRNA sequences of target gene were obtained through constructing RNAi library.2.NET-1 shRNA designed and constructed in this study showed a great inhibition of HCC cells that it could effectively down-regulated NET-1,inhibit cell proliferation and induce apoptosis.3.NET-1 shRNA resulted in related genes changing remarkably and the mainly biological function and signaling pathways of different expressed genes related to cell proliferation and cell apoptosis.4.There were differences of potential mechanism that NET-1 shRNA inhibiting effects in HCC cells with different metastatic ability.NET-1 sh RNA finally activated P53 through increased CHEK2 in high metastatic HCC cells and decreased MDM4 in low metastatic HCC cells.5.NET-1 is an effective target gene in interfering HCC.This study provides theoretical and experimental basis that NET-1 sh RNA could be used for further development on clinical treatment.