The Growth Suppression After N-ras And Epiregulin Dual Knockdown In Human Hepatoma Cells

Hepatocellular carcinomas(HCC) is a major challenge because of the complicated molecular mechanisms of hepatocarcinogenesis and the involvement of multiple oncogenes and crucial signaling pathways,multiple-targeted therapy may be a new option for HCC treatment.Combined RNAi can be a therapy specific enough to allow the use of multiple RNAi targets at the same time,without the toxic effects often observed in chemotherapeutics.Based on our former study,N-ras gene was chosen to be a target for RNA interference treatment of HCC.We hypothesize that the up-regulated genes after knockdown of N-Ras may be involved in the compensatory mechanisms of cell growth.We examined whether dual knockdown of N-ras and the compensatory gene displayed a greater inhibitory effect of HepG2 cell growth.Part 1.The alteration of gene expression profile in HepG2 cells after transient and stable transfection of N-ras-siRNA1.The RNA and protein levels of N-ras after both transient and stable transfection of N-ras-siRNA were detected by RT-PCR and Western blot.The expressions of N-Ras in both mRNA and protein levels were significantly decreased by transient and stable transfection of N-Ras-siRNA,N-Ras proteins were significantly decreased up to 60% in both experiments.2.Microarry analysis of differentially expressed genes after transient and stable transfection of N-Ras-siRNA.In microarray analysis,the increase and decrease of 1.5-fold signal intensity are regarded as a significant change in mRNA expression.In transient 24h transfected HepG2 cells,185 genes were up-regulated and 206 genes were down-regulated.After 48h transient transfection,291 genes were up-regulated and 248 genes were down-regulated.In stable transfected HepG2/N-ras(-) cells,482 genes were up-regulated and 524 genes were down- regulated.3.Gene function enrichment analysis of differentially expressed genes using DAVID Bioinformatics Resources database.There was a statistically significant overlap between differentially expressed genes after stable transfection of N-ras-siRNA and the Gene Ontology categories "cell motility","actin filament-based process" and "acin cytoskeleton organization and biogenesis".The Gene Ontology categories go with differentially expressed genes after transient transfection of N-ras-siRNA(48h) are "regulation of epithelial cell proliferation","regulation of cell proliferation" and "cell adhesion".The Gene Ontology categories go with differentially expressed genes after transient transfection of N-Ras-siRNA(24h) are "cell cycle","regulation of progression through cell cycle" and "regulation of cell cycle".4.Pathway analysis of differentially expressed genes using Pathway Miner.The altered pathways identified in stable and transient transfected(48h) HepG2 cells were focal adhesion,MAPK signaling pathway,regulation of actin cytoskeleton and regulation of actin cytoskeleton,the altered pathways identified in transient transfected (24h) HepG2 cells were focal adhesion,cell cycle,MAPK signaling pathway and wnt signaling pathway.5.Hierarchical cluster analysis of genes exhibiting significantly modulated expression after both transient and stable transfection of N-ras-siRNA.The gene function enrichment analysis showed that the Gene Ontology go with Clusterâ… -â…£are "Organ morphogenesis","Catalytic activity","Inflammatory response" and "Secretory pathway".6.RT-PCR validation of microarray results.The genes chosn for RT-PCR validation are HDGFRP3,GPNMB,VRK1,MKI67,TM4SF3,EREG and ACTG2 after N-ras stable knockdown,ACTC and FNI after N-ras transient knockdown(48h),EREG after N-ras transient knockdown(24h).The relative mRNA levels of these genes are consistent between RT-PCR and microarray experiments.7.The morphology change of HepG2 cells after N-ras-siRNA treatment is observed. Part 2.The effect of dual knockdown of N-ras and other genes on HepG2 cell growth.We picked ACTG2,EREG,R-ras and TM4SF1 for this part of the research.Among them,combined silence of N-ras and epiregulin achieved greater effect on suppressing HepG2 cell growth.SRB assay showed that the survival rates of HepG2 cells treated with Mock-siRNA,N-ras-siRNA,EREG-siRNA1 and N-ras-siRNA combined with EREG-siRNA1 were 78%,49%,41%and 15%,respectively.Part 3.The molecular mechasim of the growth suppression after dual knockdown of N-ras and epiregulin.1.Colony formation assay was used to examine the synergistic effect of combined inhibition.Compared with untreated cells,the rates of colony formation in HepG2 cells after treated with mock-siRNA(100nM),N-Ras-siRNA(100nM),EREG-siRNA1 (100nM),N-Ras-siRNA and EREG-siRNA1(50nM each),N-Ras-siRNA and EREG-siRNA1(100nM each) are 81%,55%,58%,46%and 14%respectively.The results were consistent with the SRB assay in Part 2.2.Flow cytometric analysis revealed that the treatment of HepG2 cells with N-ras-siRNA resulted in a 14.7%increase of cells at G0/G1 phase compared with control cells.Under same conditions,cells at G0/G1 phase were enhanced up to 11.9% by the treatment of EREG-siRNA1 compared to untreated cells.Moreover,dual knockdown of N-ras and epiregulin exhibited a 20.4%increase of cells at G0/G 1 phase, suggesting that dual knockdown of N-ras and epiregulin resulted in G0/G1 arrest and that the inhibition of cell growth was associated with the disturbation of cell cycle progression.3.Western blot analysis showed that N-ras-siRNA and EREG-siRNA1 significantly decreased phosphorylations of ERK1/2 and Akt with unchanged expressions of ERK1/2 and Akt proteins.The dual knockdown of N-ras and epiregulin had enhanced reductions of ERK1/2 and Akt phosphorylations.The similar effect was observed on the expression of c-myc downstream of ERK1/2.4.Western blot analysis showed that N-ras-siRNA and EREG-siRNA1 significantly decreased phosphorylations of Rb and the expression of cyclin D1.The dual knockdown of N-ras and EREG had enhanced reductions of p-Rb and cyclin D1. N-ras-siRNA and EREG-siRNA1 significantly increased the expression of p21.The dual knockdown of N-ras and epiregulin had the enhanced effect.In summary,we developed an approach that uses gene expression profile analysis to identify targets for combinatorial treatment.We demonstrate that inhibition of N-Ras and epiregulin achieves synergistic effect in suppressing hepatoma HepG2 cells growth. Our findings imply that molecular multi-targeted therapy of oncogenes can be an effective treatment for HCC.