Effect Of NOR₁ Gene On Level Of E-selectin In HepG2 Cells

Objective:1 To establish the stable transfection of hepatoma cell line pcDNA3. 1(+)/NOR₁-HepG2.2 To study the effects of the E-selectin gene expression of human hepatocarcinoma HepG2 cells after transfection of NOR₁ gene with microarray assay,RT-PCR,FQ-PCR and ELISA.Methods:1 The mammal expression vector of NOR₁(pcDNA3.1(+)/NOR₁) and eukaryotic expressing plasmid pcDNA3.1(+)were introduced into human hepatocarcinoma cell line HepG2 mediated by lipofectamin transfection.The stable G418-resistant clones were isolated.After extracting total RNA,the overexpression of NOR₁ gene were detected by RT-PCR.2 The differential expression profiles genes from NOR₁-transfected cells(HepG2-NOR₁)and empty vector cells(HepG2-PC)were determined using the human 17K cDNA expression chip with 17101 cDNA probes.3 The expression of E-selectin genes were verified by RT-PCR,FQ-PCR.Simultaneously,the levels of E-selectin in cell culture medium were measured by ELISA.Results:1 Recombinant plasmid pcDNA3.1(+)/NOR₁ can express in HepG2 cells.Stable expression of NOR₁ gene in HepG2 cells was confirmed by RT-PCR.The lever of NOR₁ mRNA increased apparently in HepG2-NO-R₁ cells compared with HepG2-PC cells.2 From the scanning results of gene chips,162 genes were differentialiy expressed in the two cells lines,among which 59 genes(or EST)expression were up-regulated and 103 genes(or EST)expression were down-regulated in the HepG2-NOR1 cells.The genes were correlated with cell apoptosis or tumor,cell cycle,cell transcription regulator activity,cell signal transducer activity,cell translation regulator activity,and so on.Among them,E-selectin genes were up-regulated in the HepG2-NOR₁ cells.3 The expression of E-selectin gene in HepG2-NOR₁ cells increased to 3.756 compared with HepG2-PC cells by RT-PCR,and increased to 3.548 by FQ-PCR.4 The levels of E-selectin in cell culture medium of NOR₁ gene transfection group were higher than those in blank group and control group(P＜0.01).Conelusion:1 Over-expression of NOR₁ gene by transfecting NOR₁ can change the gene expression profiles in HepG2 cells,leading to change of cell growth,differentiation and apoptosis in HepG2-NOR₁ cells,cDNA chip is a useful method in understanding the mechanism of cancer with its high throughput characteristics.2 The DNA microarray data suggested E-selectin was the up-regulated gene(Cy3/Cy5＞2.0).The levels of E-selectin mRNA in NOR₁ gene transfection group were higher than those in blank group and control group(P＜0.01)through RT-PCR and FQ-PCR.3 The levels of E-selectin in cell culture medium of NOR₁ gene transfection group were higher than those in blank group and control group(P＜0.01).The study laid a solid foundation for the further research of mechanism of NOR₁ gene during the occurrence and progression of hepatocarcinoma.