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Effect of NOR1 Gene on Level of E-selectin in HepG2 Cells

Author: LiYueJin
Tutor: LiDengQing
School: Central South University
Course: Clinical Laboratory Science
Keywords: NOR1 gene FQ-PCR hepatocarcinoma E-selectin cDNA chip enzyme-linked immunosorbent assay
CLC: R735.7
Type: Master's thesis
Year: 2008
Downloads: 22
Quote: 0
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Abstract


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.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Liver tumors
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