Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

Abstract

Aim: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.

Methods: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component.

Results: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.

Conclusion: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.

Figure 1

Different expression of EMP-1 detected in matched pairs of esophageal cancer tissue. The upper band represents EMP-1 (702 bp); the lower band represents GAPDH (452 bp). The expression of EMP-1 is higher in normal (N) tissue than its cancer(C) tissue. RT-PCR for GAPDH was used as equal loading control.

Figure 2

Different expression of EMP-1 cell clones transfected with pcDNA3.1-EMP-1-myc-his (-) C vector and the parental vector without cDNA insert pcDNA3.1myc-his (-) C cells as negative control. Clones 1, 2, 3, 4, 5, 6, 8 and 10 showed the increased expression of EMP-1. RT-PCR for GAPDH was used as equal loading control. B represents the parental vector as negative control.

Figure 3

Western blotting check the expression of EMP-1 after transfected into the EC9706 cell with anti-HIS monoclonal antibody. His peptide has been expressed successfully in host cell clones (C1, C2, C3, C5, C 6 and C8,). Control represents the negative control.

Figure 4

The growth curve of EC9706 cell line after transfected into EMP-1.The clones 1, 2, 3, 5, 6, 8 and 10 slow down obviously, and the clones 4, 7 and 9 are close to the vector control. Vector represents the negative control.

Figure 5

Flow cytometric analysis of cell cycle of negative con-trol (left) and cell clone 2 (right). The percentages of negative cells and the clone 2 cells in G1, G₂ and S phase are 52.2%, 6.2%, 41.7%; 64%, 5.8% and 30.2%, respectively

Figure 6

cDNA microarray analysis of parental negative con-trol expression (left) and the EMP-1 transfected cell clone 2 (Right). 35 genes show an over 2.0-fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated.

Figure 7

Verification of up-regulated and down-regulated gene expression in EC9706 induced by overexpression of EMP-1. Interleukin-6, plasminogen and P19INK4D increased their expression and COL11A1 decreased its expression. Pre represents the sample not transfected with the vector carried with the ORF of EMP-1, and post represents the sample trans-fected with EMP-1. RT-PCR for GAPDH was used as equal loading control.