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. 2011 Feb 3:11:52.
doi: 10.1186/1471-2407-11-52.

A novel tumor suppressor gene ECRG4 interacts directly with TMPRSS11A (ECRG1) to inhibit cancer cell growth in esophageal carcinoma

Lin-wei Li  1 , Yuan-yuan Li, Xiao-yan Li, Chun-peng Zhang, Yun Zhou, Shih-Hsin Lu
Affiliations

A novel tumor suppressor gene ECRG4 interacts directly with TMPRSS11A (ECRG1) to inhibit cancer cell growth in esophageal carcinoma

Lin-wei Li et al. BMC Cancer. .

Abstract

Background: The esophageal carcinoma related gene 4 (ECRG4) was initially identified and cloned from human normal esophageal epithelium in our laboratory (GenBank accession no.AF325503). ECRG4 has been described as a novel tumor suppressor gene associated with prognosis in esophageal squamous cell carcinoma (ESCC).

Methods: In this study, binding affinity assay in vitro and co-immunoprecipitation experiment in vivo were utilized to verify the physical interaction between ECRG4 and transmembrane protease, serine 11A (TMPRSS11A, also known as ECRG1, GenBank accession no. AF 071882). Then, p21 protein expression, cell cycle and cell proliferation regulations were examined after ECRG4 and ECRG1 co-transfection in ESCC cells.

Results: We revealed for the first time that ECRG4 interacted directly with ECRG1 to inhibit cancer cell proliferation and induce cell cycle G1 phase block in ESCC. Binding affinity and co-immunoprecipitation assays demonstrated that ECRG4 interacted directly with ECRG1 in ESCC cells. Furthermore, the ECRG4 and ECRG1 co-expression remarkably upregulatd p21 protein level by Western blot (P < 0.001), induced cell cycle G1 phase block by flow cytometric analysis (P < 0.001) and suppressed cell proliferation by MTT and BrdU assay (both P < 0.01) in ESCC cells.

Conclusions: ECRG4 interacts directly with ECRG1 to upregulate p21 protein expression, induce cell cycle G1 phase block and inhibit cancer cells proliferation in ESCC.

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Figures

Figure 1
Figure 1
The interaction between ECRG4 and ECRG1 was examined by binding affinity assay in vitro. FLAG-ECRG1 from EC9706/pcDNA3.1-FLAG-ECRG1 cells was bound to recombinant His-ECRG4 protein which had been pre-coated on the plate. The absorbance values of the wells (column 6) were significantly higher than those of controls (P < 0.001). And no detectable binding of Miz-1, an interaction partner of ECRG1, to His-ECRG4 was observed. *, P < 0.001, compared with EC9706/pcDNA3.1-FLAG cells.
Figure 2
Figure 2
The interaction between ECRG4 and ECRG1 was verified by co-immunoprecipitation assay in vivo. (A) Detection of ECRG4 and ECRG1 protein expression in transfected cells (pcDNA3.1, pcDNA3.1-His-ECRG4, pcDNA3.1-FLAG-ECRG1 and pcDNA3.1-His-ECRG4+FLAG-ECRG1) by Western blot. FLAG-ECRG1 or His-ECRG4 protein was detectable in EC9706/pcDNA3.1-FLAG-ECRG1 or EC9706/pcDNA3.1-His-ECRG4 cells, respectively, and both in EC9706/pcDNA3.1-His-ECRG4+pcDNA3.1-FLAG-ECRG1 cells. (B) EC9706 cells, transiently transfected with FLAG-ECRG1 and His-ECRG4, were immunoprecipitated by anti-FLAG antibody and detected by anti-His antibody. As negative control, pcDNA3.1-FLAG empty vector replaced FLAG-ECRG1. Protein lysates of EC9706/FLAG-ECRG1+His-ECRG4 (Lane 1) and EC9706/FLAG+ His-ECRG4 (Lane 2) were immunoprecipitated by anti-FLAG antibody and visualized by anti-His antibody. The rabbit IgG antibody was used as negative control, and it showed no non-specific binding of ECRG1 with IgG. (C) EC9706 cells, transiently transfected with FLAG-ECRG1 and His-ECRG4, were immunoprecipitated by anti-His antibody and detected by anti-FLAG antibody. As negative control, pcDNA3.1-His empty vector replaced His-ECRG4. Protein lysates of EC9706/FLAG-ECRG1+His-ECRG4 (Lane 1) and EC9706/FLAG-ECRG1+His (Lane 2) were immunoprecipitated by anti-His antibody and visualized by anti-FLAG antibody. The rabbit IgG antibody was used as negative control, and it showed no non-specific binding of ECRG4 with IgG.
Figure 3
Figure 3
Effect of ECRG4 and ECRG1 co-expression on p21 protein level. Representative photo (left) and statistic plot (right) of relative protein expression ratios in EC9706/pcDNA3.1, EC9706/pcDNA3.1-ECRG1, EC9706/pcDNA3.1-ECRG4 and EC9706/pcDNA3.1-ECRG4+ECRG1 cells. Analysis of cell's total proteins by Western blot showed that p21 protein expression was significantly increased in EC9706/pcDNA3.1-ECRG4+ECRG1 cells compared with in EC9706/pcDNA3.1 cells (P < 0.001). *, P < 0.001, compared with EC9706/pcDNA3.1 cells.
Figure 4
Figure 4
Effect of ECRG4 and ECRG1 co-expression on cell proliferation. EC9706/pcDNA3.1-ECRG4+ECRG1 cells proliferated significantly more slowly than EC9706/pcDNA3.1 cells as determined by the BrdU assay (P < 0.01). *, P < 0.01, compared with EC9706/pcDNA3.1 cells. Error bars represent standard deviation from mean value.
Figure 5
Figure 5
Effect of ECRG4 and ECRG1 co-expression on cell growth curve. Cell growth curves demonstrated that EC9706/pcDNA3.1-ECRG4+ECRG1 grew significantly more slowly than EC9706/pcDNA3.1 cells by MTT assay (P < 0.01). *, P < 0.01, compared with EC9706/pcDNA3.1 cells. Error bars represent standard deviation from mean value.
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