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<p>Abstract</p> <p>Background</p> <p>The esophageal carcinoma related gene 4 (ECRG4) was initially identified and cloned from human normal esophageal epithelium in our laboratory (GenBank accession no.<ext-link ext-link-id="AF325503" ext-link-type="gen">AF325503</ext-link>). ECRG4 has been described as a novel tumor suppressor gene associated with prognosis in esophageal squamous cell carcinoma (ESCC).</p> <p>Methods</p> <p>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. <ext-link ext-link-id="AF 071882" ext-link-type="gen">AF 071882</ext-link>). Then, p21 protein expression, cell cycle and cell proliferation regulations were examined after ECRG4 and ECRG1 co-transfection in ESCC cells.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>ECRG4 interacts directly with ECRG1 to upregulate p21 protein expression, induce cell cycle G1 phase block and inhibit cancer cells proliferation in ESCC.</p>