The Expression And Regulation Mechanism Of ECRG4 In Human Breast Cancer And Its Tumor-suppressing Function

ObjectivesEsophageal cancer related gene 4 (ECRG4) is a new gene that separated from human esophageal epithelial cells, and it was first found because of its role in esophageal squamous carcinoma. Its official name is C2ORF40, locating on chromosome 2ql2.2 and encoding 148 amnio acid. It was reported that ECRG4 was down-regulated or absent in certain tumors, including esophageal cancer, colorectal carcinoma, prostate cancer, T-cell leukemia, gastric cancer and glioma. And overexpression of ECRG4 results in suppression of cancer cell growth and inhibition of cancer cell migration and invasion in certain tumors. So, ECRG4 was described as a novel tumor suppressor gene in several tumors, like esophageal squamous cell carcinoma and glioma. But the exact function of ECRG4 is still unclear. Recently, it was reported that ECRG4 was associated with the promoter hypermethylation in esophageal squamous cell carcinoma, colorectal carcinoma and glioma. They think the down-regulation of ECRG4 was probably due to its promoter hypermethylation. However, the mechanism for reduced expression of ECRG4 and its functional role in breast cancers were rare reported. In this study, we first evaluated the expression of ECRG4. Then, we examined that if ECRG4 was epigenetically inactivated through promoter hypermethylation in human breast cancer cell lines and primary breast cancer tissues. Further, we investigated the anti-tumor function of ECRG4 in cell proliferation, apoptosis and migration breast cancer cells. Expecting to find new diagnostic and prognostic biomarkers and new molecular therapy targets.Methods1. The mRNA expression of ECRG4 in 17 cases of primary breast cancer tissues were evaluated with semi-quantitative reverse transcription polymerase chain reation (RT-PCR) and quantitative-polymerase chain reaction (Q-PCR).2. The ECRG4 promoter methylation status in 17 cases of primary breast cancer tissues were detected by Bisulfite sequencing PCR (BSP).3. The ECRG4 promoter methylation status and mRNA expression before and after treated with 5-Aza-CdR were evaluated with bisulfite sequencing PCR and quantitative-polymerase chain reaction (Q-PCR) in SK-BR-3 and MCF-7 breast cancer cell lines.4. We constructed ECRG4-pcDNA3.1 expression plasmid and transfected SK-BR-3 and MCF-7 cells to observe its protein expression level by Western Blotting.5. SK-BR-3 and MCF-7 cells were transfected with ECRG4-pcDNA3.1 expressing plasmid to observe the changes on cellular proliferation by MTT assay.6. SK-BR-3 and MCF-7 cells were transfected with ECRG4-pcDNA3.1 expressing plasmid to observe the changes on apoptosis by flow cytometry.7. The erasion trace test was performed to observe the migratory ability of MCF-7 cells.Results1. RT-PCR results showed that ECRG4 mRNA expression in breast cancer tissues were lower than the adjacent breast tissues (P< 0.05) in 15 cases (15/17,88.2%).2. RT-qPCR results showed that ECRG4 mRNA expression in breast cancer tissues were lower than the adjacent breast tissues (P< 0.01) in 15 cases (16/17,94.1%).3. ECRG4 promoter was hypermethylated in breast cancer tissues.4. After 5-Aza-CdR treatment for 72h, ECRG4 promoter methylation levels were decreased in SK-BR-3 and MCF-7 breast cancer cell lines. But, ECRG4 mRNA expression were recovered and up regulated and had a 5-Aza-CdR concentration dose dependent.5. Overexpressing ECRG4 can inhibit the proliferation and migration of SK-BR-3 and MCF-7 breast cancer cell lines.6. Overexpressing ECRG4 can inhibit the migration of MCF-7 breast cancer cell lines.7. Overexpressing ECRG4 can promote apoptosis of SK-BR-3 and MCF-7 breast cancer cell lines.Conclusion1. The ECRG4 mRNA expression is reduced in breast cancer.2. ECRG4 promoter is hypermethylated in breast cancer, and the down-regulation of ECRG4 is due to promoter hypermethylation.3. Overexpressing ECRG4 can inhibit the proliferation, but promote the apoptosis of SK-BR-3 and MCF-7 cell lines.4. Overexpressing ECRG4 can inhibit the migration of MCF-7 breast cancer cell lines.