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A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

Jia J, Dai S, Sun X, Sang Y, Xu Z, Zhang J, Cui X, Song J, Guo X - Mol Med Rep (2015)

Bottom Line: Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis.Flow cytometric analysis and Hoechst staining demonstrated that overexpression of ECRG4 significantly induced apoptosis.Western blot analysis confirmed that Bcl‑2‑associated X protein, cleaved‑caspase‑3 and cleaved‑poly (ADP‑ribose) polymerase were upregulated in the apoptotic process, whereas B‑cell lymphoma 2 was downregulated.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology‑Head and Neck Surgery, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China.

ABSTRACT
Human esophageal cancer‑related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep‑2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1‑ECRG4 was constructed and introduced into Hep‑2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high‑level expression of ECRG4. The 3‑(4, 5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 overexpression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpression of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl‑2‑associated X protein, cleaved‑caspase‑3 and cleaved‑poly (ADP‑ribose) polymerase were upregulated in the apoptotic process, whereas B‑cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.

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Establishment of a cell line stably overexpressing ECRG4. (A) Western blot analysis of ECRG4 protein expression. Grayscale analysis was conducted using β-actin as the internal control. (B) Reverse transcription-quantitative polymerase chain reaction analysis of ECRG4 mRNA expression. (C) Immunofluorescence examination of ECRG4 distribution. ECRG4 expression was visible under a fluorescence microscope (red). The nuclei were stained blue. Representative results of several replica experiments are shown in the figure (magnification, x600). Experimental data are expressed as the mean ± standard deviation. **P<0.01, compared with the pcDNA3.1 group. ECRG4, human esophageal cancer-related gene 4.
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f2-mmr-12-04-5058: Establishment of a cell line stably overexpressing ECRG4. (A) Western blot analysis of ECRG4 protein expression. Grayscale analysis was conducted using β-actin as the internal control. (B) Reverse transcription-quantitative polymerase chain reaction analysis of ECRG4 mRNA expression. (C) Immunofluorescence examination of ECRG4 distribution. ECRG4 expression was visible under a fluorescence microscope (red). The nuclei were stained blue. Representative results of several replica experiments are shown in the figure (magnification, x600). Experimental data are expressed as the mean ± standard deviation. **P<0.01, compared with the pcDNA3.1 group. ECRG4, human esophageal cancer-related gene 4.

Mentions: To investigate the function of the ECRG4 gene, pcDNA3.1-ECRG4 was transfected into Hep-2 cells, and ECRG4 expression in positive cells was analyzed by western blot analysis and RT-qPCR. Cells transfected with empty pcDNA3.1 vector and parental cells were used as controls. The results showed that the expression levels of ECRG4 protein and mRNA in the pcDNA3.1-ECRG4 group were increased by 3.05 (Fig. 2A, P<0.01) and 3.07-fold (Fig. 2B, P<0.01), respectively, compared with the pcDNA3.1 group. The immunofluorescence staining results showed that ECRG4 expression was obviously elevated in the pcDNA3.1-ECRG4 group compared with the other two groups (Fig. 2C), which were consistent with the western blot analysis and RT-qPCR results. Thus, a laryngeal cancer cell line stably overexpressing ECRG4 was established.


A preliminary study of the effect of ECRG4 overexpression on the proliferation and apoptosis of human laryngeal cancer cells and the underlying mechanisms.

Jia J, Dai S, Sun X, Sang Y, Xu Z, Zhang J, Cui X, Song J, Guo X - Mol Med Rep (2015)

Establishment of a cell line stably overexpressing ECRG4. (A) Western blot analysis of ECRG4 protein expression. Grayscale analysis was conducted using β-actin as the internal control. (B) Reverse transcription-quantitative polymerase chain reaction analysis of ECRG4 mRNA expression. (C) Immunofluorescence examination of ECRG4 distribution. ECRG4 expression was visible under a fluorescence microscope (red). The nuclei were stained blue. Representative results of several replica experiments are shown in the figure (magnification, x600). Experimental data are expressed as the mean ± standard deviation. **P<0.01, compared with the pcDNA3.1 group. ECRG4, human esophageal cancer-related gene 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581775&req=5

f2-mmr-12-04-5058: Establishment of a cell line stably overexpressing ECRG4. (A) Western blot analysis of ECRG4 protein expression. Grayscale analysis was conducted using β-actin as the internal control. (B) Reverse transcription-quantitative polymerase chain reaction analysis of ECRG4 mRNA expression. (C) Immunofluorescence examination of ECRG4 distribution. ECRG4 expression was visible under a fluorescence microscope (red). The nuclei were stained blue. Representative results of several replica experiments are shown in the figure (magnification, x600). Experimental data are expressed as the mean ± standard deviation. **P<0.01, compared with the pcDNA3.1 group. ECRG4, human esophageal cancer-related gene 4.
Mentions: To investigate the function of the ECRG4 gene, pcDNA3.1-ECRG4 was transfected into Hep-2 cells, and ECRG4 expression in positive cells was analyzed by western blot analysis and RT-qPCR. Cells transfected with empty pcDNA3.1 vector and parental cells were used as controls. The results showed that the expression levels of ECRG4 protein and mRNA in the pcDNA3.1-ECRG4 group were increased by 3.05 (Fig. 2A, P<0.01) and 3.07-fold (Fig. 2B, P<0.01), respectively, compared with the pcDNA3.1 group. The immunofluorescence staining results showed that ECRG4 expression was obviously elevated in the pcDNA3.1-ECRG4 group compared with the other two groups (Fig. 2C), which were consistent with the western blot analysis and RT-qPCR results. Thus, a laryngeal cancer cell line stably overexpressing ECRG4 was established.

Bottom Line: Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis.Flow cytometric analysis and Hoechst staining demonstrated that overexpression of ECRG4 significantly induced apoptosis.Western blot analysis confirmed that Bcl‑2‑associated X protein, cleaved‑caspase‑3 and cleaved‑poly (ADP‑ribose) polymerase were upregulated in the apoptotic process, whereas B‑cell lymphoma 2 was downregulated.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology‑Head and Neck Surgery, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China.

ABSTRACT
Human esophageal cancer‑related gene 4 (ECRG4) is a potential tumor suppressor gene isolated from human esophageal epithelial cells. Studies have shown that ECRG4 effectively inhibits the proliferation of tumor cells and induces apoptosis. However, the role of ECRG4 in laryngeal cancer has not yet been clearly defined. In this study, a human laryngeal cancer cell line stably overexpressing ECRG4 was established. The effect of ECRG4 on the proliferation and apoptosis of laryngeal cancer cells and the associated mechanisms were investigated. The Hep‑2 human laryngeal carcinoma cell line exhibited a low basal level of ECRG4 expression and was selected for the present study. The eukaryotic expression plasmid pcDNA3.1‑ECRG4 was constructed and introduced into Hep‑2 cells by transfection reagents. Western blot analysis, reverse transcription-quantitative polymerase chain reaction and immunofluorescence staining confirmed high‑level expression of ECRG4. The 3‑(4, 5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay and colony formation assay showed that ECRG4 overexpression suppressed the proliferative capacity of laryngeal cancer cells in vitro. Cell cycle analysis showed that ECRG4 induced cell cycle arrest at the G0/G1 phase. Flow cytometric analysis and Hoechst staining demonstrated that overexpression of ECRG4 significantly induced apoptosis. Western blot analysis confirmed that Bcl‑2‑associated X protein, cleaved‑caspase‑3 and cleaved‑poly (ADP‑ribose) polymerase were upregulated in the apoptotic process, whereas B‑cell lymphoma 2 was downregulated. In conclusion, overexpression of ECRG4 inhibited laryngeal cancer cell proliferation and induced cancer cell apoptosis. Therefore, ECRG4 exhibits potential as an effective target in gene therapy for laryngeal cancer.

Show MeSH
Related in: MedlinePlus