| BackgroundEsophageal cancer is one of the most prevalent and deadliest malignancies. Worldwide, esophageal cancer is the sixth leading cause of death from cancer, and even the most frequent malignant tumor in several regions of China. Due to metastasis and invasion of surrounding tissues in early stage, the prognosis of esophageal cancer still remains poor. Numerous genes involve in the pathogenesis of esophageal cancer, but the concrete process remains unclear. Multiple genes and proteins with abnormal expression have been found in esophageal cancer. However, a few of them can be adopted as biomarkers with clinical value. The other biomarkers related to evaluating therapeutic efficacy and prognosis need further searching and confirming. As a zinc band protein related to transcription, the ZNRD1 (Zinc ribbon domain-containing 1) gene might play important role in the process of transcription through regulating the choice of transcriptional start site, as well as the extension and termination for synthesis of RNA. Overexpression of ZNRD1 can suppress the proliferation and oncogenicity of stomach cancer cells in vitro and in vivo, and restrain its transform phenotype, thus reversing the malignant proliferation potency of some stomach cancer cells. These fingings suggest that ZNRD1 gene transduction may be a useful new strategy of gene therapy for gastric cancer. However, there is still no report about the role of ZNRD1 in esophageal cancer cells, and the precise mechanism of apoptosis regulation by ZNRD1, as well as the clinical value of ZNRD1 in evaluating therapeutic efficacy and prognosis. In this study, we explored the differential expression of ZNRD1 in esophageal cancer tissues; to understanded the relation between the level of ZNRD1 expression and clinical pathological features, as well as the prognosis of patients underwent radical esophagectomy. In addition, we transfected the eukaryotic sence expression vector of ZNRD1 into EC109 cells, and also explored the function and possible mechanisms of ZNRD1 related biological behaviours, such as proliferation, apoptosis, metastasis, as well as DNA damage and repair. All these laid a solid basis for the role of ZNRD1 in esophageal cancer.Aims: To identify the differential expression of ZNRD1 in esophageal cancer tissues and investigate the relationship between the expression of ZNRD1 and the prognosis of patients with esophageal cancer. In addition, the correlation between ZNRD1 expression and the clinical pathological characteristics was also investigated. For exploring the function and possible mechanisms of ZNRD1 related biological behaviours, such as proliferation, apoptosis, metastasis, as well as DNA damage and repair, the eukaryotic sence expression vector of ZNRD1 was transfected into EC109 cells.Methods: (1) Semiquantitative RT-PCR assay and immunohistochemistry staining technique were used to identify the differential expression of ZNRD1 mRNA and protein in esophageal cancer, normal and adjacent tissues.(2) The relationship between ZNRD1 expression and prognosis of patients with esophageal cancer was investigated by immunohistochemistry staining technique and statistical analysis.(3) The Western blot, RT-PCR assay and immunocytochemistry staining were used to detect the ZNRD1 expression in EC109 cells.(4) The eukaryotic sence expression vector of ZNRD1 was transfected into EC109 cells by liposome transfection technique, and then Western blot, RT-PCR assay and immunocytochemistry staining were used to detect the expression of ZNRD1.(5) Growth curve and drug sensitivity assay were performed using MTT assay. IC50 values of EC109 cells to anticancer drugs were calculated.(6) Invasion activity of transfected EC109 cells was investigated by Transwell loculus assay.(7) Cell cycle analysis was performed using flow cytometry.(8) The apoptosis sensitivity of transfectant and control cells was detected by DNA fragmentation assay and flow cytometry.(9) Utilizing single cell gel electrophoresis (SCGE) assay to evaluate the DNA damage and repair induced by ultraviolet light radiation.(10) The cDNA microarray analysis was performed to identify the gene expression profile mediated by ZNRD1 overexpression.(11) RT-PCR and Western blots were performed to validate the differential expression of genes identified by cDNA microarray analysis.Results: (1) the ZNRD1 expression was confirmed to be down-regulated in esophageal cancer tissues compared to normal and adjacent nonneoplastic tissues. As immunohistochemistry discovered, the positive expressing rate of ZNRD1 in esophageal cancer (29.5%) was lower than in adjacent nonneoplastic (52.3%) and normal tissues (72.7%) (p < 0.05).(2) The mRNA and protein levels of ZNRD1 were both undetectable in EC109 cells, while significantly higher expression of ZNRD1 mRNA and protein (p < 0.01) was observed in EC109/ZNRD1 cells. The results showed that ZNRD1 gene was successfully transfected into and expressed in the EC109 cells.(3) MTT and colony formation assay showed that upregulation of ZNRD1 could suppress cell growth and proliferation of EC109 cells.(4) As Transwell loculus assay showed, the invasion activity of EC109 cells was significantly suppressed by the overexpression of ZNRD1.(5) As showed by flow cytometry, ZNRD1 upregulated esophageal cancer cells EC109/ZNRD1 exhibited G1 stage blockage and less percentage of cells in S stage.(6) MTT assay showed that transfectant cell was more resistant to 5-Fu, CDDP, VCR and ADR than control cells.(7) According to the results of flow cytometry and DNA fragmentation assay, the apoptosis sensitivity of EC109/ZNRD1 was lower than control cells.(8) ZNRD1-expressing cells exhibited a significant enhanced DNA repair capacity, suggesting that upregulation of ZNRD1 could inhibit the DNA damage induced by ultraviolet light radiation.(9) As the cDNA microarray analysis discovered, there were 61 genes with different expression levels following the upregulation of ZNRD1 in EC109 cells.(10) Western blot showed that the overexpression of ZNRD1 can effectively upregulate the mRNA level of ERCC1, P-gp, MDR1 and Bcl-2 in EC109/ZNRD1, as compared with the control cells.Conclusions: ZNRD1 expression was confirmed to be down-regulated in esophageal cancer tissues compared to normal and adjacent nonneoplastic tissues by RT-PCR and immunohistochemistry. The expression of ZNRD1 in esophageal cancer was positive correlated to the prognosis of patients. The decreased expression of ZNRD1 was correlated with a worse outcome of patients with esophageal cancer. The mRNA and protein levels of ZNRD1 were both undetectable in EC109 cells, while significantly higher expression of ZNRD1 mRNA and protein was observed in EC109/ZNRD1 cells, which suggested that ZNRD1 gene was successfully transfected into and expressed in the EC109 cells. Upregulation of the ZNRD1 could partly reverse the malignant phenotype of EC109 cells, resulting in the suppressed cell growth, G1 stage blockage and the inhibited invasion activity. ZNRD1 might regulate the cell cycle by altering the expression of CDKN1A (Cyclin-dependent kinase inhibitor 1A). Moreover, upregulation of ZNRD1 could enhance the resistance of EC109 cells to chemotherapeutics and suppress the apoptosis. ZNRD1-expressing EC109 cells exhibited a significant enhanced DNA repair capacity, and the overexpression of ZNRD1 could upregulate the expression of excision repair cross-complementing 1 (ERCC1) gene. Thus suggested ZNRD1 might play an important role in the process of DNA damage and repair by regulating the expression of ERCC1. In addition, the ZNRD1 gene might be involved in the cisplatin resistance of EC109 cells by regulating the expression of ERCC1 and Bcl-2.
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