| Objective: Esophageal cancer is a common malignancy tumor, ranked eighth with 6.5/10,0000 for incidence and sixth with 5.7/10,0000 for mortality in the world. It was a serious threat to human health. The pathogenesis of esophageal cancer were not only closely related to genetic factors, but also linked with some environmental factors, such as smoking and drinking, esophageal local damage, nitrosamines foods, vitamins and micro-nutrient deficiencies and the infections of fungal and viral, but its exact pathogen has not been acknowledged. With the development of molecular biology and high-tech biotechnology, it has become a focus of medical research to explore the molecular mechanisms of the development of esophageal cancer and related biological markers.ZIP5 is the one of zinc transport proteins SLC39A(ZIP) family namely solute binding transporter protein family, there are 19 genes including ZIP5 expressed abnormally in the global gene expression profiles of esophageal cancer, which suggested that ZIP5 may act an important role in the development of esophageal cancer, but the specific mechanism has not been reported. This paper would explore the role and mechanism of ZIP5 acting on esophageal cancer development by comparing the expression of ZIP5 in esophageal carcinoma, para-carcinoma and normal tissues, as well as the correlation with COX-2. Besides, we would also to study the mechanism of ZIP5 affecting in the esophageal cancer cell proliferation and invasion at the cellular level on the basis of being established esophageal cancer cell lines KYSE170 with ZIP5 gene silencing, which would provide experimental and theoretical basis for the prevention and targeted therapy of esophageal cancer.Method:1 We collected 58 cases of esophageal carcinoma, para-carcinoma and normal tissues from patients with esophageal cancer surgery, Immunohistochemistry was used to compare the different expression of ZIP5 in esophageal carcinoma, para-carcinoma and normal tissues, as well as relationships with clinical pathology and the correlation with COX-2.2 Cultured esophageal cancer cell lines KYSE170 K with ZIP5 gene silencing and KYSE170 S transfected with empty vector. The growth curve of KYSE170 K and KYSE170 S were drafted to detect the cell viability and proliferation by MTT and CCK-8.The ability of cell migration and invasion were detected by Transwell. PCR and Western Blot were used to determine the m RNA and protein expression of genes COX-2、Cyclin D1 E-cadherin and so on,which were closely related with the development of esophageal cancer, to explore the molecular mechanism of ZIP5 affecting in esophageal cancer.Results:1 Immunohistochemical result showed that, there are 53 cases of positive ZIP5 gene expression and 36 cases of positive cox-2 gene expression in 58 cases of carcinoma; there are 30 cases of positive ZIP5 gene expression and 28 cases of positive cox-2 gene expression in 58 cases of para-carcinoma; there are 6 cases of positive ZIP5 gene expression and 1 cases of positive cox-2 gene expression in 58 cases of normal tissues,The score of ZIP5 were 4.64±3.67,1.14 ±1.29 and 0.19±0.66,meanwhile, 2.09±2.23,1.14±1.57 and 0.02±0.13 for the COX-2 in the esophageal carcinoma, para-carcinoma and normal tissues, respectively. The expression of ZIP5 was positively correlated with lymph node metastasis,depth of invasion and the degree of differentiation. The expression of ZIP5 and COX-2 were positively correlated.2 MTT results showed that: after 12 h, 24 h, 48 h and 72 h, the OD value of KYSE170 S transfected with empty vector were 0.206±0.034, 0.417±0.076, 0.64±0.045 and 1.130±0.053; For KYSE170 K with ZIP5 silencing, the OD values were 0.120±0.052,0.185± 0.048,0.291±0.060 and 0.512±0.071, ZIP5 silencing group reproduced significantly slowly than in empty vector group(P <0.05).3 CCK-8 results showed that: after 12 h, 24 h, 48 h and 72 h, the OD value of KYSE170 S transfected with empty vector were 0.477 ±0.085, 1.043±0.092,1.143±0.115 and 1.343±0.087; For KYSE170 K with ZIP5 silencing, the OD values were 0.239± 0.051,0.492±0.083,0.581±0.054 and 0.777±0.059, ZIP5 silencing group reproduced significantly slowly than in empty vector group(P <0.05).4 Transwell results showed that: after 24 h, the number of cells passed through membrane without matrigel chamber was about 3500±206.23,for empty vector group and 1500±127.68 for ZIP5 silencing group. The number was about 1200±106.49 for empty vector group and 500±78.74 for ZIP5 silencing group when the membrane was added matrigel chamber. The ability of cell migration and invasion decreased 57.2% and 58.4% after silencing ZIP5.5 Real-time PCR results showed that: The relative m RNA expression of COX-2, Cyclin D1 and E-cadherin in the empty vector KYSE170 S were 0.892±0.075, 0.703±0.034 and 0.365±0.036; which were 0.314±0.044, 0.229±0.042 and 0.732±0.051 in ZIP5 silencing group KYSE170 K, the expression of COX-2 and Cyclin D1 decreased by 64.8% and 67.4%,while E-cadherin increased by 50.2% at the m RNA level after ZIP5 silencing, and the difference was statistically significant(P <0.05).6 Western Blot results showed: The relative protein expression of COX-2, Cyclin D1 and E-cadherin in the empty vector KYSE170 S were 0.654±0.036, 0.873±0.075 and 0.256±0.064,which were 0.284±0.051, 0.31±0.044 and 0.437±0.082 in ZIP5 silencing group KYSE170 K. The expression of COX-2 and Cyclin D1 decreased by 56% and 63%,while E-cadherin increased by 41.5% at the protein level after ZIP5 silencing, and the difference was statistically significant(P <0.05).Conclusions:1 ZIP5 express highly in human esophageal cancer tissues and lowly in para-carcinoma and normal tissues. The expression of ZIP5 are positively correlated with lymph node metastasis, depth of invasion and the degree of differentiation.2 The expression of ZIP5 and COX-2 were positively correlated.3 ZIP5 silencing could inhibit the proliferation and invasion of esophageal cancer cell KYSE170.4 ZIP5 silencing could down-regulate the expression of COX-2 and Cyclin D1, but up-regulate E-cadherin at the level of m RNA and protein. |
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