The Effects Of The Silence And Overexpression Of EPS-8 On The Biological Behaviors Of Human Melanoma A375 Cells

Backgrounds and ObjectivesMelanoma, one of the most common malignant cutaneous cancers, has an increasing morbidity and it harms the health of human. The malignant grade of melanoma is very high relatively and local invasion and distant metastasis usually appear early. So the early discovery, diagnosis and treatment are the key to treat melanoma. However, the mechanism of the occurrence and development of melanoma remains unclear what attenuates the treatment of melanoma. Therefore, the study concerning the effects of the genes and their proteins related to the occurrence and development of melanoma contributes to clarify the specific mechanisms of the occurrence and development of melanoma and provide theoretical basis for the diagnosis and treatment of melanoma.Epidermal growth factor receptor pathway substrate 8（EPS-8） is one of the substrates of epidermal growth factor receptor（EGFR） and could be phosphorylated by several tyrosinases and involves into EGFR signaling pathway. EPS8, which is located in 12q23-q24, expresses in the cytoplasm and cytomembrane of epithelial cells, leydig cells and hematopoietic cells. It has been shown that the expression of EPS-8 plays important roles in the proliferation, apoptosis and migration of several different kinds of cancer cells.However, the expression of EPS-8 in melanoma and roles of it plays in the occurrence and development of melanoma are unclear. In the first chapter of this study, we used tissue microarray and immunohistochemistry staining to analyze the expressions of EPS-8 in normal skin tissues and melanoma tissues. In the second chapter, we established silence and expression plasmid of EPS-8 and used RT-PCR,Western blot to detect their interference efficient. In the third chapter, we used CCK-8, Hochest33258, Western blot and Transwell to examine the relationships of the expression of EPS-8 with proliferation, apoptosis and migration of A375 cells.Methods1 Tissue microarray and immunohistochemistry staining were used to detect the expression of EPS-8 in 18 normal skin tissues, 62 melanoma tissues and 20 melanoma metastatic tissues and discussed the relationships between the expression of EPS-8 and the clinicopathologic data of melanoma patients.2 Four shRNA of EPS8（GenBank accession number:NM₀04447） were designed and synthesized and GV248 was chosen to establish EPS-8 sh RNA plasmid and control plasmid; GV144 was used to establish EPS-8 expression plasmid. RT-PCR and Western blot were used to detect the levels of EPS-8 m RNA and protein. The EPS-8 sh RNA plasmid which influenced the levels of EPS-8 m RNA and protein most obviously was chosen.3 CCK-8, colony formation assay, Hochest33258, Western blot and Transwell to examine the relationships of the expression of EPS-8 with proliferation, apoptosis and migration of A375 cells.Results1 In the 62 melanoma tissues, EPS-8 expressed positively in 38 tissues（61.2%）, and in the 20 melanoma metastatic tissues, EPS-8 expressed positively in 17 tissues（85.0%）, in the 18 normal skin tissues, EPS-8 expressed positively in 3 tissues（16.7%）. The results showed that the positive expressions of EPS-8 in melanoma tissues were higher than that in normal skin tissues（P < 0.05）. In addition, the positive expressions of EPS-8 in melanoma tissues were associated with the metastasis（P < 0.05）, but not with age, gender and TNM of patients（P > 0.05）.2 Four shRNA（EPS-8-SR1, EPS-8-SR2, EPS-8-SR3 and EPS-8-SR4） were established and after sequencing and identification, screening, EPS-8-SR3 was chosen. Meanwhile, EPS-8 expression plasmid was also established, and after sequencing and identification, they were transferred into A375 cells.3 The results of CCK-8 showed the proliferation rates of A375 group, A375-NC group, A375-RNAi group and A375-EPS-8 group were 65.31% ± 11.2%, 61.29% ± 9.8%, 28.74% ± 6.9% and 85.67% ± 8.7% respectively. The results of colony formation assay showed that the coliny numbers of A375 group, A375-NC group, A375-RNAi group and A375-EPS-8 group were 44 ± 4.5, 42 ± 3.0, 34 ± 2.7 and 58 ± 3.6. The results of Hochest33258 indicated that apoptotic rate of cells in A375 group, A375-NC group, A375-RNAi group and A375-EPS-8 group were 19.1% ± 2.9%, 18.8% ± 1.3%, 42.3% ± 3.5% and 11.27% ± 3.5%. The results of Western blottingshowed the expression of Bcl-2 in A375-EPS-8 group was enhanced. The results of Transwell showed that the number of cells passed though the membrance in A375 group, A375-NC group, A375-RNAi group and A375-EPS-8 group were 21.50 ± 3.62, 23.20 ± 4.13, 13.70 ± 2.85 and 39.80 ± 5.45.Conclusions1 EPS-8 overexpressed in melanoma tissues and the the positive expressions of EPS-8 in melanoma tissues were associated with the metastasis.2 The silence and overexpression of EPS-8 could influence the the levels of EPS-8 m RNA and protein.3 The overexpression of EPS-8 could promote the proliferation and invasion of A375 cells and inhibit the apoptosis of A375 cells. On the contray, the silence of EPS-8 could inhibit the proliferation and invasion of A375 cells and induce the apoptosis of A375 cells. EPS-8 was related to the proliferation, invasion and apotosis of A375 cells and it may refer to the development of tumor.