Study On The Inhibition Of Tyrosinase Activity With The SiRNA Technique In Huamn Melanoma Cells

Objects:Melanoma is a kind of malignant skin tumor which has often been seen. This highly malignant melanoma has a strong anti-effect to the normal radio-chemo-therapy and is very difficult to cure. With the development of modern molecular biology, the gene therapy of melanoma is becoming a promising therapy. The tyrosinase plays a key role in the biosynthesis of melanin, over-expression of the tyrosinase activity can lead to the color spot and melanoma. Therefore, the tyrosinase gene (TYR) was chosen as the target gene , and the siRNA expression cassette was constructed through the two-step PCR method to inhibit the tyrosinase activity and melanin content of the human melanoma cell strain A375 and the tumor on the nude mice model, which provides a new idea and method for the gene therapy of melanoma.Methods: The coding region of tyrosinase gene was chosen as the target to design three siRNA sequences, construct the TYR siRNA expression cassette by the two-step PCR method, and then experiments were carried out in vitro and in vivo, respectively. In vitro, transfect the TYR siRNA expression cassette into the human melanoma cell strain A375, since the expression cassette contains the green fluorescence protein(GFP) gene with the CMV promoter as its reporter gene, the transfection effect of the expression cassette can be detected by the fluorescence microscope, and then the cytoxicity, adhesion and migration effect of the TYR siRNA expression cassette to A375 cells can also be detected. In vivo, first construct the nude mice model with the tumor of human melanoma cell strain A375 on its back, transfect the TYR siRNA expression cassette into the tumor of the nude mice model, detect the transfection in vivo through the frozen histological section , and test the effects of siRNA expressioncassette to the tyrosinase activity and melanin content.Results: The three TYR siRNA expression cassettes and their negative controls have been successfully constructed. In vitro, siTYR-1 ,siTYR-3,siTYR-1,siTYR-1,2,3 and their negative controls can be effectively expressed in A375 cells, their transfection rate varies from 5% to 50%, which is sufficient for the further study; The cytoxicity result indicates that TYR siRNA expression cassettes and their negative controls all can inhibit the viability of A375 cells , and shows significant dependency between dose and inhibition rate ; Compared with the blank control, the migration and adhesion ability of the A375 cells that transfect the siTYR and their negative controls decrease significantly; The result of the tyrosinase activity and melanin content indicates that all the TYR siRNA expression cassettes can significantly inhibit the tyrosinase activity and its melanin content of A375 cells, and shows a significant dependency between dose and inhibition rate, the result of the comparision of TYR siRNA and its negative controls with the rank test shows that only siTYR-3 and its negative control has a significant difference (p<0.05), the other siTYR and its negative control has no significant difference (p>0.05). In vivo, construct the nude mice model with the tumor of human melanoma cell strain A375 on its back successfully, and transfect the TYR siRNA expression cassette into the tumor , the result indicates that compared with the blank control, in the groups that transfect siTYR-1 negative control, siTYR-3, siTYR-2 negative control, siTYR-3 negative control, siTYR-1,2,3, the volume and mass of their tumor decrease significantly, TYR siRNA can be expressed in tumor according to the result of fluorescence microscope; the result of the tyrosinase activity and melanin content tests shows that compared with the blank control, the tyrosinase activity and melanin content of all the groups that transfect the TYR siRNA have no significant difference.Conclusion: (1)Three TYR siRNA expression cassettes and their negative controls were constructed successfully. (2)In vitro, they can inhibit the tyrosinase activity and melanin content of human melanoma cell strain A375, among those, the tyrosinase activity and melanin content of the group transfected with siTYR-3 has significant difference with those transfected with siTYR-3 negative control, indicating that siTYR-3 may have more specific inhibition effect to TYR. (3)In vivo, none of the TYR siRNA has significant inhibition effect to the tyrosinase activity and melanin content of the tumor, which suggests that the gene therapy of the human melanoma by siRNA technique has a long way to go.