The Effect of Antisense TRP-1 on Proliferation and Functions of Melanocytes and Malignant Melanoma Cells / 中华皮肤科杂志

ABSTRACT

Objective To construct the antisense eukaryotic vector of human TRP-1 (tyrosinase related protein 1) encoding gene, and transfect it into TRP-1 highly expressed melanocytes and malignant melanoma cell line, in order to further study the effects of antisense TRP-1 on the proliferation and functions of those cells. Methods TRP-1 cDNA was amplified by polymerase chain reaction (PCR) and the PCR products were subcloned into eukaryotic expression vector pcDNA3.1 on the opposite direction. Antisense recombinant vector was transfected into melanocytes and melanoma cell line. TRP-1 mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR). TRP-1 protein level was detected by Western blot. Cell cycle was determined by flow cytometry. The activity of tyrosinase was valued by L-dopa reaction. Results The recombinant antisense vector pcDNA3.1/TRP-1(-) was constructed. Positive transfected cells could steadily express TRP-1 antisense RNA. It was showed that there was a low level of TRP-1 mRNA as indicated by RT-PCR, and a low level of TRP-1 protein as indicated by Western blot. Cell cycles were blocked in G1 stage. The suppress rates of tyrosinase was 46% in transfected melanocytes and 54% in malignant melanoma cells, respectively. Conclusions TRP-1 plays an important role in the proliferation and functions of melanocytes and melanoma cells. Antisense TRP-1 could block the cell cycles and decrease the activity of tyrosinese in those cells.