RNAi Silencing Of The WT1 Gene Reverses Drug Resistance Of Malignant Melanoma

Objective: Malignant melanoma is the most aggressive form of skin cancer and its prognosis is poor due to the development of drug resistance. Thus,one of the main goals for melanoma treatment is to reverse chemo- resistance of tumor cells. Studies have shown that antiapoptosis is the primary mechanism of melanoma chemoresistance, and downregulation of antiapoptotic genes to promote apoptosis of cells can improve sensitivity of tumor cells to chemotherapeutic drugs. WT1 gene was originally isolated as a tumor-suppressor gene that was inactivated in a subset of Wilms' tumors. Rrecent years,WT1 is gaining increasing attention as a therapeutic target molecule due to its common expression in many human cancers. Studies show that WT1 is expressed in malignant melanoma in 80% of the tumor cells, but not in normal skin or benign melanocytic nevi,and silencing of WT1 gene can inhibit proliferation and promote apoptosis of cells.RNA interference is a efficient and specific gene silencing technology.In this study, we transfected WM451 cells with siRNA eukaryotic expression vector against WT1,to study the effect of downregulation of WT1 on the proliferation,apoptosis,and chemoresistance of melanoma cells.Methods:1.Construction of the eukaryotic expression vector of siRNA targeting to WT1 gene.2.WM451 cells was transfected with siRNA eukaryotic expression vector against WT1 by liposomes.3.The mRNA and protein expression of WT1 in WM451 cells was detected by the method of RT-PCR, Western-blot and immunocytochemistry to detect the effect of WT1 gene silencing. 4.Tumor cells growth curve was detected by MTT assay.5.Morphology of apoptotic cells is observed by electron microscope.6.Sensitivity of WM451 cells to chemotherapeutic drugs is detected by MTT assay.Results:1. The eukaryotic expression vector of siRNA targeting to WT1 gene was successfully constructed.2. WT1 was highly expressed in malignant melanoma cell line WM451 cells by RT-PCR;3.WM451 cells was transfected with siRNA eukaryotic expression vector against WT1 by liposomes,and the expression of WT1 were signify- cantly inhibited. The transfection rate was 73%.4. Silencing of WT1 by siRNA can inhibit cell proliferation and promote apoptosis.5. Apoptotic cells were seen with nuclear pyknosis and chromosome margination by electron microscope.6. IC50 of cisplatin in WM451 cells transfected with siRNA decrease from 9.37ug/ml to 4.13ug/ml,and the sensitivity of cells to cisplatin was improved.Conclusion:1.Sequence-specific siRNA targeting to WT1 can significantly inhibit WT1 gene expression.2.Downregulation of WT1 gene inhibits cell proliferation and promotes apoptosis of WM451 cells.3. RNAi silencing of theWT1 gene can improve sensitivity to cisplatin in WM451 cells,and RNA interference provides a novel therapeutic strategyfor treatment of malignant melanomas.