Effect of transfection of CDK2-shRNA on chemotherapy sensitivity of melanoma B16-F1 cells to dacarbazine

JIN Jialu; ZHU Renshu; XIE Yuyuan; LIU Hongchun

doi:10.3969/j.issn.1002-266X.2017.23.003

Abstract：Objective To explore the effects of cyclin-dependent protein kinase 2-short hairpin RNA (CDK2-shRNA) on the chemotherapy sensitivity of B16-F1 cells to dacarbazine (DTIC) and its mechanism.Methods B16-F1 cells were divided into the observation group, empty plasmid group, and control group.The recombinant lentiviral vector pUL-CDK2-shRNA was transfected into the observation group, the recombinant plasmid pUL-NC-shRNA was transfected into the empty plasmid group, and the control group was not transfected with any plasmid.After 12 hours, every group was treated with 250 μmol/L dacarbazine for 72 h.MTT assay was used to detect the absorbance values in the three groups.Colony forming assay was used to detect colony forming rate (CFR).AnnexinV-FITC/PI double staining method was used to detect the apoptosis.Western blotting was used to detect the protein expression levels of Bcl-2, Bax, and Caspase-3 in each group, then we calculated Bax/Bcl-2 ratio.Results The absorbance values in the observation group, the empty plasmid group, and the control group were 0.221±0.019, 0.684±0.049, and 0.699±0.051, respectively.The CFR in the above three groups were 33.00%±1.80%, 67.50%±7.26%, and 69.83%±5.25% respectively.The apoptosis rates in the above three groups were 43.6%±4.1%, 17.6%±3.8%, and 15.1%±4.0%.The expression ratios of Bax/Bcl-2 in the above three groups were 2.56±0.28, 0.84±0.04, and 0.86±0.11, respectively.The relative expression of Caspase-3 was 0.92±0.07, 0.69±0.08, and 0.57±0.05, respectively.Significant differences were found in the absorbance value, CFR, apoptosis rate, and expression of Bax/Bcl-2 and Caspase-3 between the observation group and the empty plasmid group, the control group (all P<0.05).Conclusion The transfection of CDK2-shRNA can improve the chemotherapy sensitivity of melanoma B16-F1 cells to DTIC, and the mechanism may be that B16-F1 melanoma cells transfected with CDK2-shRNA in advance can increase the expression levels of Bax/Bcl-2 and Caspase-3, and then promote the apoptosis of melanoma B16-F1 cells.