转染CDK2-shRNA对黑色素瘤B16-F1细胞达巴卡嗪敏感性的影响及其机制

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

目的探讨转染周期素依赖性蛋白激酶2短发夹RNA(CDK2-shRNA)对黑色素瘤B16-F1细胞达巴卡嗪敏感性的影响及其机制。方法将黑色素瘤B16-F1细胞分为观察组、空质粒组和对照组,观察组转染重组慢病毒pUL-CDK2-shRNA质粒,空质粒组转染重组慢病毒pUL-NC-shRNA质粒,对照组不转染。转染12 h后用250μmol/L的达巴卡嗪孵育三组细胞72 h。采用MTT法检测三组细胞吸光度值,采用集落形成实验检测三组细胞集落形成率(CFR),采用Annexin V-FITC/PI双染法检测三组细胞凋亡率,采用Western blotting法检测三组细胞Bcl-2、Bax、Caspase-3蛋白相对表达量,计算Bax/Bcl-2。结果观察组、空质粒组和对照组细胞吸光度值分别为0.221±0.019、0.684±0.049、0.699±0.051。观察组细胞吸光度值与空质粒组和对照组相比,P均<0.05。观察组、空质粒组和对照组细胞CFR分别为33.00%±1.80%、67.50%±7.26%、69.83%±5.25%。观察组细胞CFR与空质粒组和对照组相比,P均<0.05。观察组、空质粒组和对照组细胞凋亡率分别为43.6%±4.1%、17.6%±3.8%、15.1%±4.0%。观察组细胞凋亡率与空质粒组和对照组相比,P均<0.05。观察组、空质粒组和对照组细胞Bax/Bcl-2分别为2.56±0.28、0.84±0.04、0.86±0.11,Caspase-3相对表达量分别为0.92±0.07、0.69±0.08、0.57±0.05。观察组细胞Bax/Bcl-2、Caspase-3相对表达量与空质粒组和对照组相比,P均<0.05。结论转染CDK2-shRNA可以提高黑色素瘤B16-F1细胞对达巴卡嗪的敏感性。其机制是转染CDK2-shRNA可以提高达巴卡嗪共培养的黑色素瘤B16-F1细胞Bax/Bcl-2和Caspase-3表达水平,促进其凋亡。

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.