miR-205通过调控PKC蛋白影响黑色素瘤细胞的增殖和凋亡

MiR-205 affects the proliferation and apoptosis of melanoma cells by regulating PKC protein

目的探讨miR-205通过调控PKC蛋白影响黑色素瘤细胞的增殖和凋亡行为的作用和机制。方法实验分为miR-NC组(转染阴性对照慢病毒)、miR-205-mimic组(转染miR-205过表达慢病毒)、miR-205-mimic+PKC-siRNA组(同时转染miR-205过表达慢病毒和敲减PKCsiRNA)。qPCR试验检miR-205在不同黑色素瘤细胞中的表达水平,Western blotting检测PKC蛋白在不同黑色素瘤细胞(C8161,A375,WM115和B16)中的表达水平;双荧光素酶实验检测miR-205和PKC蛋白的相互关系;MTT细胞增殖实验检测miR-205和PKC对黑色素瘤细胞株增殖能力的影响;流式细胞术实验检测miR-205和PKC对黑色素瘤细胞株凋亡行为的影响。结果 miR-205在B16细胞株中表达水平相对细胞株C8161,A375,WM115较低,PKC蛋白在B16细胞中表达水平较低(P<0.05)。双荧光素酶实验证实miR-205可以直接靶向调控PKC蛋白的表达活性。miR-205-mimic组的B16细胞增殖速率明显低于miR-NC组,凋亡率明显高于miR-NC组(P<0.05);同时抑制PKC蛋白的表达后,miR-205-mimic+PKC-siRNA组B16细胞的增殖速率明显高于miR-205-mimic组,凋亡率明显低于miR-205-mimic组(P<0.05)。结论 miR-205可以通过调控PKC的表达影响黑色素瘤细胞的增殖和凋亡,同时PKC蛋白对miR-205有一定的反馈调节作用。

Objective To investigate the effect and mechanism of miR-205 on the proliferation and apoptosis of melanoma cells by regulating PKC protein. Methods The expression of miR-205 in different melanoma cells was detected by q PCR（ C8161,A375,WM115 and B16）. Western blotting was used to detect the expression of PKC protein in different melanoma cells. The relationship between miR-205 and PKC protein was detected by double luciferase assay. MTT cell proliferation assay was used to detect the effect of miR-205 and PKC on the proliferation of melanoma cell lines. Flow cytometry was used to detect the effect of miR-205 and PKC on the apoptosis of melanoma cell lines. Results The expression level of miR-205 in B16 cell line was relatively low,and the expression level of PKC protein in B16 cells was higher（ P〈0. 05）. Double luciferase assay confirmed that miR-205 could directly target the expression of PKC protein. The proliferation rate of B16 cells in miR-205-mimic group was significantly lower than that in miR-NC group,and the apoptosis rate was significantly higher than that in miR-NC group（ P〈0. 05）. After inhibiting the expression of PKC protein,miR-205-mimic ＋ PKC group the proliferation rate of B16 cells in siRNA group was significantly higher than that in miR-205-mimic group,and the apoptosis rate was significantly lower than that in miR-205-mimic group（ P〈0. 05）. Conclusion miR-205 can regulate the expression of PKC and affect the proliferation and apoptosis of melanoma cells,and the PKC protein has a certain feedback effect on miR-205.