沉默pim-3基因对黑色素瘤细胞B16的抑制作用

Inhibitory effect of target silencing Pim-3 gene on melanoma B16 cell line

目的:观察靶向沉默原癌基因pim-3对小鼠黑色素瘤细胞B16的抑制作用。方法:用脂质体将特异性靶向沉默pim-3载体(pim-3-shRNA)和pim-3过表达载体(pim-3-MIGR1)转染到黑色素瘤细胞B16中,荧光定量PCR和Western blotting法检测pim-3 mRNA和蛋白表达的变化,Annexin V/PI染色和TUNEL染色检测细胞凋亡情况,荧光定量PCR和Western blotting法检测pBad、Bcl-2、Bcl-xl、Bax、caspase-3等凋亡相关分子表达的变化,MTT法检测细胞增殖情况,流式细胞术检测细胞周期,RTCAxCELLigence系统检测细胞迁移情况,Transwell侵袭实验检测细胞侵袭的变化。结果:转染pim-3-sh RNA组B16细胞的pim-3mRNA及蛋白表达较对照组明显下降(P<0.05),转染pim-3过表达质粒组B16细胞的pim-3 mRNA及蛋白表达较对照组显著增加(P<0.05)。转染pim-3-shRNA后,B16细胞的凋亡明显增加,在此基础上共转pim-3过表达质粒后则明显逆转沉默pim-3所引起的凋亡(P<0.05);进一步检测发现沉默pim-3明显下调凋亡相关分子p-Bad、Bcl-2、Bcl-xl的表达,上调Bax的表达,最终引起caspase-3活性增加(P<0.05)。沉默pim-3后B16细胞增殖和迁移受到抑制(P<0.05),而细胞周期无明显变化。结论:靶向沉默pim-3基因能促进黑色素瘤细胞的凋亡,抑制细胞的增殖和迁移,提示pim-3基因可以作为黑色素瘤基因治疗的新靶点。

Objective： To observe the inhibitory effect of target silencing pim-3 on mouse melanoma B16 cell line. Methods： Pim-3-shRNA vector andpim-3 over-expressing vector （pim-3-MIGR1） were respectively transfect- ed into B16 melanoma cells by liposome, and the changes in mRNA and protein expression ofpim-3 was measured by qPCR and Western blotting assay, respectively; the apoptosis of B 16 cells was detected by AnnexinV/PI staining and TUNEL staining; the expressions of apoptosis-related factors, such as p-Bad, Bcl-2, Bcl-xl, Bax, caspase-3, were evaluated by qPCR and Western blotting; MTT assay was applied to confirm the proliferation of B16 cells; Flow cytometry was used to detect cell cycle; RTCAxCELLigence system was performed to confirm cell migration; and Transwell assay was applied to examine cell invasion. Results： pim-3-shRNA significantly reduced while pim-3- MIGR1 significantly enhanced pim-3 expression in B 16 cells at both mRNA and protein levels （all P〈0.05）. Pim-3- shRNA also effectively induced the apoptosis of B16 cells; however, co-transfection with pim-3-MIGR1 obviously reversed the apoptosis induced by Pim-3 silencing （P〈0.05）. Further studies found pim-3-shRNA significantly down- regulated the expressions of apoptosis-related molecules （p-Bad, Bcl-2 and Bcl-xl）, but remarkably up-regulated Bax expression, and finally resulted in increased caspase-3 activity （all P〈0.05）. The proliferation and migration of B 16 cells were significantly inhibited by pim-3 silencing （all P〈0.05）; however, there was no obvious change in cell cy- cle. Conclusion： Pim-3-specific gene silencing effectively promoted the apoptosis and inhibited both the proliferation and migration of B 16 cells, suggesting that pim-3 may serve as a potential effective gene therapy target for melanoma.