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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 pim-3 targeting silencing on B16 in mouse melanoma cells. METHODS: Specific targeting and silencing pim-3 vector (pim-3-shRNA) and pim-3 overexpression vector (pim-3-MIGR1) were transfected into melanoma B16 cells by lipofectamine. The changes of pim-3 mRNA and protein were detected by Western blotting. The apoptosis of cells was detected by Annexin V / PI staining and TUNEL staining. The expressions of pBad, Bcl-2, Bcl-xl, Bax and caspase- 3 and other apoptosis-related molecules. The cell proliferation was detected by MTT assay. The cell cycle was detected by flow cytometry. The cell migration was detected by RTCAxCELLigence system. The cell invasion was detected by Transwell invasion assay. Results: pim-3mRNA and protein expression in B16 cells transfected with pim-3-sh RNA were significantly decreased (P <0.05), pim-3 mRNA and protein expression in B16 cells transfected with pim-3 overexpression plasmid Than the control group increased significantly (P <0.05). After transfection with pim-3-shRNA, the apoptosis of B16 cells was significantly increased. On the basis of pim-3 overexpression plasmid, pim-3 overexpression significantly reversed the apoptosis induced by pim-3 silencing (P <0.05) The results showed that silencing pim-3 significantly down-regulated the expressions of p-Bad, Bcl-2 and Bcl-xl and up-regulated the expression of Bax, leading to an increase in the activity of caspase-3 (P <0.05). The proliferation and migration of B16 cells were inhibited after silencing pim-3 (P <0.05), but the cell cycle did not change significantly. Conclusion: Targeting silencing of pim-3 gene can promote the apoptosis of melanoma cells and inhibit the proliferation and migration of melanoma cells, suggesting that pim-3 gene may serve as a new target for gene therapy of melanoma.