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p-Bad、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，RTCAx-CELLigence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-shRNA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cas-pase-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 and pim-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 of pim-3 was measured by qPCR and Western blotting assay, respectively; the apoptosis of B16 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-shRNAsignificantly reduced while pim-3-MIGR1 significantly enhanced pim-3 expression in B16 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-shRNAsignificantly 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 B16 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 prolifera-tion and migration of B16 cells, suggesting that pim-3 may serve as a potential effective gene therapy target for mel-anoma.

国家自然科学基金资助项目（No.81472646, No.81273220, No.91442114）