沉默MCM7基因介导AKT信号通路参与黑色素瘤细胞增殖及凋亡的实验

MCM7 Knockdown Inhibits Migration and Invasion While Induces Apoptosis of Cutaneous Malignant Melanoma Cells Through AKT Signaling Pathway

目的探讨MCM7基因沉默介导AKT信号通路参与人皮肤黑色素瘤细胞的增殖及凋亡。方法构建MCM7基因和沉默MCM7基因表达的慢病毒RNA(LV-shRNA-MCM7)的表达载体;将A375细胞分为Control组、Emptyvector转染组(空载质粒转染组)、siRNA(LV-shRNA-MCM7)转染组、siRNA NC(LV-shRNA-MCM7 negative control)转染组;MTT法检测每组细胞增殖;流式细胞术检测细胞周期、细胞凋亡情况;划痕实验法检测细胞迁移能力;qRT-PCR法测定细胞中MCM7基因、AKT信号通路相关基因、Cyclin D1及凋亡相关基因Bcl-2、Bax、caspase-3的相对表达量;Westernblot法测定蛋白相对表达量。结果沉默MCM7后,黑色素瘤细胞中的MCM7基因、Cyclin D1、Bcl-2、AKT信号通路相关基因AKT3的mRNA和蛋白相对表达量显著下调(P<0.05);凋亡相关基因Bax、caspase-3的mRNA和蛋白表达量显著上调(P<0.05);siRNA转染组凋亡率显著上升,G_0/G_1期细胞数目明显增多,S期细胞数目明显减少;细胞增殖、迁移能力显著下降(P<0.05)。结论沉默MCM7基因抑制AKT信号通路的激活,从而抑制A375黑色素瘤细胞增殖、迁移,促进A375黑色素瘤细胞凋亡。

Objective To investigate the functional impact of minichromosome maintenance protein 7（MCM7） silencing on cutaneous malignant melanoma（CMM） cell apoptosis and proliferation via AKT signaling pathway. Methods Lentivirus vector（LV）-shRNA-MCM7 was constructed, and A375 cells were assigned into control, empty vector, siRNA and siRNA negative control（NC） groups. The viability, migration, apoptosis and cell cycle entry of A375 cells in response to MCM7 knockdown were detected by MTT assay, scratch test and flow cytometry. qRT-PCR and Western blot were conducted to measure the expression of MCM7, AKT3, Cyclin D1, Bcl-2, Bax and caspase-3. Results Compared with control, empty vector and siRNA NC groups, MCM7, AKT3, Cyclin D1 and Bcl-2 expression were decreased in siRNA group after MCM7 silencing（P〈0.05）, but Bax and caspase-3 expression were increased（P〈0.05）, cell apoptosis and the number of cells at G0/G1 phase were increased, but cell migration and proliferation and the number of cells at S phase were decreased（P〈0.05）. Conclusion The functional suppression of MCM7 could inactivate AKT signaling pathway, thus inhibiting the migration, proliferation and promoting apoptosis of CMM A375 cells.