慢病毒介导SHCBP1低表达对黑色素瘤B16细胞增殖的影响

The effect of lentivirus-mediated down-regulation of SHCBP1 on the proliferation of melanoma B16 cells

构建Shcbp1慢病毒干扰载体,探究SHCBP1在黑色素瘤B16细胞中的生物学功能。通过设计靶向干扰小鼠Shcbp1基因的shRNA-1和shRNA-2以及对照序列shRNA-NC,连接至慢病毒骨架载体,利用HEK293T细胞包装慢病毒颗粒,侵染B16细胞,检测干扰效率,并通过CCK-8和流式细胞术检测细胞活力和细胞周期。利用Western Blot检测增殖相关基因的表达。结果显示:敲低Shcbp1显著抑制B16细胞增殖;流式细胞术检测结果发现,敲低Shcbp1将B16细胞阻滞在G1期;ERK1/2的磷酸化水平降低,P21的表达水平升高。结果表明SHCBP1可能通过ERK1/2信号通路抑制P21的转录,加速B16细胞从G1到S期进程。

To construct Shcbp1 lentiviral interference vector to explore the biological function of SHCBP1 in melanoma B16 cells,first,the shRNA-1 and shRNA-2 that target to interfere with the mouse Shcbp1 gene and the control sequence shRNA-NC were designed and connected to the lentiviral backbone vector,HEK293 cells were used to package the lentiviral particles to infect B16 cells,the interfer-ence efficiency was tested and CCK-8 and flow cytometry were used to detect cell viability and cell cycle.Western Blot was used to de-tect the expression of proliferation-related genes.The results showed that knockdown of Shcbp1 significantly inhibited the proliferation of B16 cells;flow cytometry results showed that knockdown of Shcbp1 blocked B16 cells in the G1 phase;the phosphorylation level of ERK1/2 decreased and the expression level of p21 increased.These results indicate that SHCBP1 may inhibit the transcription of P21 through the ERK1/2 signaling pathway and accelerate the progress of B16 cells from G1 to S phase.