Beclin-1基因shRNA慢病毒载体的构建及其对B16F10细胞自噬及活力的影响

利用RNA干扰技术构建稳定沉默Beclin-l基因的B16F10小鼠黑色素瘤细胞系,分析稳转细胞系的自噬水平和活力,为探讨Beclin-1基因、自噬与肿瘤三者关系奠定基础。构建小鼠Beclin-l基因干扰载体,转染至293T细胞,获得慢病毒颗粒,将病毒颗粒感染B16F10细胞,通过加压筛选、有限稀释、细胞驯化得到稳定转染m-Beclin-1-shRNA1的B16F10细胞系。采用RT-PCR和免疫荧光技术检测对Beclin-l的干扰效果,Western blot和透射电镜分析稳转细胞系中自噬标志物LC3蛋白表达及自噬小体形成情况,CCK-8法检测稳转细胞系的活力。结果表明,成功构建了靶向抑制Beclin-l基因表达的shRNA,纯化的慢病毒滴度为1×10⁸ TU·mL^-1,建立的细胞系中Beclin-1 mRNA和蛋白表达都受到不同程度的抑制,mRNA的沉默效率约为75%（P<0.01）,发现干预Beclin-1表达能够抑制LC3-Ⅱ蛋白的表达及自噬小体的形成数量,降低B16F10细胞活力。以上结果表明,干扰Beclin-1表达能够有效降低B16F10细胞自噬活性,促进B16F10细胞死亡,这为探讨Beclin-1基因功能及自噬在抗肿瘤中的作用奠定重要基础。

Construction and Influence of Beclin-1 Gene shRNA Lentiviral Vector on Autophagy and Virability of B16F10 Cell

In this study, we constructed a mouse melanoma cell line B16F10 with stable interference targeting Beclin-1 gene by RNA interference, and to explore autophagy and cell viability, which is the foundation for analysis of the relationship between Beclin-1 gene, autophagy, and tumor. Firstly, we designed the shRNA of mouse Beclin-l and constructed interference vectors. Then we transfected the lentiviral vector into 293T cells to obtain lentiviral particles, and B16F10 cells were infected with lentivirus packaging. To obtain the monoclonal cell line, the transfected cells were selected with the methods of pressurized screening and limited dilution. And then the monoclonal cells were cultured in fed-batch mode for quality evaluation. The interference effect of Beclin-l in the stable cell line was detected by RT-PCR and immunofluorescence technique, the expression of autophagy protein LC3 and the formation of autophagosomes were analyzed by Western blot and transmission electron microscopy, and cell viability were determined by CCK-8 assay. The results showed that the lentiviral vector targeting Beclin-l was successfully constructed, and the titer of lentivirus purified by packaging was 1×10⁸TU·mL^-1. Beclin-1 mRNA and protein were inhibited in the stable cell line, and the interference efficiency was around 75% (P<0.01). The results demonstrated that intervention of Beclin-1 expression could inhibit the expression of LC3-Ⅱ protein and the number of autophagosomes, and reduce the viability of B16F10 cells. These data indicated that the silence of Beclin-1 gene could effectively reduce the autophagy intensity and promote cell death in B16F10 cells, which lays the foundation for exploring the function of Beclin-1 gene and the role of autophagy in anti-tumor.