RNAi沉默HMGN5基因对肺癌A549细胞增殖的影响

Experimental study of the effect of RNAi to silence HMGN5 gene expression on proliferation of A549 cells

目的构建HMGN5基因的shRNA慢病毒载体,并在肺癌A549细胞上鉴定其沉默效率,观察其对细胞增殖能力的影响。方法筛选HMGN5基因特异性siRNA靶点,合成短发卡结构shRNA,与LentiLox3.7慢病毒载体重组形成shRNA表达载体,包装shRNA慢病毒颗粒,感染肺癌A549细胞,设阴性组及干涉组,应用Real-time PCR和Western印迹的方法检测HMGN5在mRNA和蛋白水平的沉默效率,MTT和克隆形成实验观察HMGN5基因沉默对A549细胞增殖的影响。结果构建的shRNA慢病毒颗粒感染A549细胞后,干涉组HMGN5基因的mRNA表达量较阴性对照载体慢病毒感染组下降了71.7%,显著抑制其蛋白表达;MTT结果表明细胞增殖能力明显降低,干涉组克隆形成能力较阴性组明显减弱。结论成功构建了HMGN5基因的shRNA慢病毒表达载体,其能够在肺癌A549细胞上有效沉默靶基因。HMGN5基因具有影响肺癌细胞恶性增殖的能力,为肺癌的基因治疗奠定了实验基础。

Objective To construct shRNA lentiviral vector targeting HMGN5 gene,detect the efficiency of gene silence and its effect on proliferation of A549 cells.Methods Specific siRNA targets using short hairpin in frame were designed and synthesized according to the mRNA sequence of HMGN5 gene.DNA oligo was cloned into LentiLox 3.7 lentiviral expression vector.ShRNA lentivirus was harvested by virus packaging system.The negative group and RNAi group were set up.Both Real-time PCR and Western blotting were performed to determine the expression level of HMGN5 in the infected A549 cells.MTT assay and colony formation assay were used to detect its effect on cell proliferation.Results HMGN5 expression in A549 cells was knock down at both mRNA and protein level by virus infection.It showed mRNA decreased 71.7% vs negative control group,and the protein was inhibited significantly.Results of MTT assay and colony formation showed the proliferation of A549 cells was inhibited,and the number of cell coloning was decreased.Conclusions The recombinant lentiviral shRNA expressing vector targeting HMGN5 gene is successfully constructed and packaged.Both mRNA and protein can be effectively down-regulated in A549 cells.It is proved that HMGN5 gene can affect the proliferation of A549 cells.It lays a foundation of gene therapy of lung cancer.