重组干扰质粒对卵巢癌COC1细胞HDAC1表达的影响

摘要

目的：构建重组 HDAC1基因的短发夹 RNA （shRNA）慢病毒干扰质粒并研究其对卵巢癌 COC1细胞HDAC1表达的影响。方法针对 HDAC1基因设计特异性 siRNA 靶点，构建慢病毒干扰质粒载体，筛选获得的有效shRNA 慢病毒干扰序列，将其转染卵巢癌 COC1细胞。采用 Real-time PCR 方法检测靶基因在 mRNA 水平的沉默效果，Western blot 方法检测靶基因在蛋白质水平的沉默效果。结果构建的慢病毒载体 shRNA 的 PCR 鉴定和测序正确。shRNA 慢病毒干扰质粒转染 COC1细胞后 HDAC1基因的 mRNA 表达量和蛋白质水平较阴性对照质粒转染组均显著下调（P ＜0．05）。结论成功构建 HDAC1基因的 shRNA 慢病毒干扰质粒，该慢病毒干扰质粒能够在细胞水平有效沉默靶基因。%Objective To construct the shRNA lentiviral plasmid targeting HDAC1 gene and detect its effect of gene silence in COC1 cells.Methods The specific siRNA sequences targeting HDAC1 gene were cloned into pRNAT-U6.2/Lenti lentiviral plasmid.After screening for the valid siRNA ,HDAC1 specific shRNA was transfectted into COC1 cells.Then ,real-time PCR and Western Blot was performed to determine the expression level of HDAC1.Results PCR and sequencing results revealed that shRNA plasmids were correctly constructed.The HDAC1 expressions in COC1 cells were down regulated at mRNA and protein level by plasmid transfection.The expression of HDAC1 was decreased in mRNA levels and protein compared with negative control.Conclusion The recombinant lentiviral shRNA plasmid targeting HDAC1 gene has been successfully constructed.HDAC1 mRNA and protein can be down-regulated availably in COC1 cells.