慢病毒介导磷酸二酯酶7A基因沉默细胞株的构建及三磷酸腺苷结合盒转运体A1的表达变化

Construction of PDE7A gene silencing THP-1 cell lines by lentivirus-mediated RNA interference and expression of ABCA1

摘要:

目的  利用慢病毒介导构建磷酸二酯酶7A（PDE7A）基因沉默人单核巨噬细胞（THP-1）株，并分析沉默细胞株的三磷酸腺苷结合盒转运体A1（ABCA1）的表达变化。方法  以PDE7A基因为靶点，设计合成3段shRNA片段，与慢病毒载体PmiRzip连接，构建重组质粒。测序鉴定后进行病毒包装，感染THP-1细胞，实时荧光定量聚合酶链反应（qPCR）进行分析，确定最佳干扰片段。嘌呤霉素筛选得到PDE7A基因沉默稳转细胞株。qPCR和Western blot检测鉴定所构建的细胞株，将其诱导成为泡沫细胞后鉴定ABCA1基因的表达。结果  转染shRNA1、shRNA2、shRNA3细胞的PDE7A相对表达量分别为（0.480±0.028）、（0.561±0.016）和（0.377±0.013），故选择shRNA3作为干扰PDE7A基因的最佳片段。采用1.4 g/L嘌呤霉素成功筛选出PDE7A沉默细胞株，qPCR和Western blot检测PDE7A基因的干扰效率，其抑制效率>70%。将其诱导成巨噬泡沫细胞后，Western blot检测显示，ABCA1的表达量增加40%。结论  成功构建PDE7A shRNA慢病毒干扰载体，并筛选出PDE7A基因沉默的THP-1细胞株，且ABCA1的表达量增加。

Abstract:

Objective To construct phosphodiesterase 7A (PDE7A) gene silence human acute monocytic leukemia (THP-1) cell lines by lentivirus-mediated RNA interference technique, and to analyze the expression of ATP-binding cassette transporter A1 (ABCA1) in THP-1 cell lines. Methods Three human PDE7A gene targeted shRNA fragments (shRNA1, shRNA2 and shRNA3) and shRNA-NC fragment were designed and synthesized, then connected with lentiviral vector PmiRzip to construct recombinant plasmids. After DNA sequencing, the lentivirus was packaged, and infected the THP-1 cells. The inhibitory effect of PDE7A shRNAs was analyzed by qPCR. Subsequently, the THP-1 cells were screened with Puromycin to get stable PDE7A gene silencing cell lines which were then identified by qPCR and Western blot. The expression of ABCA1 was determined after THP-1 cells were induced to develop macrophage foam cells. Results The relative expression of PDE7A in the cells transfected with shRNA1, shRNA2 or shRNA3 was (0.480 ± 0.028), (0.561 ± 0.016) and (0.377 ± 0.013) respectively; then shRNA3 was chosen as interferent PDE7A gene fragment. PDE7A silence cell lines were successfully screened with 1.4 g/L Puromycin. PDE7A gene interference efficiency was identified by qPCR and Western blot, and inhibition efficiency was more than 70%. The expression of ABCA1 wasincreased by more than 40% after THP-1 cells were induced into macrophage foam cells. Conclusions PDE7A silencing lentivirus interference vector has been successfully constructed, and PDE7A gene silencing THP-1 cell lines have been screened out in which ABCA1 expression is increased.