hsa-miR-26b沉默载体的构建及验证

Construction and identification of hsa-miR-26b silencing vector

目的构建hsa-miR-26b沉默载体,与其靶基因PTEN共转染HEK-293T细胞验证其沉默效果。方法用miRNA海绵体技术设计并合成与hsa-miR-26b成熟序列反向互补的3个重复序列的双链DNA,将短发卡状RNA(shRNA)克隆入LV3-GFP-Puro载体,与靶基因PTEN共转染HEK-293T细胞,于24 h后收集细胞RNA,用real-time PCR检测hsa-miR-26b表达水平,同时用双荧光素报告系统检测其对靶基因的作用。结果成功构建了有效的hsa-miR-26b沉默载体,经测序验证与设计序列一致;real-time PCR验证hsa-miR-26b表达水平降低了75%;双荧光素报告系统验证其对靶基因无抑制作用(P>0.05)。结论成功构建hsa-miR-26b沉默载体,并验证其对靶基因PTEN无抑制作用。

Objective：To construct a recombinant vector carrying hsa-miR-26b sponges and co-transfect with its target gene PTEN into HEK293T cells to validate the silencing efficiency. Methods：The double-stranded DNAs of three repetitive sequences which were antisence complementary to mature sequence of hsa-miR-26b were designed and synthesized by miRNA sponge. The short hairpin RNA （shRNA） was cloned into the LichV3-GFP-Puro vector and validated exactly by sequencing. The expressed plasmid and target gene PETN plasmid were co-transfected into HEK-93T cells. The cellular RNA was collected after 24 hours. The hsa-miR-26b silencing level and the effects on target gene were respectively detected by real-time quantitative PCR and dual-Luciferase reporter assay system. Results：The constructed hsa-miR-26b sponge vector was validated to be consistent with the designed sequence. The expression level of hsa-miR-26b reduced by 75%. No inhibitory effect of hsa-miR-26b on its target gene was shown by dual-Luciferase reporter assay （P〉0.05）. Conclusion：The silencing vector carrying hsa-miR-26b sponge was successfully constructed. It should be helpful for the further study on the function and mechanism of hsa-miR-26b in human adipocytes.