Speedy A蛋白调控相关miRNA的筛选及miR-23b靶标的验证

Screening the microRNA associated with Speedy A and validation of miR-23b targets

目的筛选和鉴定调控Speedy A蛋白表达的miRNAs。方法通过在线数据库TargetScan、miRNA和miRwalk预测与Speedy A相互作用的miRNAs,并结合本实验室前期建立的精原细胞和精母细胞小RNA数据库,筛选出评分较高的候选miRNAs。实时定量PCR检测小鼠7、14、21、35、64d睾丸中候选miRNAs和Speedy A基因的表达,筛选出表达趋势呈显著负相关的miRNAs。构建野生型Speedy A基因3’端非翻译区荧光素酶报告基因载体(WT-Speedy A-3’UTR)及miR-23b靶序列突变型载体(mut-Speedy A-3’UTR),分四组转染HEK-293T细胞株:野生型实验组,WT-Speedy A-3’UTR质粒+miR-23bmimic;野生型对照组,WT-Speedy A-3’UTR质粒+miR-23b mimic NC;突变型实验组,mut-Speedy A-3’UTR质粒+miR-23bmimic;突变型对照组:mut-Speedy A-3’UTR质粒+miR-23bmimic NC,双荧光素酶报告基因系统检测荧光素酶活性。结果通过生物信息学方法结合本实验室前期建立的精原细胞和精母细胞小RNA数据库筛选出4个评分较高的miRNA:miR-23b、miR-143、miR-345-3p和miR-881。实时荧光定量PCR检测不同发育时期小鼠睾丸中以上4种miRNAs和Speedy A基因的表达水平,结果显示在不同发育时期的小鼠睾丸中,miR-23b的表达趋势与Speedy A呈显著负相关(P<0.01)。成功构建了重组野生型/突变型(WT/mut)Speedy A3’-UTR载体,通过双荧光素酶报告基因检测显示野生型实验组相比对照组吸光值明显降低,差异有统计学意义(P<0.01)。结论野生型实验组对其它3组对照组荧光活性有明显的抑制作用,证实miR-23b能够靶向调控Speedy A基因的表达。

Objective： To screen and identify the miRNAs regulated the expression of speedy A. Methods： The potential candidate miRNAs associated with speedy A were screened by predicted the possible target miRNAs associated with speedy A based on online database,such as TargetScan,miRNA, miRwalk, and combined with utilizing the small RNA database of spermatogonia and spermatocyte,which was established in our laboratory. Real-time quantitative PCR was used to detect the expression of candidate miRNAs and Speedy gene in 7, 14, 21,35, 64 days old mouse testis, and then the candidate miRNAs which were significantly negatively correlated with Speedy A were screened and identified. The 3＇-untranslated regions （3＇-UTR） of the wild type Speedy A and mutant Speedy A sequence were cloned into luciferase reporter vector pmirGLO. The HEK-293T cells were divided into four groups randomly：wild type experimental group（wild-type plasmid＋miR-23b group）;Wild type control group （wild-type plasmid ＋ miRNA negative control group） ;Mutant type experimental group （mutant plasmid ＋ miR-23b group） ; Mutant type control group （mutant plasmid＋miRNA negative control group）. The relevant plasmids and miRNAs were transfected into the HEK-293T cells of each group. The luciferase activity was detected by dual luciferase reporter gene system. Results. Four miRNAs were screened by bioinformatics methods combined with utilizing the database of spermatogonia and spermatocyte established in our laboratory. The four miRNAs were miR-23b, mir- 143,mir-345-3p and mir-881. The results of real-time quantitative PCR for different developmental stages testes showed that the expression of miR-23b was negatively correlated with speedy A （P〈0.01）. The recombinant wild-type/mutant （WT/mut） Speedy A3 ＇-UTR vector was successfully constructed. The lueiferase activity of wild-type plasmid＋ miR-23b group was significantly lower than that of wild-type plasmid＋miRNA negative control group （P〈0.01）. Conclusions： The wild type experimental group had obvious inhibitory effect on the fluorescence activity. It is confirmed that mir-23b can be used to regulate the expression of speedy A.