绵羊INHBB、SMAD4和FGF18基因的双荧光素酶载体构建及其与miR-370-3p的靶向验证

为研究绵羊输卵管功能相关的oar-miR-370-3p与抑制素亚基βB （inhibin subunit beta B，INHBB）、SMAD家族成员4（SMAD family member 4，SMAD4）和成纤维细胞生长因子18（fibroblast growth factor 18，FGF18）基因之间的靶向关系，本研究对多个物种（绵羊、人、小鼠、大鼠、猕猴、豚鼠和兔）中miR-370-3p的序列进行了比对，利用RNAhybrid程序预测绵羊miR-370-3p与INHBB、SMAD4和FGF18基因的3'UTR可能存在的结合位点，随后分别构建INHBB、SMAD4和FGF18基因3'UTR的野生型和突变型双荧光素酶载体，并将其与miR-370-3p mimics、mimics NC共转染至HEK293T细胞，检测双荧光素酶活性。结果表明，绵羊miR-370-3p序列与其他6个物种不同，但具有一定的保守性。RNAhybrid程序预测到oar-miR-370-3p与INHBB、SMAD4和FGF18基因的3'UTR存在结合位点。PCR扩增结果和测序结果表明，INHBB、SMAD4和FGF18基因3'UTR的野生型和突变型载体构建成功。共转染INHBB、SMAD4和FGF18野生型载体和miR-370-3p mimics的双荧光素酶活性极显著或显著低于相应的对照组（P<0.01；P<0.05）；而3种突变型载体和miR-370-3p mimics共转染的双荧光素酶活性均与相应的对照组无显著差异（P>0.05）。表明INHBB、SMAD4和FGF18基因的3'UTR区域均能与miR-370-3p结合并抑制双荧光素酶活性，验证了INHBB、SMAD4和FGF18基因均是miR-370-3p的靶基因，为进一步研究oar-miR-370-3p影响绵羊输卵管功能与绵羊繁殖力的分子机制提供依据。

Construction of Ovine INHBB,SMAD4 and FGF18 Genes Dual-luciferase Reporter and Validation of Their Targeting Relationship with miR-370-3p

To investigate the targeting relationship between miR-370-3p and inhibin subunit beta B (INHBB), SMAD family member 4 (SMAD4) and fibroblast growth factor 18 (FGF18) genes that were associated with oviductal function in sheep, in this study, the sequences of miR-370-3p in multiple species (Ovis aries, Homo sapiens, Mus musculus, Rattus norvegicus, Macaca mulatta, Cavia porcellus and Oryctolagus cuniculus) were compared and RNAhybrid was used to predict the potential binding sites of ovine miR-370-3p to the 3'UTR of INHBB, SMAD4 and FGF18 genes. Then wild type and mutant type dual-luciferase reporter vectors for INHBB, SMAD4 and FGF18 gene 3'UTR were constructed and co-transfected with miR-370-3p mimics and mimics NC into HEK293T cells to detect dual-luciferase activity, respectively. The results showed that the ovine miR-370-3p sequence was different from the other six species but was somewhat conserved, and was predicted by RNAhybrid to have binding sites to the 3'UTRs of the INHBB, SMAD4 and FGF18 genes. The electrophoresis results and sequencing results indicated that the wild type and mutant type vectors of INHBB, SMAD4 and FGF18 gene 3'UTR were successfully constructed. The dual-luciferase activities of the co-transfected INHBB, SMAD4 and FGF18 wild type vectors and miR-370-3p mimics were extremely significantly or significantly lower than the corresponding controls (P<0.01;P<0.05). While the dual-luciferase activities of the three mutant type vectors and miR-370-3p mimics co-transfected were not significantly different from the corresponding controls (P>0.05). It was shown that the 3'UTR region of INHBB, SMAD4 and FGF18 genes could all bind to miR-370-3p and inhibit dual-luciferase activity, which verified that INHBB, SMAD4 and FGF18 genes were both target genes of miR-370-3p and provided a basis for further study on the molecular mechanism of oar-miR-370-3p affecting oviductal function and sheep fecundity.