牛GHR基因与miR-139靶向关系的验证

Targeting Verification between Bta-GHRand miR-139

旨在构建牛生长激素受体(Growth hormone receptor,GHR)基因3′非翻译区(Untranslated region,UTR)双荧光素酶载体,并确定miR-139与牛GHR基因的靶向关系。本研究采用生物信息学方法预测miR-139与牛GHR基因3′UTR的结合位点;人工合成牛GHR基因3′UTR及突变型片段并克隆至双荧光素酶载体psiCHECK-2上,构建野生型(psiCHECK2-GHR-W 3′UTR)及突变型(psiCHECK2-GHR-M 3′UTR)双荧光素酶报告质粒。将培养的Hela细胞分为4组,分别共转染miR-139mimic或阴性对照和psiCHECK2-GHR-W 3′UTR重组质粒或psiCHECK2-GHR-M 3′UTR重组质粒,然后用双荧光素酶检测试剂盒检测4组细胞中荧光素酶活性。之后培养奶牛乳腺上皮细胞,分别设对照组、阴性对照组、miR-139 mimic及miR-139inhibitor组,对阴性对照组、miR-139mimic组及miR-139inhibitor组分别转染阴性对照、miR-139mimic或miR-139inhibitor,利用qPCR(Realtime quantitative PCR)进一步检测各组miR-139及GHR基因的mRNA表达。结果,通过双酶切试验证实成功构建了野生型及突变型重组双荧光素酶报告质粒psiCHECK2-GHR-W 3′UTR和psiCHECK2-GHR-M 3′UTR;当野生型重组质粒psiCHECK2-GHR-W 3′UTR和miR-139mimic共转染Hela细胞后,双荧光素酶报告质粒表达的荧光素酶活性显著低于其他处理组(P<0.05)。qPCR进一步证实miR-139能显著下调奶牛乳腺上皮细胞中GHR基因mRNA的表达(P<0.05)。本研究成功构建了牛野生型及突变型GHR基因3′UTR双荧光素酶报告质粒;双荧光素酶试验及qPCR证实miR-139与牛GHR基因3′UTR存在靶向关系。

The aim of this study was to construct a dual luciferase reporter plasmid containing the3′untranslated region（UTR）of bta-GHR,and verify the targeting relationship between miR-139 and bta-GHR.The binding site of miR-139 to bta-GHR were predicted using bioinformatics.The synthetic 3′UTR fragment of bta-GHR was cloned into psiCHECK-2vector to construct wild type（psiCHECK-2-GHR-W 3′UTR）or mutant type（psiCHECK2-GHR-M 3′UTR）dual luciferase reporter plasmid.Cultured Hela cells were divided into 4groups,they were co-transfected with miR-139 mimic or negative control and psiCHECK2-GHR-W 3′UTR or psiCHECK2-GHRM 3′UTR.The relative luciferase activity was detected.The cultured bovine mammary epithelial cells were transfected with miR-139 mimic,negative control or miR-139 inhibitor.Using realtime quantitative PCR（qPCR）,we measured the mRNA level of GHRand miR-139 in cow.The results showed that,by digestion of restriction enzymes,the wild type recombinant plasmid（psiCHECK2-GHR-W 3′UTR）and mutant type recombinant plasmid（psiCHECK2-GHR-M 3′UTR）were proved to be successfully constructed.After psiCHECK2-GHR-W 3′UTR and miR-139 mimic co-transfected Hela cells,the luciferase activity was significantly lower than other treatment groups（P〈0.05）.Using qPCR,we further confirmed that the mRNA level of GHR was down-regulated by miR-139（P〈0.05）.The dual luciferase reporter plasmid containing 3′UTR of wild type or mutant type of bta-GHR was constructed successfully;The targeting relationship between miR-139 and bta-GHR was confirmed by dual luciferase assay and qPCR assay.