牛let-7a与CC趋化因子受体7基因靶向关系的验证

Verification of targeting relationship between bovine let⁃7a and CC chemokine receptor 7 gene

为了验证小分子RNA let-7a与其预测靶基因CC趋化因子受体7(CCR7)之间的关系,采用生物信息学软件预测CCR7的3′非编码区(3′UTR)是否存在与let-7a的靶向结合位点;利用PCR扩增出包含靶向结合位点区域的CCR7-3′UTR,并构建其荧光素酶表达载体;将验证后的阳性重组子瞬时共转染人胚胎肾细胞293(293T),进行双荧光素酶报告基因检测;通过将合成的let-7a模拟物和let-7a抑制剂分别转染奶牛乳腺上皮细胞MAC-T,利用qRT-PCR和ELISA检测let-7a与CCR7基因mRNA和蛋白表达水平的变化,验证两者之间的靶向调控关系。菌液PCR和双酶切结果表明,成功构建了重组双荧光素酶报告质粒pMIR-REPORTTM-CCR7-3′UTR。双荧光素酶报告基因检测结果表明,let-7a与CCR7基因之间确实存在靶向调控关系。qRT-PCR和ELISA进一步证实了过表达let-7a能显著下调MAC-T细胞中CCR7基因mRNA和蛋白的表达量(P<0.05),两者存在负调控关系。本研究结果证实了let-7a与CCR7基因之间存在靶向调控关系,为进一步探究其分子机制提供参考。

To verify the targeting relationship between microRNA let⁃7a and CC chemokine receptor 7 gene(CCR7),the binding site of let⁃7a to the 3′untranslated region(3′UTR)of CCR7 was predicted using bioinformatics.The predicted binding site in CCR7⁃3′UTR was amplified by PCR and then cloned to a luciferase expression vector.The verified positive recombinants were transi⁃ently co⁃transfected into human embryonic kidney 293(293T)cells for dual⁃luciferase reporter assay.In addition,the bovine mam⁃mary epithelialcells MAC⁃T were transfected with let⁃7a mimic or let⁃7a inhibitor to further verify the targeting relationship between let⁃7a and CCR7 by detecting the expression levels of CCR7 via qRT⁃PCR and ELISA.The results of PCR and double enzyme diges⁃tion showed that the recombinant dual luciferase reporter plasmid pMIR⁃REPORTTM⁃CCR7⁃3′UTR was successfully constructed.Dual⁃luciferase reporter assay confirmed the targeting relationship between let⁃7a and CCR7.qRT⁃PCR and ELISA results further confirmed that the overexpression of let⁃7a significantly down⁃regulated the mRNA and protein expression levels of CCR7 in MAC⁃T cells(P<0.05),indicating a negative regulatory relationship between them.This study confirms the targeted regulation between let⁃7a and CCR7,providing a reference for further exploration of its molecular mechanism.