【摘要】 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。更多还原

【Abstract】 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 transiently co-transfected into human embryonic kidney 293(293T) cells for dual-luciferase reporter assay. In addition, the bovine mammary 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 digestion 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.更多还原