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目的结合TargetScan软件预测,体外验证小鼠microRNA-135a(miR-135a)与信号传导及转录激活因子6(signal transducer and activator of transcription 6,STAT6)的靶点结合作用。方法将聚合酶链反应(polymerase chain reaction,PCR)扩增的靶基因STAT6的非编码3’UTR和突变基因分别克隆到双荧光素酶报告载体中,构建野生型和突变型载体。分别将miR-135a模拟物、模拟对照物与靶基因野生型、突变型质粒载体共转染体外培养的293T细胞,测定载体的校正荧光hluc值及报告荧光hRluc值。结果与模拟对照物相比较,miR-135a模拟物与靶基因STAT6的3’UTR野生型质粒的相互作用能明显降低hRluc/hluc的相对荧光比值,差异有统计学意义(P<0.01);而miR-135a模拟物与突变型质粒相作用则对hRluc/hluc的相对荧光比值比较差异无统计学意义(P>0.05)。结论小鼠miR-135a与STAT6的3’UTR体外特异性结合作用,为进一步探讨小鼠miR-135a对变应性鼻炎的免疫调控机制提供了依据。
Objective To predict the binding of mouse microRNA-135a (miR-135a) to target transducers and signal transducers and activators of transcription 6 (STAT6) in vitro using TargetScan software. Methods The non-coding 3’UTR and the mutated gene of target gene STAT6 amplified by polymerase chain reaction (PCR) were cloned into luciferase reporter vector to construct wild-type and mutant vectors, respectively. The 293T cells cultured in vitro were cotransfected with the miR-135a mimics, the mock control and the target gene wild-type and mutant plasmid vectors respectively, and the corrected fluorescence hluc values and the reporter fluorescence hRluc values of the vectors were determined. Results The relative fluorescence of hRluc / hluc was significantly lower than that of the control (P <0.01), but the interaction between the miR-135a mimic and the 3’UTR wild-type plasmid of target gene STAT6 significantly decreased The relative fluorescence ratio of hRluc / hluc between miR-135a mimics and mutant plasmids had no significant difference (P> 0.05). Conclusion The in vitro specific binding of miR-135a and STAT6 to 3’UTR in mice provides the basis for further study on the mechanism of miR-135a immunostimulation in allergic rhinitis.