MiR30b与小鼠Bach2相互作用的研究

Study of Direct Interaction Between MiR30b and mRNA of Mouse Bach2

目的研究B细胞末端分化过程中miR30b新的作用靶点。方法经生物信息学分析,利用特异性引物以及定点突变引物从小鼠c DNA中分别扩增大小为530、361和189bp三个目的片段,胶回收并对361和189bp进行拼接,获得野生型、突变型小鼠Bach2 mRNA特异性片段,分别与pmir GLO载体连接,构建野生型、突变型重组荧光素酶报告基因质粒pmir GLO-m Bach2、pmir GLO-m Bach2 mt;与miR30b共转染至HEK293 T细胞,分析其荧光素酶活性。结果重组荧光素酶报告基因质粒经双酶切及测序证实构建正确;共转染后,与pmir GLO+miR30b组相比,pmir GLO-m Bach2+miR30b组的荧光素酶活性明显降低(P<0.05),而pmir GLO-m Bach2 mt+miR30b组的荧光素酶活性则无明显改变(P>0.05)。结论小鼠Bach2 mRNA是miR30b的靶点。

Objective To analyze the novel target of miR30b in the B cell terminal differentiation. Methods Three target gene fragments, at lengths of 530,361 and 189 bp, were amplified from mouse eDNA by PCR u- sing specific primers and site-direct mutant primers, respectively, and recovered with gel. The complete mu- tant fragment was obtained through jointing the 361 and 189 bp. The wild/mutant fragment of mouse Bach2 was inserted into pmirGLO vector, respectively to construct wild/mutant recombinanr lueiferase reporter gene plasmid, pmirGLO-mBach2 and pmirGLO-mBach2 rot. They and miR30b eo-transfected into HEK293 T cells. The Dual-Lueiferase Reporter Assay System was performed to determine the Firefly-Renilla luciferase activity among groups. Results Restriction enzyme analysis and sequencing proved that recombinant plasmids, pmir- GLO-mBach2 and pmirGLO-mBach2 mr, were constructed successfully. Compared with pmirGLO ＋ miR30b group, luciferase activity in pmirGLO-mBach2 ＋ miR30b group was significant decreased （ P 〈 0.05 ） , while no significant change in pmirGLO-mBach2 mt ＋ miR30b group （ P 〉 0.05 ）. Conclusion Mouse Bach2 mRNA is a novel tarzet gene of miR30b.