中文摘要：

目的：构建能高效表达成熟miR-508-5p小分子的慢病毒过表达载体,研究其对MAPK1/ERK信号通路靶向调控作用.方法：利用化学合成miR-508-5p茎环结构RNA,并将其克隆入线性化的pSicoR 质粒中,经双酶切及测序鉴定;同时构建与miR-508-5p互补靶基因MAPK1的3＇非翻译区,将其克隆入线性化的Report载体中.利用脂质体转染试剂将鉴定阳性的pSicoR-miR-508-5p重组质粒转染HEK-293T细胞,进一步通过Relative luciferase activity、Western blot及real-time PCR试验检测MAPK1蛋白和mRNA相对表达水平.结果：酶切及测序结果证明成功构建pSicoR-miR-508-5p 及Report-MAPK1 3＇-UTR重组质粒,并通过实验证实miR-508-5p过表达明显抑制MAPK1的蛋白表达水平及mRNA相对含量（P〈0.05）;抑制miR-508-5p的表达,又明显上调MAPK1的蛋白表达水平及mRNA相对含量（P〈0.05）.结论：成功构建pSicoR-miR-508-5p慢病毒过表达载体,证实miR-508-5p直接靶标MAPK1的表达,在转录后水平对其进行负调控,为进一步研究miRNA对细胞通路和细胞周期的调控作用奠定了基础.

英文摘要：

Objective：To construct the lentiviral vector which can over-expression miR-508-Sp and explore its targeted effects on MAPK1/ERK signaling pathways. Methods： Based on chemical technology to synthetic stem-loop structure RNA of miR-508-Sp, and cloned it into the linearized pSicoR plasmid which was digested by restriction enzyme and sequenced, the positive recombinants were transfected into HEK-293T cells; To validate the targeted regulatory relationship between miR-508-Sp and MAPK1 3＇-UTR through the relative luciferase activity, Western blot and real-time PCR （qRT-PCR） test. Results： The PCR, restriction enzyme digestion analysis and DNA sequencing demonstrated that the recombinant plasmids of pSicoR-miR-508-Sp and Report-MAPK1 3＇-UTR were constructed successfully, and confirmed that the over-expression of miR-508-5p suppressed the expression level of MAPK1 protein and mRNA significantly（ P 〈0.05 ） ; suppression of miR-508-Sp expression increased the expression level of MAPK1 protein and mR- NA significantly（ P 〈 0.05 ）. Conclusion： The recombinant plasmid of pSicoR-miR-508-5p has been constructed successfully, demon- strating that miR-508-5p can directly target on the gene expression of MAPK1. It is negatively regulated at the post-transcriptional level, construct the foundation of miRNA for regnlation of the further research on signaling pathways and cell cycle.