家蚕miR-14的上调表达与靶基因的预测分析

Upregulation and Expression of Bombyx mori bmo-miR-14 and Prediction of Its Target Genes

【目的】了解家蚕细胞中上调miRNA的方法及靶基因预测,为家蚕miRNA的功能研究提供方法参考。【方法】构建家蚕miR-14的真核表达载体pSK-hr3-ie1-EGFP-pri-mir-14并在家蚕BmN细胞中表达,同时通过转染化学合成的miRNA mimics上调BmN细胞中miR-14的水平。首先通过PCR扩增miR-14的前体及其侧翼序列(pri-mir-14)并克隆至真核表达载体pSK-hr3-ie1-EGFP的EGFP基因下游。分别将重组质粒pSK-hr3-ie1-EGFP-pri-mir-14、对照质粒pSK-hr3-ie1-EGFP、bmo-miR-14 mimics和negative control mimcs转染至家蚕BmN细胞,观察EGFP及FAM在细胞中的荧光情况,以检测其转染效率,qRT-PCR方法检测bmo-miR-14在细胞中的水平。分别采用RNAhybrid和miRanda预测bmo-miR-14的靶基因。【结果】重组表达载体pSK-hr3-ie1-EGFP-pri-mir-14与miRNA模拟物bmo-miR-14 mimics都能上调BmN细胞中的miR-14水平,与对照相比分别提高了2.1、984倍。利用生物信息学方法预测bmo-miR-14靶基因,RNAhybrid和miRanda分别预测得到153和171个潜在bmo-miR-14靶基因,其中两者共同预测的靶基因有49个。GO分析结果显示,预测的49个靶基因的分子功能集中于结合和催化;而生物学过程集中于细胞过程和代谢过程。【结论】通过转染重组质粒及化学合成miRNA mimics,使细胞中相应的miRNA上调,可用于bmo-miR-14后续的功能研究。

【Objective】The objective of this study is to offer a method for upregulating miRNA and predicting its targets in BmN cells,and to provide a methodological reference for studying the function of miRNAs in BmN cells.【Method】An eukaryotic expression vector pSK-hr3-ie1-EGFP-pri-mir-14 for expressing Bombyx mori bmo-miR-14 was constructed and the miRNA gene was expressed in BmN cells.Additionally,the level of bmo-miR-14 in BmN cells was also up-regulated by transfecting chemically synthesized miRNA mimics.Firstly,the fragment containing pri-mir-14 was amplified by PCR and inserted into the downstream of EGFP of eukaryotic expression vector pSK-hr3-ie1-EGFP.The recombinant plasmid pSK-hr3-ie1-EGFP-pri-mir-14,control plasmid pSK-hr3-ie1-EGFP,bmo-miR-14 mimics and negative control mimics were transfected into BmN cells,respectively.The fluorescent light was observed and used to identify the efficiency of transfection.The miRNA levels in transfected BmN cells were identified by qRT-PCR.The RNAhybrid and miRanda were used to predict the targets of bmo-miR-14,respectively.【Result】The miR-14 level was improved in BmN cells by transfecting with pSK-hr3-ie1-EGFP-pri-mir-14 and bmo-miR-14 mimics,and the level was improved as 2.1 and 984 times as the control,respectively.Further,the targeted genes of bmo-miR-14 were identified by bioinformatics tools and a total of 153 and 171 potential targeted genes were identified by RNAhybrid and miRanda software,respectively.In the predicted target genes,a total of 49 genes were predicted by RNAhybrid and miRanda together.The 49 targeted genes were assigned to GO terms of molecular function ontology,and binding and catalytic were overrepresented.In the biological process ontology,cellular process and metabolic process overrepresented among the 49 genes.【Conclusion】The approach to upregulate the level of bmo-miR-14 by transfecting with recombinant expression vector or miRNA mimics has laid a good foundation for the further studies on bmo-miR-14 in BmN cells.