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《Scientia Agricultura Sinica》 2013-06
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Upregulation and Expression of Bombyx mori bmo-miR-14 and Prediction of Its Target Genes

LIU Yue1,YANG Lan-cui2,NIE Zuo-ming2,LU Xuan1,Lü Zheng-bing2,CHEN Jian2,YU Wei2,WU Xiang-fu2,ZHANG Yao-zhou2(1Departmen of Applied Engineering,Zhejiang Economic & Trade Polytechnic,Hangzhou 310018;2Institute of Biochemistry,School of Life Science,Zhejiang Sci-Tech University,Hangzhou 310018)  
【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.
【Fund】: 浙江省自然科学基金项目(LY12C06003 Y3090304);; 国家高技术研究发展计划“863”项目(2011AA100603)
【CateGory Index】: S881.26
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