Bioinformatics analysis of hsa-miR-34a target genes and construction of green fluorescence protein reporter gene vectors

Objective To predict the hsa-miR-34a target genes and construct green fluorescence protein（GFP） reporter gene vectors containing autophagy-associated target genes. Methods Biological targets of hsa-miR-34a were predicted using the online program TargetScan Release 5.1 and RNA22.Their molecular function was analyzed using software Gominer.Oligonucleotide sequences of hsa-miR-34a binding sites were synthesized and cloned into pEGFPC2 after digestion with EcoR I and BamH I.Recombinant plasmids was confirmed by DNA sequencing.Plasmids were transfected into Hela cells with Lipofectamine 2 000.Expression of GFP in Hela cells was observed under a fluorescence microscope. Results A total of 2 904 targets were found in hsa-miR-34a.The functions of hsa-miR-34a included three knowledge domains:molecular function,biological process and cellular component.Two and three miR-34a target islands were observed in autophagy relative genes,beclin1 and sestrin 2 mRNA,respectively.Sequence analysis showed the insert sequence was correct.Gene transfection was mediated by Lipofectamine 2 000 24h later.Fluorescence microscopy displayed that the GFP was expressed in Hela cells. Conclusion The construction of GFP reporter gene vectors has laid a foundation for studying the role of hsa-miR-34a in the molecular mechanism of autophagy.