title: The Effect of Different microRNA Backbones on Artificial miRNA Expression and Knockdown Activity Against HIV-1 Replication.
abstract: Artificial microRNAs (miRNAs) are designed to develop an RNAi-based gene therapy. Recently, it has been suggested that the flanking sequences and terminal loop structure play a critical role in RNAi biogenesis and target recognition, but no extensive study regarding the different miRNA backbone for artificial miRNAs optimization has been conducted.
  
  
  
  We tested three artificial miRNAs with human hsa-miR30a (common miRNA), hsa-miR150 (T cell specific miRNA), and hsa-miR122 (liver specific miRNA) backbones in HEK-293T and Jurkat cell lines.
  
  
  
  Artificial miRNA processing and knockdown efficiency were analyzed by stem-loop RT-PCR, qRT-PCR, luciferase assay and target challenging.
  
  
  
  We identified strikingly different RNAi activities among these different artificial miRNAs. Our results demonstrated that expression and function of art-miR150 was more than art-miR30 and artmiR122 in both HEK-293T and Jurkat cell lines. Since the main difference in these artificial miRNAs was flanking sequences and terminal loop structure, the change between the expression and function of artificial miRNAs can be attributed to these structures.
  
  
  
  This study showed that expression of cell-specific artificial miRNA in target and nontarget cells is not different, but variation in flanking sequences and terminal loop can be involved in expression and function of artificial miRNAs. These results can be important for improving artificial miRNA design in RNAi-based gene therapy.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Nejati
authors.forename: Masoud
authors.initials: M
authors.lastname: Soleimani
authors.forename: Ehsan
authors.initials: E
authors.lastname: Arefian
authors.forename: Sayed M
authors.initials: SM
authors.lastname: Marashi
authors.forename: Hamideh
authors.initials: H
authors.lastname: Tabatabaie
authors.forename: Mohammad
authors.initials: M
authors.lastname: Farahmand
authors.forename: Zabihollah
authors.initials: Z
authors.lastname: Shoja
authors.forename: Mahmood
authors.initials: M
authors.lastname: Mahmoodi
authors.forename: Rakhshandeh
authors.initials: R
authors.lastname: Nategh
authors.affiliation: Virology Department, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran. shahmahmoodi@tums.ac.ir.
authors.forename: Shohreh
authors.initials: S
authors.lastname: Shahmahmoodi
mesh_terms: D045744:Cell Line, Tumor; D015316:Genetic Therapy / Q000379:methods; D057809:HEK293 Cells; D015658:HIV Infections / Q000628:therapy; D015497:HIV-1 / Q000235:genetics* / Q000254:growth & development*; D006801:Humans; D019169:Jurkat Cells; D035683:MicroRNAs / Q000235:genetics*; D034622:RNA Interference; D014779:Virus Replication / Q000235:genetics*
chemical_list: D035683:MicroRNAs; C531907:MIRN122 microRNA, human; C533097:MIR150, human; C546086:MIRN30b microRNA, human