title: Lentiviral miR30-based RNA interference against heparanase suppresses melanoma metastasis with lower liver and lung toxicity.
abstract: OBJECTIVE
  To construct short hairpin RNAs (shRNAs) and miR30-based shRNAs against heparanase (HPSE) to compare their safety and their effects on HPSE down-modulation in vitro and in vivo to develop a more ideal therapeutic RNA interference (RNAi) vector targeting HPSE.
  
  
  METHODS
  First, we constructed shRNAs and miR30-based shRNAs against HPSE (HPSE-shRNAs and HPSE-miRNAs) and packed them into lentiviral vectors. Next, we observed the effects of the shRNAs on knockdown for HPSE expression, adhesion, migration and invasion abilities in human malignant melanoma A375 cells in vitro. Furthermore, we compared the effects of the shRNAs on melanoma growth, metastasis and safety in xenograft models.
  
  
  RESULTS
  Our data showed that these artificial miRNAs targeting HPSE could be effective RNAi agents mediated by Pol II promoters in vitro and in vivo, although these miRNAs were not more potent than the HPSE-shRNAs. It was noted that obvious lung injuries, rarely revealed previously, as well as hepatotoxicity could be caused by lentivirus-mediated shRNAs (LV shRNAs) rather than lentivirus-mediated miRNAs (LV miRNAs) in vivo. Furthermore, enhanced expression of pro-inflammatory cytokines IL-6 and TGF-β1 and endogenous mmu-miR-21a-5p were detected in lung tissues of shRNAs groups, whereas the expression of mmu-let-7a-5p, mmu-let-7b-5p and mmu-let-7c-5p were down-regulated.
  
  
  CONCLUSIONS
  These findings suggest that artificial miRNAs display an improved safety profile of lowered lung injury or hepatotoxicity relative to shRNAs in vivo. The mechanism of lung injuries caused by shRNAs may be correlated with changes of endogenous miRNAs in the lung. Our data here increase the flexibility of a miRNA-based RNAi system for functional genomic and gene therapy applications.
authors.affiliation: Department of Dermatology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China.
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Liu
authors.forename: Qiu-su
authors.initials: QS
authors.lastname: Tang
authors.forename: Hong-chao
authors.initials: HC
authors.lastname: Chen
authors.forename: Xiao-ling
authors.initials: XL
authors.lastname: Jiang
authors.forename: Hong
authors.initials: H
authors.lastname: Fang
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D005966:Glucuronidase / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008099:Liver / Q000187:drug effects*; D008168:Lung / Q000187:drug effects*; D008545:Melanoma / Q000473:pathology*; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D009362:Neoplasm Metastasis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000633:toxicity*; D060888:Real-Time Polymerase Chain Reaction; D000099175:Heparanase
chemical_list: D005966:Glucuronidase; D035683:MicroRNAs; D034741:RNA, Small Interfering; D000099175:Heparanase
keywords: RNA interference; heparanase; metastasis; microRNA(miRNA); safety