title: miR-224 is critical for celastrol-induced inhibition of migration and invasion of hepatocellular carcinoma cells.
abstract: OBJECTIVE
  The molecular mechanisms of celastrol on hepatocellular carcinoma (HCC) cells migration and invasion ability is the major problem that prompted the study.
  
  
  METHODS
  We first evaluated the effect of celastrol on migration and invasion ability of HepG2 cells using transwell migration and matrigel invasion assays. Next, we assessed the effect of celastrol on NF-κB transcriptional activity in hepatocellular carcinoma cells using western blot and luciferase reporter assay. We also performed real-time PCR to measure miR-224, MMP-2 and MMP-9 expression. Western blot was used to measure protein expression of MMP-2 and MMP-9. Furthermore, we used miR-224 inhibitor to evaluate whether down-regulation of miR-224 expression can affect MMP-2 and MMP-9 expression. The binding ability of p65/NF-κB on the miR-224 promote has been assessed by chromatin immunoprecipitation and quantitative real-time PCR (ChIP-qPCR). Finally, we evaluated the effect of miR-224 on celastrol-induced anti-tumor activity using miR-224 precursor.
  
  
  RESULTS
  Celastrol significantly impaired migration and invasion of HepG2 cells and inhibited the activation of NF-κB and Akt in dose-dependent manner. IGF (the strong stimulator of Akt) inhibited the transcriptional activity of NF-κB in cells treated with celastrol. Besides, celastrol efficiently decreased the expression of miR-224 and protein expression of MMP-2 and MMP-9. ChIP-qPCR showed that p65/NF-κB binding to the miR-224 promoter sharply decreased after exposure to celastrol in time-dependent manner. Furthermore, inhibition of miR-224 expression can decrease MMP-9 protein level. Most importantly, miR-224 precursor can reverse the effect of celastrol on impairment of migration and invasion in HepG2 and Huh7 cells.
  
  
  CONCLUSIONS
  Celastrol treatment inhibits migration and invasion of HCC cell and that the effect is partly due to NF-κB regulating miR-224 expression.
authors.affiliation: Department of General Surgery, Shengjing Hospital Affiliated to China Medical University, China Medical University, Shenyang.
authors.forename: Hangyu
authors.initials: H
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.forename: Hongzhi
authors.initials: H
authors.lastname: Sun
authors.forename: Jingang
authors.initials: J
authors.lastname: Liu
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000188:drug therapy; D002465:Cell Movement / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D008113:Liver Neoplasms / Q000188:drug therapy; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D035683:MicroRNAs / Q000494:pharmacology* / Q000627:therapeutic use; D016328:NF-kappa B / Q000378:metabolism; D009361:Neoplasm Invasiveness; D053978:Pentacyclic Triterpenes; D060888:Real-Time Polymerase Chain Reaction; D014315:Triterpenes / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; C546140:MIRN224 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; C050414:celastrol