miR-224 is critical for celastrol-induced inhibition of migration and invasion of hepatocellular carcinoma cells.

BACKGROUND/AIMS: 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.

CONCLUSION

Celastrol treatment inhibits migration and invasion of HCC cell and that the effect is partly due to NF-κB regulating miR-224 expression.