MiR-198 overexpression leads to its vesicular release from hepatocellular carcinoma cells

Background and Aim:

With the progression of liver diseases, miR-198 is significantly downregulated in hepatocellular carcinoma (HCC). Previous studies have shown that miR-198 is a tumor suppressor in various cancer types, including liver. Therefore, we focused on molecular mechanisms of miR-198's reduction in hepatoma cells.

Methods:

For conditional, doxycycline-induced miR-198 expression, the miR198 encoding sequence has been inserted in a Tet-on vector system and a stable, transgenic Huh7 hepatoma cell line was generated. Vesicles were isolated from medium supernatant by ultracentrifugation. RNA from cells, supernatants, and from exosomal fractions was quantified by qPCR. Impact of miR-198 on protein expression was studied by immunoblotting.

Results:

Under the Tet-on inducible system, after doxycycline treatment intracellular miR-198 was massively overexpressed within the first 12 hours. Interestingly, miR-overexpression was followed by a marked decrease in the next 36 hours. Meanwhile, in the supernatant there was a significant higher amount of extracellular miR-198. Isolation of exosomes from the supernatants demonstrated that extracellular miR-198 was enormously enriched in the exosomal fractions, characterized by the exosome markers, CD63 and HSP70. Both Ago2 and ubiquitin immunoprecipitation have shown that in HCC cells, miR-198 was interacting with Ago2 proteins and strongly associated with ubiquitin. Furthermore, treatment with various inhibitors proved vesicular release of miR-198 by an ubiquitin-associated pathway.

Conclusion:

In hepatoma cells, intracellular levels of tumor suppressor miR-198 are tightly controlled. The loss of the tumor suppressor miR-198 during hepatocarcinogenesis is suggested to be partly due to vesicular release from HCC cells during the transformation process.