Overexpression of tumor suppressor miR-198 in hepatoma cells leads to its spontaneous and active export via vesicles

Background and Aim:

As a potent tumor suppressor, miR-198 expression is downregulated in various cancer types including liver cancer. Previous reports have shown that circulating miR-198 was detected in serum samples from patients with hepatocellular carcinoma. In the present study, we focused on molecular mechanisms of miR-198"s decrease in hepatoma cells.

Methods:

For conditional, doxycycline-induced miR-198 expression, the miR198 encoding sequence was cloned in a Tet-on vector system and stable, transgenic Huh7 and Hep3B derived hepatoma cell lines was generated. Vesicles were isolated from cell culture supernatants by ultracentrifugation. miR-198 of NA preparations, obtained from cells, supernatants, and from exosomal fractions, was quantified by qPCR. The miR-198 interacting proteome was analysed by pull down experiments followed by mass spectrometry and validated by immunoblotting. The impact of ubiquitination on the miR-198 vesicle export was studied functional transgenic mutants.

Results:

Under the Tet-on inducible system, after doxycycline treatment intracellular miR-198 was massively overexpressed within the first 8 hours. Interestingly, miR-198 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 ubiquitin-associated pathway.

Conclusion:

In hepatoma cells, intracellular expression levels of miR-198 were tightly modulated. The decrease of the tumor suppressor miR-198 during hepatocarcinogenesis is proposed to be due to, at least partly, vesicular release.