Tumor suppressor microRNA-198 is actively transported out of liver cancer cells

Background and Aim: microRNA-198(miR-198) has been proven as a tumor suppressor in liver cancer cells, inhibiting cell growth and proliferation. Previous studies have shown that miR-198 is the most downregulated miRNA during liver cancer progression. Therefore, we aimed to study the mechanism of miR-198 decrease in liver cancer cells.

Methods: A miR-198 expression cassette was cloned downstream to a tet-on promoter and then stably transfected into huh7 liver cancer cells. Isolated total RNA was applied to real time PCR miR-198 quantification. Furthermore, vesicles were isolated from cell culture supernatants by serial centrifugation and the vesicular proteins and RNA were characterized by proteomics, immunoblot and real time PCR. MTT cell growth tests and migration assays were performed in order to study the function of miR-198 overexpression.

Results: In contrast to treatment of liver cancer cells with miR-198 mimics, miR-198 overexpression induces no cell growth inhibition. However, it leads to an immense miR-198 release from cancer huh7 cells, which is associated with release of vesicles into the cell supernatants. Characterization of vesicles proved the exosome markers, CD63 and HSP70. Importantly, we show that exosomes, released from liver cancer cells and carrying miR-198, can be up-taken by other cell types. Furthermore, cells, without endogenous miR-198 expression, respond with growth inhibition after exosome treatment.

Conclusion: In liver cancer cells, tumor suppressor miR-198 is tightly controlled and is preferably sequestered in vesicles and transported into medium via exosomes. This altogether can provide new insights in exosomal miRNA release and intercellular communication pathways.