Eurycomalactone switched hepatocellular carcinoma cells into quiescence through 5’tRFAla/DVL/β-catenin pathway inhibition

Although tsRNA has been demonstrated to modulate various physiological processes analogous to miRNA, the potential regulatory functions and mechanisms of tsRNAs related to the pharmacological effects of small molecule drugs remain unclear. Herein, it is shown that eurycomalactone (ELT), a natural product, can reversibly switch hepatocellular carcinoma (HCC) PLC/PRF/5 and HUH7 cells into a quiescent state. This quiescence is characterized by cell proliferation inhibition without cytotoxicity, cell cycle arrest at the G0/G1 phase, and cell reactivation following the removal of ELT. Given the established role of β-catenin activity in mediating cancer cellular quiescence or proliferation, a notable reduction in total, cytoplasmic, and nuclear β-catenin expression, along with its downstream targets Survivin, c-myc, and Cyclin D1, was observed in ELT-treated cells. Subsequently, two new tsRNAs, namely 5'tRF^Ala and 5'tiRNA^Ala, which match well with the mRNAs of two pivotal upstream regulators (DVL2 and DVL3) of β-catenin based on bioinformatics analyses, were detected to be significantly decreased in ELT-treated PLC/PRF/5 cells using Arraystar small RNA microarray analyses. Consistently, the concentrations of the DVL2 and DVL3 proteins were also found to be reduced by ELT. The mimic of 5'tRF^Ala could increase the relative expression of DVL2 and DVL3 mRNA and rescue their decrease induced by ELT, while the mimic of 5'tiRNA^Ala could not. It therefore seems that ELT could down-regulate the expression of 5'tRF^Ala, leading to the suppression of DVL2 and DVL3 mRNA translation, consequently inhibiting the β-catenin signaling pathway and reversibly switching HCC cells into a quiescent state. Conclusively, our findings imply that tsRNAs, like miRNAs, might activate the translation of their matched mRNAs in non-dividing cells and provide a possible potential for repressing tumor cell growth, although further evidence is still needed.