Eurycomalactone switches hepatocellular carcinoma cells into a quiescence state through 5'tRFAla&5'tiRNAAla/DVL/β-catenin pathway inhibition

Although tsRNA was reported to have the ability to modulate a variety of physiological processes analogous to miRNA, the possible regulatory functions and mechanisms of tsRNAs related to the pharmacological effects of small molecules are still obscure. Herein it is shown that the natural product eurycomalactone (ELT) can reversibly switch hepatocellular carcinoma (HCC) PLC/PEF/5 and HUH7 cells into a quiescence state characterized by cell proliferation inhibition without cell death, cell cycle arrest at G0/G1 phase and cell reactivation after drug withdrawal. On the basis that β-catenin activity might mediate the proliferation or quiescence of cancer cells, the total, cytoplasmic and nuclear β-catenin, as well as its target proteins, c-myc, CD44 and cyclin D1, are all shown to be significantly weakened by ELT. Subsequently, two new tsRNAs, namely 5'tRFAla and 5'tiRNAAla, which match well with the mRNAs of Dishevelled proteins DVL2 and DVL3, two vital upstream regulators of β-catenin, are found to be decreased significantly in ELT-treated PLC/PEF/5 cells. The concentrations of the DVL2 and DVL3 proteins are similarly reduced. Accordingly, it is suggested that ELT decreases 5'tRFAla and 5'tiRNAAla levels and so suppressing the translation of their matched DVL2 and DVL3 mRNAs that thereby inhibits the β-catenin pathway and so reversibly switching HCC cells into a quiescence state. As such, this study suggests that, like miRNAs, tsRNAs might activate the translation of their matched mRNAs in non-dividing cells and so providing a possible means for suppressing tumor cell growth tsRNAs’ expression in hepatocellular carcinoma (HCC) PLC/PEF/5 cell was measured at 48h after exposure to doses of 8 μM ELT. The total cellular RNA of each sample was respectively extracted by Trizol and was mixed by three cell culture dishes to minimize errors.