Vault RNAs partially induces drug resistance of human tumor cells MCF-7 by binding to the RNA/DNA-binding protein PSF and inducing oncogene GAGE6.

Abstract

Vault is the largest nonicosahedral cytosolic nucleoprotein particle, which is widely involved in induction of chemoresistance and lead to failure in long-term chemotherapy. Vault contains three different major vault proteins (MVPs) and four vault RNAs paralogues (vtRNAs, vtRNA1-1, vtRNA1-2, vtRNA1-3 and vtRNA2-1). Disruption of the MVPs do not induce hypersensitivity while expression of vtRNAs contributes to cells' drug resistance, indicates that vtRNAs, but not MVPs play an important role in causing drug resistance. Polypyrimidine tract binding protein associated splicing factor (PSF) contributes to cell sensitivity to chemotherapy by its transcriptional activity, promotes us to figure out its potential association with vtRNAs. We investigate the interaction between PSF and vtRNAs by electrophoretic mobility shift assays (EMSA) and RNA-immunoprecipitation (IP), and showed the binding between PSF and vtRNAs. Chromatin Immunoprecipitation (ChIP) was performed to detect the effects of vtRNAs on the interaction of PSF with GAGE6 promoter. The role of vtRNAs on chemoresistance in MCF-7 was detected by CCK-8 and EdU staining. The independent role of vtRNAs with MVP is detected by MVP or vtRNAs knockdown. The complex with vtRNA1-1 releases PSF, allowing transcription of GAGE6 to proceed. Then we showed that induction of GAGE6 caused drug resistance by promoting cell proliferation and colony formation in soft agar. Ectopic expression of shRNA targets to vtRNA1-1 further confirmed the role of vtRNA1-1 in regulating PSF transcriptional activity independent with the expression of MVP. By vtRNA1-1 or MVP knockdown, it is revealed that vtRNA1-1 caused chemoresistance independent of MVP. Furthermore, knockdown of GAGE6 does not cause drug resistance, indicates the GAGE6 is directly involved in cell proliferation, but not the drug resistance. These results suggest that vtRNAs regulates cell proliferation, drug resistance, and possibly other physiological processes of humans, by complex formation with PSF.