Translation of yes-associated protein (YAP) was antagonized by its circular RNA via suppressing the assembly of the translation initiation machinery

摘要

The circYap expression in tissues and cells. The expression of circYap in the tumor and paracancerous tissues of breast cancer patients.  = 12. The expression of circYap in immortalized non-cancerous cell lines (BEAS2B, HaCaT, HGF, HEK293T) and tumor cell lines (MDA-MB231, MCF-7, MDA-MB468, SKBR3, BoM1833, HepG2, JHH1, SNU449) were examined by real-time PCR analysis.  = 4–10 **    0.01 compared to the expression in HaCaT cells. The Yap protein levels in immortalized non-cancerous cell lines (BEAS2B, HaCaT, HGF, HEK293T) and tumor cell lines (MDA-MB231, MCF-7, MDA-MB468, SKBR3, BoM1833, HepG2, JHH1, SNU449) were examined by western immunoblotting. The density of bands were quantified and analyzed with Quantity One program (Bio-Rad).  = 4. **  d Upper, expression of circYap after the cells transiently transfected with circYap plasmid or vector in B16 mouse melanoma cells, MDA-MB231 human breast cancer cells and HepG2 human liver cancer cells. Lower, expression of circYap was examined with the junction primers (forward primer spanned the back-splicing junction) or non-junction primers (forward and reverse primers were located at the two side of the junction). The levels of circYap were compared to the levels of Yap mRNA and housekeeping gene U6 in HEK293 cells and MDA-MB231 cells. Structure of circYap. The existence of circYap was validated by Sanger sequencing. Red letters and square represent “head to tail” junction of circYap. The expression of circYap (left) and Yap mRNA (right) in MDA-MB231 cells that stably overexpressing circYap was compared to those in vector control cells. An equal amount of RNA was also incubated with or without RNase R for 15 min at 37 °C. The spike-in RNA was added after treatment to serve as internal control.  = 6. **  ##  g Vector or plasmid containing circYap or its linear precursor were transient transfected to HEK293T cells. The junction primers and non-junction primers were used to amplify circYap in wide-type (wt), vector, circYap overexpressed and its linear precursor overexpressed cells. The lower panel of bar graph shows the circYap expression detected by qPCR.  = 3 **  h The vector control and circYap overexpressed cells were cultured in serum-free medium for 3 days and re-culture the cells in 10% FBS medium for 24, 32, 40, 48, 56, 64 and 72 h before sample collection. Yap protein expression was examined by western immunoblotting (left). The density of bands was quantified with Quantity One program (left).  = 3 *  p