Fig. 4

The RPTOR-regulated SPHK2/S1P pathway via binding to transcription factor YY1. A Dual-luciferase reporter assays indicated that RPTOR enhanced SPHK2 expression in H1299 and PC9 cell lines. B Transcription factor YY1 was screened to significantly enhance the transcriptional activity of SPHK2 when compared with other candidate transcription factors like HIF1A and ETV7, when co-transfected with pGL3-SPHK2. C-F qRT-PCR assays revealed a positive correlation between SPHK2 and YY1 expression, with both YY1 and SPHK2 expression down-regulated after treatmenting PC9 and H1299 cells with pcDNA-YY1 or YY1 siRNA. G Co-IP assays showed mutual interactions between RPTOR and YY1 in lung cancer cell lines. H–K The JASPAR database was used to predict YY1-binding sites in the SPHK2 promoter sequences. BS1 and BS2 were predicted to be YY1-binding sites, with BS2 at -353 ~ -365 nt of SPHK2 reporting a greater DNA enrichment by ChIP assays. The enrichment of SPHK2 DNA was reduced by mutation of BS2, confirming that BS2 is the site in the SPHK2 promoter bound by YY1. L-M Effect of YY1 expression on SPHK2 promoter sequences. YY1 enriched fewer SPHK2 promoter sequences after si-YY1 1# transfection but more sequences after pcDNA-YY1 transfection