Sal-like protein 2 upregulates p16 expression through a proximal promoter element.
Bottom Line: Promoter-reporter assays of p16(INK4A) and several other tumor-related genes indicated that the Sall2 regulation of these promoters was not significantly different between the two major forms of Sall2 with alternative exon 1 or exon 1A.Finally, to confirm the significance of Sall2-activated p16 expression in cell cycle regulation, we co-transfected the SKOV3 cells with a Sall2 expression construct and a p16 minigene and also co-transfected the ES-2 cells with a Sall2 expression construct and the siRNA against p16 for flow cytometry analysis.Our results showed that Sall2 enhanced the p16 minigene blocking of cell cycle progression and p16 knockdown with siRNA abolished most of the Sall2 inhibition of cell cycle progression.
Affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.Show MeSH
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Mentions: To confirm that the cell cycle regulation functions of Sall2 also include the promotion of p16 expression, we studied the effect of Sall2 on the cell cycle progression by Sall2-induced p16 expression (Fig.5). To take advantage of the defective p16 background in SKOV3 cells for comparison, we transfected a p16 expression plasmid under p16 promoter control, named p16 minigene (Fig.5a) and a Sall2 expression construct, either separately or in combination, to SKOV3 cells co-transfected with EGFP to mark the transfected cells in flow cytometry assays (Fig.5b). Cell population at G0/G1, G2/M or S phase were analyzed (Fig.5c) and showed that the percentage of cell population in G2/M phase changed significantly with different transfections. Compared with pcDNA3 empty vector control, the transfection of p16 minigene alone reduced the G2/M population from 6.97% to 4.06%, a 0.58 fold of the control cells (Fig.5d); similarly, Sall2 transfection alone reduced the G2/M population to 4.43%, a 0.64 fold of the control level. However, the co-transfection of both Sall2 and p16 minigene reduced G2/M level to 1.02% or 0.15 fold of the control level, less than half of the expected 0.37 fold if no interaction between p16 minigene (0.58 fold of control) and Sall2 (0.64 fold of control). The additional G2/M reduction caused by co-transfection of Sall2 and p16 minigene is consistent with the upregulation of p16 minigene expression by the co-transfected Sall2.
Affiliation: School of Pharmacy, Shanghai Jiao Tong University, Shanghai, China.