Role of YAP1 in regulating lens epithelial cell proliferation and cell cycle by binding to the SOX2 promoter

Objective  To investigate the role of Yes-associated protein 1 (YAP1) in regulating the proliferation of human lens epithelial cells (LECs) and elucidate its possible mechanism of transcriptional regulation of SRY-box transcription factor 2 (SOX2).

Methods  The human lens epithelial cell line SRA01/04 was used. Cells were transfected with small interfering RNA (siRNA) targeting YAP1 or a negative control (siNC). YAP1 mRNA and protein expression levels were detected by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot to select effective interfering sequences. Cell proliferation was assessed using the cell counting kit-8 (CCK-8) assay and EdU incorporation assay. Western blot was used to detect the expression changes of cell cycle-related proteins Cyclin D1 and Cyclin E1. LECs were divided into empty vector group and YAP1 group, transfected with empty plasmid and the YAP1 plasmid, respectively. The mRNA expression levels of YAP1 and SOX2 were detected by qRT-PCR. Cleavage under targets and tagmentation (Cut&Tag) assay was performed to detect the binding enrichment of YAP1 in the SOX2 promoter region. A SOX2 promoter luciferase reporter system was constructed to verify the effect of YAP1 on SOX2 transcriptional activity. Furthermore, transfection with siSOX2 was conducted to observe the effects of SOX2 knockdown on cell proliferation and the expression of cell cycle-related proteins.

Results  The mRNA and protein expression levels of YAP1 in the siYAP1-1 group were significantly lower than those in the siNC group (both P<0.05). The EdU positive rate in the siYAP1-1 group was (23.2±2.7)%, which was significantly lower than (80.2±0.9)% in the siNC group ( t=31.853, P<0.001). The absorbance values at 24, 48, and 72 hours in the siYAP1-1 group were significantly lower than those in the siNC group (all P<0.05). The relative protein expression levels of Cyclin D1 and Cyclin E1 in the siYAP1-1 group were significantly lower than those in the siNC group ( t=12.857, P<0.001; t=9.432, P=0.001). Cut&Tag sequencing results indicated significant binding enrichment of YAP1 in the SOX2 promoter region in LECs. The relative SOX2 mRNA expression level was significantly lower in the siYAP1-1 group than in the siNC group and significantly higher in the YAP1 group than in the empty vector group ( t=9.914, 11.185; both P<0.001). The relative luciferase activities in the YAP1 group at different doses were significantly higher than those in the empty vector group (all P<0.001). Knockdown of SOX2 significantly inhibited cell proliferation and down-regulated the expression of Cyclin D1 and Cyclin E1, exhibiting phenotypic changes consistent with YAP1 knockdown.

Conclusions  YAP1 promotes the proliferation and cell cycle progression of LECs. Its mechanism may involve YAP1 binding to and transcriptionally activating SOX2, thereby up-regulating the expression of key G1/S phase cell cycle proteins.