Retinoblastoma ( Rb ) promotes senescence of corneal endothelial cells by inhibiting the activation of E2F2

Abstract

Background Senescence-like changes occur in aging Corneal endothelial cells (CECs), and these changes are associated with decreased vision and age-related corneal diseases. such as Fuchs endothelial dystrophy (FED), chronic corneal allograft dysfunction (CCAD). Such changes have also been shown to cultured cell in vitro after passaging. Therefore, studying the mechanism of CEC senescence would aid in the development of anti-senescence treatment, which would benefit FED and CCAD. The tumor suppressor retinoblastoma (RB) gene product pRB triggers senescent growth arrest when inactivated. In this study, we used siRNA treatment to evaluate whether RB knockdown could suppress CEC senescence, and we investigated relevant molecular mechanisms.Methods RCECs were obtained and were cultured with or without siRb. Senescent cells were detected with a β-galactosidase senescence staining kit. The gene p21, which is associated with a senescent phenotype, was measured by RT-PCR. The morphology and migration of cultured RCECs were examined by phase-contrast microscopy. ZO-1 and N-Cadherin, which are involved in pump and barrier functions, were assessed by immunofluorescence. Cell cycle assessment was performed using a flow cytometer (BD FACSCalibur).Results As the cells were passaged, the number of senescent RCECs, the levels of the senescence-related gene p21, and the levels of senescence-associated secretory factors increased. SiRNA-mediated knockdown of RB led to suppression of cell senescence and the SASP. Furthermore, RB intervention increased the numbers of cells at the G2/M and S phase but did not influence the cell function or migratory ability. Knockdown of RB promoted the activation of E2F2.Conclusions We demonstrated that as the cells increased in passage number, the number of senescent RCECs, the levels of the senescence-related genes p21, and the levels of senescence-associated secretory factors increased. Retinoblastoma (Rb) promoted the senescence of corneal endothelial cells by inhibiting the activation of E2F2.