Downregulation of P16 promotes cigarette smoke extract-induced vascular smooth muscle cell proliferation via preventing G1/S phase transition

The proliferation of vascular smooth muscle cells (VSMCs) serves an important role in cigarette smoking-associated vascular diseases; however, the underlying mechanisms responsible for this remain unclear. The aim of the present study was to elucidate the role of P16 in cigarette smoke extract (CSE)-induced VSMC proliferation and the underlying mechanism responsible. Human aortic smooth muscle cells (HAOSMCs) were exposed to CSE, and an MTT assay and flow cytometry were performed to evaluate cell proliferation and cell cycle distribution. Western blotting was conducted to examine protein expression and bisulfite genomic sequencing polymerase chain reaction was used to determine the methylation status of the P16 promoter CpG island. It was demonstrated that treatment with CSE significantly promoted the proliferation of HAOSMCs in a concentration- and time-dependent manner and induced a downregulation in P16 expression (all P<0.05). A luciferase reporter gene assay data demonstrated that CSE treatment induced hypermethylation of the P16 promoter, which led to a significant decrease in its transcriptional activity and significantly reduced P16 protein expression in HAOSMCs (both P<0.01). Furthermore, P16 downregulation induced a significant increase in the expression of cyclin-dependent kinase (CDK) 4, CDK6 and phosphorylated retinoblastoma (p-Rb) protein (all P<0.001) and significantly increased the ratio of cells in S phase in CSE-treated HAOSMCs (P<0.001). Overexpression of P16 inhibited CSE-induced cell proliferation through inducing cell cycle arrest in G1 phase (P<0.001), and led to decreased levels of CDK4 (P<0.01), CDK6 (P<0.01) and p-Rb (P<0.001) in HASMCs. The results of the present study therefore demonstrate that P16 may be associated with the CSE-induced proliferation of VSMCs, suggesting that P16 serves a role in the development of cigarette smoke-associated vascular diseases.