Objectives To investigate the relationship and molecular mechanism of lincRNA-p21 in the development of atherosclerosis (AS).
Methods Firstly, we detected the expression of lincRNA-p21 expression in the aortic plaque of apoE-/- mice fed with high-fat diet and peripheral blood mononuclear cells of clinical coronary disease patients. Then we observed the role of lincRNA-p21 in cell proliferation and apoptosis using mice macrophage cell line RAW264.7 and human vascular smooth muscle cell line HA-VSMCs by loss-of-function and gain-of function approaches. Meanwhile, the expression of mRNA and protein levels of apoptosis-related downstream targeting of p53 also been detected. Furthermore, we preformed bioinformatics prediction, RNA-Immunoprecipitation (RIP), RNA-pulldown and deletion mapping experiments to test the potential interaction and specific interaction pattern between lincRNA-p21 and MDM2. Then co-Immunoprecipitaiton (co-IP) and Chromatin- Immunoprecipitation (ChIP) assays were preformed to verify the possible influence on p53 transcriptional activity of this binding. We further investigated whether or not lincRNA-p21 was involved in the formation of neointimal hyperplasia in vivo, therefore recombinant lentivirus vector expressing Si-RNA against lincRNA-p21 was injected into the injured area of mouse carotid arteries.
Results In this study, we have indentified lincRNA-p21 was down-regulated in apoE-/- AS model mice. By loss-of-function and gain-of-function approaches, we found that both lincRNA-p21 and p53 could repress proliferation and induce apoptosis inVSMCs. LincRNA-p21 knockdown blocked P53 signalling and the effect of p53 on VSMC proliferation and apoptosis. MDM2 is a key factor to regulate p53 activity by prevention of P300-induced p53 acetylation. We found that lincRNA-p21 can binding to MDM2 directly, which makes the MDM2 dissociation with p53, therefore, leads to p53 acetylation by P300, increases p53 activity. This finding is of significance, because after treatment with SiRNA against lincRNA-p21, the neointimal hyperplasma is increased dramatically in the injured carotid artery in mouse. Therefore, lincRNA-p21 might be a switch of MDM2, via indirectly effect on p53 activity, affect VSMC proliferation and apoptosis, which is involved in the pathogenesis of atherosclerosis.
Conclusions It is well-known that p53 plays an important role on the pathogenesis of AS. Both the quantity and transcriptional activity of p53 can be regulated by multiple ways. MDM2, a p53 positive responsive gene, take part in both this two pathways, and the P300-MDM2-p53 complex plays a key important role in the regulation of p53. LincRNA-p21 could bind to the RING domain of MDM2 through its 5’ end 728-2057nt region, prevent the binding of MDM2 with p53, therefore, there would be more p53 binding with P300, which leads to p53 acetylation, increased p53 activity. After treatment with siRNA of lincRNA-p21, the neointimal hyperplasma is increased in the injured carotid artery in vivo.
In conclusion, lincRNA-p21 might be a switch of MDM2, the binding between lincRNA-p21 and MDM2 would release more free P53, which would be binding with P300, leads to P53 acetylation, indirectly affects on P53 activity, increases VSMC proliferation and decreases VSMCs apoptosis, involved in the pathogenesis of atherosclerosis.