| Background and Objective:Atherosclerosis (AS) is one of the most common vascular disorders. Proliferation of vascularsmooth muscle cells (VSMCs) and the formation of neointima dominate atherosclerosis lesiondevelopment. p53, an essential molecule in cell cycle and apoptosis control, also plays a centralrole in atherosclerosis. Inactivation of p53stimulates the development of atherosclerosis.More than90%of the human genome is transcribed. Whereas protein-coding genesaccount for less than2%of the human genome, non-coding RNAs (ncRNAs) are importantcomponents of the mammalian transcriptome， including the well-known microRNAs(miRNAs), which are~21-23nt long and have been proven to play a key role in the regulationof gene expression and function in a variety of biological and pathological processes Longnon-coding RNAs (lncRNAs), also known as long intergenic non-coding RNAs (lincRNAs),constitute another class of ncRNAs. Defined as non-coding transcripts longer than200nucleotides, at least a subset of lncRNAs are likely to have biological activity. Numerousstudies have already shown the involvement of lncRNAs in cancer development. However,the role of lncRNAs in cardiovascular system is less understood.LincRNA-p21was initially identified as a direct transcriptional target of p53.LincRNA-p21appears to function as a component of the p53pathway, at least in part, byphysically interacting with a p53repressive complex to downregulate many p53target genes.lincRNA-p21also acts as a suppressor of translation by directly associating with targetmRNAs. Despite these studies, the biological function of lincRNA-p21remains elusive. Baseon the above knowledge, in this study, we examined the function of lincRNA-p21in thepathogenesis of atherosclerosis, look into the role of lncNRAs in cardiovascular system.Methods:Part I:(1) We first examined the expression of lincRNA-p21using qRT-PCR in aortic atherosclerotic plaques of ApoE-/-mice fed a high-fat diet, a widely used animalmodel of atherosclerosis.(2) Next, we used the mouse macrophage cell line RAW264.7and the human vascularsmooth muscle cell line HA-VSMC and designed small interfering RNA (siRNA) toinhibit mouse and human lincRNA-p21expression in above2cell lines. Theefficiency of siRNA transfection was tested by qRT-PCR. Then we investigated thefunction of lincRNA-p21in cell proliferation and apoptosis using Cell CountingKit-8and Annexin V-FITC apoptosis detection.(3) In order to understand the molecular mechanism by which linRNA-p21regulates cellproliferation and apoptosis, we performed unbiased gene array analysis to measuregene expression changes in lincRNA-p21knockdown cells and then verified usingqRT-PCR and western-blotting.Part II:(1) To explore possible MDM2binding to lincRNA-p21, we used computationalapproaches to assess the likelihood of protein-RNA interaction. Next, we examinedthe direct binding and binding site between lincRNA-p21and MDM2through RNA-Immunoprecipitation (RIP) and deletion-mapping experiments in human HA-VSMCand mouse RAW264.7cells. To understand how the lincRNA-p21/MDM2interactionaffects the formation of the p53/p300/MDM2complex, co-immunoprecipitation(Co-IP) experiments were performed.(2) We next investigated if inhibition of lincRNA-p21, influenced p53binding to thepromoters/enhancers of its target genes. To this aim, we performed p53chromatinimmunoprecipitation (ChIP) followed by high-throughput DNA sequencing(ChIP-Seq) experiments in HA-VSMCs treated with control or lincRNA-p21siRNA.The observations from ChIP-seq were further verified via ChIP-qPCR.(3) Next, to investigate the interplay of p53and lincRNA-p21on cell proliferation,apoptosis and atherosclerosis, we overexpressed p53in human VSMC cells, with orwithout lincRNA-p21knockdown.(4) It is well established that p53regulates cell proliferation and apoptosis in response tostress. So doxorubicin (Dox), an anti-cancer chemotherapy drug which also causescardiotoxicity, was further induced to examined apoptosis and cell proliferation under stress using TUNEL staining and Ki67labeling. Subsequently，we tested whetherknockdown of lincRNA-p21might affect p53protein level and its acetylation statusunder stress condition by western-blot using anti-p53and-Acetyl-p53antibody. Next,the binding of p53to the promoters/enhancers of its target genes was observed usingChIP-qPCR.Part III:(1) We next investigated the involvement of lincRNA-p21in the formation of neointimain vivo, using the classic murine carotid artery injury model. Recombinant lentivirusvector expressing lincRNA-p21siRNA or control siRNA was injected into the injuredarea of mouse carotid arteries. These mice were then fed with high fat diet for onemonth, then neointima formation was examined. We performed immunostaining onvessel sections to detect the proliferation marker Ki67, apoptosis was also assessedusing TUNEL assay. Then further uncover the role of lincRNA-p21on the interactionof p53-p300-MDM2proteins in vivo via Co-IP.(2) Lastly, we examined the expression of human lincRNA-p21by qRT-PCR assays,using total RNAs isolated from coronary artery tissues of coronary artery diseasepatients and artery tissues of control patients. Similarly, lincRNA-p21level was alsotested in another set of coronatry artery disease and control patients using total RNAsisloated from peripheral blood mononuclear cells.Results：Part I:(1) We found that the expression of lincRNA-p21was substantially lower in the aorticplaques of ApoE-/-mice when compared with that of wild type control mice.(2) Inhibition of lincRNA-p21substantially increased total cell numbers in bothRAW264.7and HA-VSMC cells. Increased cell proliferation and viability in thesecells. Furthermore, lincRNA-p21knockdown decreased apoptosis in both cell lines.(3) We found that331and274genes were up-and down-regulated more than two folds,respectively, after linRNA-p21was knocked down. Gene ontology (GO) analysisindicated that the down-regulated genes are over-represented for functional termsrelated to apoptosis, cell death and the p53signaling pathway. However, we detectedno change in the expression of either p53transcript or protein when lincRNA-p21 was inhibited. We then confirmed that lincRNA-p21depletion in HA-VSMCsdecreased the mRNA and protein levels of p53downstream target genes. Conversely,overexpression of lincRNA-p21induced the expression of these p53target genes inRAW264.7cells.Part II:(1) The online predictor-CatRAPID, predicted nt700-1500of the lincRNA-p21bindingto the RING domain (amino acid residues400-480) of MDM2with thediscriminative power of82%. RIP results showed that lincRNA-p21interacts withMDM2protein in both cell lines. Deletion-mapping experiments showed that thent728-2057region of the lincRNA-p21mediates the interaction with the RINGdomain of the MDM2protein in mouse RAW264.7cells. Through Co-IP experiments,we confirmed the interaction of p300/p53and MDM2/p53increased in p53overexpressing cells. LincRNA-p21knockdown decreased p300/p53interaction andincreased MDM2/p53interaction.(2) We obtained36-53million sequence reads from each experimental sample viaChIP-seq, and greater than95%of them uniquely aligned to the human genome. Incontrol siRNA-treated samples, we identified more than4,800p53-bound regions.Intriguingly, lincRNA-p21knockdown diminished p53binding at many of theseregions. We then compared the peak distributions of known p53-regulated genes, andfound that knockdown of lincRNA-p21dramatically reduced the association of p53to the promoters/enhancers of these genes. ChIP-qPCR assays was further performedto confirm the above observation.(3) Overexpression of p53inhibited VSMC proliferation, inhibition of lincRNA-p21suppressed p53-mediated inhibition of VSMC proliferation. Similarly, p53-inducedapoptosis was markedly repressed in lincRNA-p21knockdown cells. Moreover,lincRNA-p21inhibited the stimulatory effect of p53overexpression on levels ofdown-stream genes.(4) Si-LincRNA-p21partially suppressed the Dox-induced reduction of cell number. Wefurther found that both the induction of cell apoptosis and the reduction of cellproliferation can be rescued after lincRNA-p21knockdown. Next, qPCR datashowed that whereas Dox treatment increased the expression of p53target genes, knocking down endogenous lincRNA-p21partially suppresses such increase of geneexpression. Then, we found that Dox treatment induced the level of acetylated p53significantly, which was reduced when endogenous lincRNA-p21was inhibited. Next,ChIP-qPCR showed that Dox enhances the binding of p53to its targets, which wasreduced when lincRNA-p21was knocked down.Part III:(1) Quantification of intima-media thickness confirmed a significant neointimalhypertplasia after siRNA-lincRNA-p21injection. Cell proliferation was induced andapoptosis was reduced in the LincRNA-p21siRNA-treated vessels in vivo. Consistentwith in vitro studies, there was a reduced binding of p300and p53in si-lincRNA-p21samples. Conversely, the association of MDM2and p53was increased insi-lincRNA-p21treated carotid tissues. As a result, the expression levels of p53targets were repressed in si-lincRNA-p21treated vessels in vivo.(2) The expression of lincRNA-p21was more than50%lower in coronary artery tissuesof coronary artery disease patients compared to control patients. Similarly, we foundthat lincRNA-p21level was also decreased in the peripheral blood mononuclear cellsof coronatry artery disease patients.Conclusion:In this study,(1) We identified lincRNA-p21as a key regulator of cell proliferation and apoptosis,showed that it represses cell proliferation and induces apoptosis in vitro and in vivo.(2) Knockdown of endogenerous lincRNA-p21accelerated neointima formation ininjured carotid arteries.(3) Mechanistically, we found that lincRNA-p21directly binds to MDM2, leading to p53release from MDM2and binding to p300, which thereby enhances p53activity.This finding is significant because it implicates non-coding RNAs in cardiovasculardiseases such as atherosclerosis, and suggests that modulation of the activity of non-codingRNAs such as lincRNA-p21may be a novel therapeutic approach to treat humancardiovascular disease. |
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