Icariin Attenuates Atherosclerosis Through Long Noncoding RNA H19-mediated Regulation Of PKC??

Atherosclerosis?AS?,characterized by progressing atherosclerotic plaques formation and luminal narrowing of arteries,underpins serious complications with high morbidity worldwide.Although the application of statins was carried out over the past decades,the potential adverse effects can not be ignored.Hence,identifying novel curative strategies as well as new biomarkers for the therapeutic targeting in AS are desired.Noncoding RNAs?ncRNAs?are emerging as powerful regulators of many life activities.Recent study demonstrated that long noncoding RNAs?lncRNAs?play vital roles in development and are closely associated with the pathogenesis of diseases.Studying functional roles of lncRNAs in pathophysiology may provide new insights into causal pathogenic mechanism and opportunities for novel diagnostic and therapeutic strategies in human diseases.Icariin is the main bioactive pharmaceutical ingredient of Herba Epimedii.Evidence indicates that icariin possesses beneficial actions for protecting cardiovascular system.Moreover,icariin is reported to attenuate the formation of atherosclerosis,which suggests its therapeutic potential in preventing or treating cardiovascular diseases.However,the concrete molecule mechanisms of icariin's protective effect in atherosclerosis need to be further investigated.Previous studies mainly focused on target coding genes of icariin,the understanding about the effect of icariin on the lncRNAs was not enough.In this study,the anti-atherosclerotic properties and molecular mechanism of icariin mediated by lncRNAs were explored through animal model and cellular experiments,in order to investigate the function of lncRNA in therapeutic effect of icariin against atherosclerosis.The aim of our study is explaining the pathological mechanisms of atherosclerosis from the perspective of lncRNAs,and elucidating the possible mechanisms of icariin against atherosclerotic development.In vivo experiments,as we established the atherosclerotic model in ApoE^-/-mice with high-fat diet,the AS model group developed obvious atherosclerotic lesions with Oil Red O in the aortic tree.Compared with the AS model group,lipid metabolism was significantly improved with icariin treatment.En face analysis revealed that icariin administration remarkably reduced the size of atherosclerotic lesions,and the pathological damage of aorta was also alleviated significantly.LncRNA and mRNA integrated microarrays were performed on aortic tissues of ApoE^-/-mice.Through differentially expressed genes profile screening and bioinformatics analysis,lncRNA H19 and PKC??were identified as the core genes for further research.Quantitative real-time PCR?qPCR?was performed to further validate the expressions of selected lncRNAs and mRNAs.Subsequently,immunohistochemistry was used to detect the expression of PKC??in aortas of each group.The results showed that lncRNA H19was up-regulated and PKC??was down-regulated in icariin-treated groups,the identified genes might play critical roles in anti-atherosclerotic by effect of icariin.Proliferation and migration of vascular smooth muscle cells are central events in atheroma formation.To simulate high-fat internal environment of atherosclerosis,human aortic smooth muscle cells?HA-VSMCs?were stimulated by oxidized low density lipoprotein?ox-LDL?in vitro.Flow cytometry,MTS assay and Transwell methods were applied to demonstrate the inhibition of icariin on cell cycle,cell proliferation and migration in HA-VSMCs.QPCR and Western blot demonstrated that the expression of PKC??,Cyclin D1 and MMP-9 were down-regulated in icariin-treated HA-VSMCs.Icariin might suppress cell proliferation and migration via down-regulating of PKC??,Cyclin D1 and MMP-9 in HA-VSMCs.According to the core position of PKC??in the Gobal Signal Transduction Network of microarray analysis,we tested the role of PKC??in regulating key events linked to cell proliferation and migration.Inhibition of PKC??induced the down-regulation of Cyclin D1 expression,blockade of the cell cycle progression,and reduction of cell proliferation.Meanwhile,inhibition of PKC??induced the down-regulation of MMP-9 and attenuated cell migration in HA-VSMCs.Consistent with these findings,activation of PKC??could partly restore the inhibition of icariin on the expression of Cyclin D1 and MMP-9,and partly reverse the suppression of icariin on proliferation and migration in HA-VSMCs.PKC??was identified as an important gene of icariin's anti-atherosclerotic effects.These results indicated a critical role of PKC??in suppressing HA-VSMCs proliferation and migration.ICA could inhibit the expression of Cyclin D1 and MMP-9,block cell proliferation and migration via down-regulating PKC??.In order to explore the mechanism of icariin in atherosclerotic progression,we initially verified the correlation between icariin and lncRNA H19 expression level in HA-VSMCs.Furthermore,to investigate the molecular function of lncRNA H19 in HA-VSMCs,the expression level of lncRNA H19 was modified by over-expression vector of lncRNA H19?pLVX-H19?or small interfering RNA H19?si-H19?.Cell proliferation and migration were observed,the expression of PKC??,Cyclin D1 and MMP-9 were also evaluated.LncRNA H19 could negatively regulate cell cycle progression,cell proliferation and migration.Meanwhile,the over-expression of lncRNA H19 was accompanied by the inhibition of PKC??,Cyclin D1 and MMP-9expression.Hence,lncRNA H19 served as a regulatory factor for PKC??,Cyclin D1and MMP-9 in HA-VSMCs.Moreover,the regulatory relationship between lncRNA H19 and PKC??was demonstrated.The inhibition of PKC?partially reduced the promoted effect of si-H19 on proliferation and migration in HA-VSMCs,accompanied by a decrease in Cyclin D1 and MMP-9 expression.Consistent with the hub role in intracellular signal transduction of PKC??,lncRNA H19 inhibited the expression of Cyclin D1 and MMP-9 via down-regulating the expression of PKC??,resulted in suppressing of cell proliferation and migration in HA-VSMCs.On the basis of identified regulatory function of lncRNA H19,knock-down of lncRNA H19significantly reversed the inhibitory effect of icariin on cell proliferation and migration,and also restored the expression levels of PKC??,Cyclin D1 and MMP-9.Our results suggest that icariin inhibits proliferation and migration of HA-VSMCs via up-regulating lncRNA H19 and subsequent inhibition of PKC??,which is the upstream regulator of Cyclin D1 and MMP-9.Taken together,lncRNA H19 is associated with the expression of PKC??,which is involved in regulating cell proliferation and migration.Blocking the signaling pathway might have a cumulative effect on inhibiting cell proliferation and migration in HA-VSMCs,which might be a meaningful measure for suppressing the progress of AS.LncRNA H19 is an important target of icariin in anti-atherosclerotic effect,the regulatory relationship of lncRNA H19-PKC??interferes with the effect of icariin on cell proliferation and migration in HA-VSMCs.This study provides an important complement to the biological function of lncRNA H19,and also provides new theoretical basis for icariin used to treat atherosclerosis.