| ?Background? Estrogen re Placement thera Py(ERT)has long been believed to reduce the risk of cardiovascu Lar diseases in Postmeno Pausal women.However,the Women's Health Initiative declared no cardiovascu Lar benefits from ERT.The controversy continues and has led to the “timing hy Pothesis”.As a su PPort,the recent Danish Osteo Porosis Prevention Study showed that women receiving ERT early after meno Pause had significantly beneficial cardiovascu Lar outcomes.Thus,the cardiovascu Lar effects and the underlying mechanisms of estrogen need to be further investigated.Estrogen retards atherogenesis in numerous in vivo and in vitro ex Periments.Mechanistically,17?-estradiol(E2)im Proves li Pid Profile,induces vasorelaxation by increasing nitric oxide and hydrogen su Lfide release,accelerates endothelial re Pair by Promoting endothelial Proliferation and migration,inhibits vascu Lar smooth muscle cell Proliferation and migration,alleviates vascu Lar inflammatory res Ponse by modu Lating functions of immune cells,etc.Among these,the direct effects of E2 on vascu Lar endothelial cells a PPear to be critical since endothelial dysfunction is the initial ste P of atherosclerosis(AS).However,the molecu Lar mechanisms of estrogen's actions in vascu Lar endothelial cells are not fu Lly elucidated.Micro RNAs(mi RNAs)are short nucleotides and non-coding RNA molecu Les that base-Pair with the 3' untranslated region of target mRNAs,leading to the block of their translation.In recent years,mi RNAs are efficiently studied as im Portant candidates for involvement in vascu Lar endothelial functions and AS.For instance,mi R-126 maintains a Proliferative reserve in vascu Lar endothelial cells and Prevents atherosclerotic lesion formation.mi R-26 Prevents endothelial cell a Po Ptosis and retards atherogenesis.Thus,the identification of mi RNAs may Provide novel molecu Lar insight and new thera Peutic strategies to treat AS.Estrogen is the Powerfu L regu Lator of mi RNAs.For exam Ple,E2 stimu Lates MCF-7 breast cancer cell migration and invasion via downregu Lation of mi R-124.Notwithstanding,these studies were Performed exclusively in re Productive organs including mammary gland and uterus.To our knowledge,currently there is no evidence showing the functions of estrogen-regu Lated mi RNAs and their im Plications in AS.Recent evidence shows that mi R-126-3P,the micro RNA highly enriched in the endothelium,Plays crucial role in endothelial Proliferation,migration and tube formation.In mice model,the atherosclerotic lesion area in mi R-126-/-Apo E-/-group was significantly increased when com Pared to mi R-126+/+ Apo E-/-control group.Therefore,in this study,we selected to investigate if E2 regu Lates mi R-126-3P ex Pression.Firstly we measured the serum level of mi R-126-3P in cycling women and revealed a Positive relationshi P between mi R-126-3P and E2.Then we used Apo E-/-female mice as atherosclerosis model to observe the role of mi R-126-3P in estrogen's anti-atherogenesis effects.Since endothelial dysfunction is critical for AS,we utilized human vein umbilical endothelial cells(HUVECs)as cell model to confirm the effects of E2 on mi R-126-3P ex Pression and we identified the critical role of Ets-1(a transcri Ption factor for mi R-126-3P)in it.Spred1(a Protein that inhibits mitogenic signaling)is the well-known target of mi R-126-3P and it regu Lates endothelial Proliferation,migration,tube formation and monocyte adhesion,so finally we observed the effects of E2 on Spred1 Protein ex Pressions via mi R-126-3P and its im Pacts on endothelial functions.?Objectives? Endothelial micro RNA 126(mi R-126)attenuates the develo Pment of atherosclerosis(AS).However,there is no evidence showing the role of mi R-126 in estrogen's anti-atherogenic effects.We hy Pothesized that 17?-estradiol(E2)modu Lates mi R-126 ex Pression,thus imProves endothelial function and retards AS develo Pment.?Methods and Results? 1.17?-estradiol reduced aortic atherosclerotic lesion via mi R-126-3p 1.1 The serum level of E2 was positively associated with mi R-126-3p level As ex Pected,hormones levels varied across the menstrual cycle.The relative level of serum mi R-126-3P expression was increased during ovu Lation phase and midluteal Phase when compared to its expression during early follicu Lar phase.Moreover,the serum level of E2 was positively associated with serum mi R-126-3P level.1.2 E2 reduced aortic atherosclerotic lesion and Spred1 expression via mi R-126 1.2.1 Aorta sinus plaque was extensively found in OVX group when compared to SHAM group of Apo E-/-mice.The size of plaque was significantly diminished in OVX + E2 group(P<0.001)and this effect was largely reversed when mice were injected with antago-mi R-126-3p(OVX + E2 + antago-mi R126 group,P<0.01).The injection with mi R-126-3p mimics alone(OVX + mi R-126)also reduced the lesion area.In addition,negative control of antago-mi R and mimics had no effect on the size of Plaque.1.2.2 The staining of the histopathologic slides of aortic roots showed that in OVX group,Spred1 was extensively found in plaque area and they were reduced in OVX+E2 or mi R-126-3P mimics alone groups.The antago-mi R-126-3p reversed E2 effect on the ex Pression of Spred1.2.The mechanistic exploration of the effect of E2 on mi R-126-3p expression 2.1 E2 increased mi R-126-3p expression in HUVECs 2.1.1 Treatment with E2(1 n M-1?M)for 24 h all increased pri-mi R 126 and mature mi R-126-3P expression,with the increasing magnitude of(176.5±20.2)%(n = 5,P<0.05),(321.5±38.6)%(n = 5,P<0.05),(266.9±31.4)%(n = 5,P<0.05),(203.2±39.6)%(n=5,P<0.05),res Pectively.The maximal effect was achieved by E2 at the concentration of 10 n M.2.1.2 E2(10 n M)increased mi R-126-3P ex Pression from 12 to 72 h,with the increasing rate of(265.3±28.5)%(n=5,P<0.01),(341.6±35.7)%(n=5,P<0.01),(228.6±42.1)%(n=5,P<0.01),(196.5±25.6)%(n=5,P<0.01).2.2 E2 increased mi R-126-3p ex Pression via ER?.To identify the subtype of ER involved,HUVECs were treated with selective ER? agonist PPT or ER? agonist DPN.It was found that PPT,while not DPN,mimicked the effect,which was inhibited by the Pure ER antagonist ICI 182,780.2.3 E2 increased mi R-126-3p ex Pression via Ets-1.Genome bioinformatics Promoter analysis revealed that SP1 and Ets-1 binding motifs are Prevalent in mi R-126 host EGLF7 gene.In order to identify their roles,we silenced these two transcri Ption factors by using si RNAs.E2(10n M)failed to increase mi R-126-3p expression when Ets-1 was knockdown,while this effect was not altered by the silencing of SP1.2.4 The underlying mechanism of E2 on Ets-1 expression.2.4.1 E2 increased Ets-1 transcriptional activity.To examine the role of E2 on Ets-1 transcriptional activity,HUVECs were transiently transfected with an Ets-1 binding site(EBS)-luciferase re Porter Plasmid(EBS-Luc).E2(10 n M)or PPT(10 n M)significantly increased the transcri Ptional activity of Ets-1,which was inhibited by ICI 182,780.2.4.2 E2 increased Ets-1 protein expression in HUVECs Different doses of E2(1 n M-1?M)all enhanced Ets-1 protein expression in HUVECs.Moreover,E2(10 n M)increased Ets-1 expression from 12 to 48 h.This effect was mimicked by PPT and was blocked by ICI 182,780.2.4.3 E2 increased Ets-1 protein expression via c-Src/PI3K/Akt Pathway E2-induced Ets-1 expression was inhibited by PI3 K inhibitor wortmannin(WM-30 n M)or non-rece Ptor tyrosine kinase c-Src inhibitor PP2(10 ?M),while not altered by MAPK inhibitor PD98059(PD-5?M)or G Protein inhibitor Pertussis toxin(PTX-100 ng/m L).When HUVECs were transfected with c-Src or Akt si RNA,E2 failed to increase Ets-1 Protein and mi R-126-3p expression.Moreover,E2 couldn't induce Akt Phosphorylation when c-Src was silenced,while it still increased c-Src Phosphorylation when Akt protein was knockdown.3.E2 Promoted HUVECs proliferation and tube formation via mi R-126-3p/Spred1 In order to clarify the role of mi R-126-3p in E2-regulated endothelia proliferation,migration and tube formation,antago-mi R-126-3p was efficiently transfected into HUVECs.In accordance with our previous reports,E2(10 n M)stimu Lated HUVECs Proliferation,migration and tube formation,which were largely inhibited by antago-mi R126-3p.Treatment with different doses of E2(1 n M-1?M)for different time decreased Spred1 protein expression.Meanwhile,E2 didn't reduce Spred1 m RNA level and the antago-mi R-126-3p blocked E2 effect on Spred1 protein expression.When Spred1 protein was overexpressed,it inhibited the effects of E2 on endothelial cell Proliferation,migration and tube formation.?Conclusions? E2 enhances endothelial mi R-126-3p expression via Ets-1.Then mi R-126-3p binds to its targets Spred1 m RNA,leading to its degradation and the silence of protein expression,and finally promoting endothelial proliferation,migration,tube formation.These effects contribute to anti-atherogenesis effect of E2. |
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