血管内皮细胞miR-125和miR-126的功能研究
摘要
[研究背景]
血管内皮细胞既是循环和组织器官间的第一道屏障,也是许多心血管活性物质的主要来源,直接参与了心血管活动的调节,其功能是维持循环系统平衡和稳定的基础。内皮细胞的功能调节又有赖于一系列心血管活性物质,如血管内皮生长因子、内皮素、一氧化氮和血管紧张素Ⅱ等等。近年来研究发现的一种小分子调节物质microRNA,能够通过调控这些与内皮细胞功能密切相关的基因的表达而参与内皮细胞和血管功能的调节。尤其是一些血管内皮细胞特异性和高表达的microRNA,在血管的生理和病理生理过程中发挥着重要的调控功能。miR-125和miR-126是目前报道的与血管内皮细胞及血管功能密切相关的microRNA。研究表明,miR-125家族的两个成员：miR-125a和miR-125b在球囊损伤内皮引起内膜增生的血管中表达显著下降；而在单核巨噬细胞中,miR-125a能够为氧化低密度脂蛋白刺激所诱导表达增加,并能抑制单核巨噬细胞对脂质的摄取以及炎症因子的分泌,提示miR-125a/b参与了动脉粥样硬化疾病的血管炎症反应和血管内皮损伤及内膜增生等血管病理过程。miR-126是一种血管内皮细胞特异性表达的microRNA,它能够通过调控Spred-1、VCAM-1、HoxA9、v-Crk、EGFL-7和VEGF等基因的表达参与调节血管发育、新生血管形成以及血管炎症反应等血管的生理和病理生理过程。通过生物信学预测,我们发现：miR-125a和miR-125b可能调控内皮素-1 (Endothelin-1,ET-1)这一重要的心血管活性物质的基因表达,并可能藉此影响动脉粥样硬化和高血压等心血管疾病的发病过程；而由一种microRNA前体：pre-miR-126剪切生成的两种血管内皮特异性表达的microRNA:miR-126和miR-126*可能共同调控一种干细胞趋化因子：间质源性因子-1 (Stromal derived factor-1, SDF-1)的基因表达,影响组织干细胞向缺血组织和器官的迁移,进而参与心脑急性缺血损伤后的机体自身修复和重建过程。本课题利用多种microRNA的表达和功能研究方法,研究和探讨了这些科学问题。
[实验方法]
一、MicroRNA-125a/b抑制血管内皮细胞内皮素-1基因表达及机制
1、利用microRNA定量PCR技术检测miR-125a和miR-125b在小鼠不同组织和细胞的表达分布,同时利用Western blot检测ET-1基因初级翻译产物：前内皮素原(ppET-1)在这些组织细胞中的表达。
2、在H5V血管内皮细胞内转染pSuper-miR-125a和pSuper-miR-125b分别过表达miR-125a和miR-125b, Western Blot检测前内皮素原(ppET-1)的表达；利用miR-125a inhibitor和miR-125b inhibitor在血管内皮细胞分别下调miR-125a和miR-125b后,检测ppET-1表达,研究miR-125a/b对血管内皮细胞ppET-1表达的影响。
3、构建携载ET-1基因mRNA 3'非翻译区的pGL3重组萤光素酶报告基因质粒,分别与pSuper-miR-125a和pSuper-miR-125b共转染293A细胞,检测萤光素酶的活性；明确miR-125a和miR-125b调控ET-1基因表达的机制。
4、分别利用10μg/ml氧化低密度脂蛋白刺激H5V和b.END3两种血管内皮细胞6、12、24、36和48小时,定量PCR检测miR-125a和miR-125b的表达变化,同时利用Western Blot检测ppET-1的表达变化。通过转染pSuper-miR-125a和pSuper-miR-125b在H5V血管内皮细胞内分别过表达miR-125a和miR-125b后,再利用10μg／ml氧化低密度脂蛋白刺激上述转染的细胞24小时,检测细胞总蛋白中ppET-1的表达以及培养上清液中ET-1的含量,明确miR-125a/b对氧化低密度脂蛋白诱导血管内皮细胞ET-1表达的影响。
5、检测6周龄SHR-SP大鼠和WKY大鼠主动脉中miR-125a、miR-125b和ET-1的表达。
二、血管内皮miR-126／126*通过抑制SDF-1的表达调控骨髓间充值干细胞的迁移
1、利用microRNA定量PCR方法检测miR-126和miR-126*在小鼠不同组织和部分细胞中的表达分布；并检测低氧培养和氯化钴两种模拟缺氧的理化刺激条件下H5V血管内皮细胞中miR-126和miR-126*的表达变化,以及小鼠急性脑缺血模型脑组织中miR-126和miR-126*的表达变化。
2、在血管内皮细胞,通过转染pSuper-miR-126同时过表达miR-126和miR-126*随后对血管内皮细胞进行低氧培养,Western blot检测SDF-1蛋白表达变化,研究miR-126和miR-126*对低氧诱导SDF-l表达的影响；通过共转染miR-126 inhibitor和miR-126* inhibitor同时下调miR-126和miR-126*的表达,检测SDF-1蛋白的表达,研究在生理条件下下调血管内皮细胞miR-126和miR-126*对SDF-1表达的影响。同时利用ELISA检测上述实验中细胞培养上清液SDF-1的含量。
3、构建携载SDF-1基因mRNA 3'非翻译区的pGL3重组质粒,并分别对SDF-1基因mRNA 3'非翻译区上预测的miR-126和miR-126*的作用靶序列进行突变,得到两种突变型的pGL3重组质粒。通过将pSuper-miR-126、miR-126 mimic以及miR-126* mimic分别与这三种pGL3的重组质粒共转染293A细胞,检测共转染后重组萤光素酶报告基因的活性,研究miR-126和miR-126*对SDF-1基因mRNA3’非翻译区上各自预测靶位点的功能作用。
4、分别利用miR-126 mimic和miR-126* mimic转染血管内皮细胞同时或单独过表达miR-126和miR-126*,检测SDF-1的表达变化。利用miR-126 inhibitor和miR-126*inhibitor在血管内皮细胞同时或单独下调miR-126和miR-126*,检测SDF-1的表达变化。研究单独或同时过表达和下调miR-126及miR-126*对SDF-1表达的影响及二者的相互关系。
5、鉴于SDF-1对干细胞的趋化功能,在H5V血管内皮细胞过表达miR-126和miR-126*后对其进行低氧培养,利用体外Transwell迁移实验研究miR-126和miR-126*对骨髓间充质干细胞(MSC)向缺氧血管内皮细胞迁移的影响
[结果]
一、MicroRNA-125a/b抑制血管内皮细胞内皮素-1基因表达及机制
1、miR-125a和miR-125b主要表达于小鼠的肺、脑和主动脉这些富含血管和血管内皮细胞的组织。在小鼠不同细胞中,miR-125a和miR-125b均高表达血管内皮细胞,miR-125b还高表达于血管平滑肌细胞。ppET-1主要表达于肺和主动脉组织,相对高表达于成纤维细胞和血管内皮细胞。
2、在H5V血管内皮细胞内转染pSuper-miR-125a和pSuper-miR-125b分别过表达miR-125a和miR-125b后,能够显著抑制ppET-1蛋白的表达；而利用miR-125ainhibitor和miR-125b inhibitor在血管内皮细胞分别下调miR-125a和miR-125b,能够诱导ppET-1的表达。
3、将pSuper-miR-125a和pSuper-miR-125b两种microRNA表达质粒分别与携载ET-1基因mRNA 3'非翻译区的pGL3重组萤光素酶报告基因质粒共转染293A细胞,二者均能够显著抑制重组萤光素酶报告基因的活性。
4、利用氧化低密度脂蛋白刺激H5V血管内皮细胞后,miR-125a在刺激后6小时显著升高(>4倍),随后逐渐回落至正常水平；而miR-125b则在刺激后显著降低,于刺激24小时后降低最为明显；ppET-1则在刺激后6小时表达增加,于刺激后24小时升高最为明显。在H5V血管内皮细胞内分别过表达miR-125a和miR-125b均能够显著抑制氧化低密度脂蛋白诱导的ET-1表达和分泌的增加。
5、与WKY相比,miR-125a和miR-125b在SHR-SP的主动脉中表达显著降低,而ET-1的表达显著升高。
二、血管内皮miR-126／126*通过抑制SDF-1的表达调控骨髓间充值干细胞的迁移
1、验证了miR-126是一种血管内皮细胞特异性高表达的microRNA,同时也证明miR-126*也是一种血管内皮细胞特异性高表达的microRNA;并且发现低氧培养和氯化钴刺激均不影响HSV血管内皮细胞中miR-126和miR-126"的表达,而在小鼠急性脑缺血模型脑组织miR-126和miR-126'表达均下调。
2、在血管内皮细胞同时过表达miR-126和miR-126*能够显著抑制低氧所诱导的SDF-1表达增加和分泌,而同时下调miR-126和miR-126*则能够显著诱导SDF-1的表达和分泌。
3、利用pSuper-miR-126和miR-126/126* mimic同时和分别过表达miR-126和miR-126'均能够抑制携载野生型SDF-1基因mRNA 3'非翻译区的萤光素酶报告基因表达,而将SDF-1基因mRNA 3'非翻译区上miR-126和miR-126*的靶位点突变后,miR-126和miR-126"对重组萤光素酶报告基因的抑制作用消失。
4、在血管内皮细胞单独过表达miR-126和miR-126*并不能抑制低氧所诱导的SDF-1表达增加,而单独下调miR-126和miR-126"也不能诱导SDF-1的表达,只有同时过表达或下调miR-126和miR-126*才能够抑制或诱导SDF-1的表达；同时过表达miR-126和miR-126*对携载野生型SDF-1基因mRNA 3'非翻译区的重组萤光素酶报告基因的抑制作用比分别单独过表达二者更为显著。
5、在血管内皮细胞利用pSuper-miR-126和miR-126/126* mimic同时过表达miR-126和miR-126*能够显著抑制低氧诱导的MSC向缺氧血管内皮细胞的迁移；同时下调miR-126和miR-126*则能够诱导MSC向血管内皮细胞的迁移。
[结论]
1、miR-125a和miR-125b均为血管内皮细胞高表达的microRNA。
2、miR-125a和miR-125b能够通过作用于ET-1基因mRNA 3'非翻译区上特异性靶位点抑制ET-1基因的表达,并可能藉此参与ox-LDL刺激对血管内皮细胞ET-1的表达调控以及SHR-SP大鼠血压的调节。
3、miR-126和miR-126*均为血管内皮特异性高表达的microRNA。
4、miR-126和miR-126*能够作用于SDF-1基因mRNA 3'非翻译区上各自特异性靶序列抑制血管内皮细胞SDF-1的表达和分泌,二者作用具有协同效应。
5、miR-126／126*能够通过抑制SDF-1的表达调控骨髓间充质干细胞向缺氧内皮细胞的迁移。
Background
Vascular endothelial cells (VECs) were not only the barrier between the circulation and the organs, but also the resource of many cardiovascular active substrates. They directly participated in the regulation of cardiovascular activity and maintained the balance and stabilization of our circulatory system. Meanwhile, the regulation of the endothelial cell function also depended on various cardiovascular active substrates, such as vascular endothelial growth factor (VEGF), Endothelin, NO, Angiotensin II and so on. A small regulatory RNA which was called MicroRNA, was found to influence the function of endothelial cells and vessels by regulating the expression of genes which were significant to endothelial cells. Particularly, some microRNAs which were specially and highly expressed in vascular endothelial cells play great roles in the physiological and pathological processes of vessel. It was reported that miR-125 and miR-126 were critical for the function of vascular and vascular endothelial cells. Some studies show that the expression of miR-125a and miR-125b were decreased in the vessels with endometrial hyperplasia induced by saccules injury. Forthermore, the expression of miR-125a could be induced by ox-LDL stimulation in monocyte macrophages, as well as inhibited the uptake of lipid and secretion of inflammatory factors. These results suggested that miR-125a/b may participate in the inflammation of atherosclerosis, the dysfunction of vascular endothelium and the endometrial hyperplasia. miR-126 was specially expressed in vascular endothelial cells and took part in the processes of vascular development, angiogenesis and vessel inflammation by targeting genes of Spred-1、VCAM-1、HoxA9、v-Crk、EGFL-7 and VEGF. Bioinformatics analysis show that miR-125a and miR-125b may regulate the expression of endothelin-1 (ET-1), a key cardiovascular active peptide for atherosclerosis, hypertension and some other cardiovascular disease. While, vascular endothelial cell-specific microRNAs:miR-126 and miR-126* which were processed from the same precursor:pre-miR-126, have different target sites in 3'UTR of stromal derived factor-1 (SDF-1) which was a very important chemokine for stell cell, and impact on the migration of tissue stem cells to the ischemia areas in tissue repairment and rebuilding after heart infraction and stroke.
Method
MicroRNA-125a/b inhibits endothlin-1 expression in vascular endothlial cells
1. First of all, distribution of miR-125a and miR-125b in different tissues and cells of mice were determined by Real-time PCR. At the same time, the expression of the primary translated product of ET-1, ppET-1 was detected by Western blot.
2. Expressing plasmid of miR-125a and miR-125b, and pSuper-miR-125a/b was constructed; inhibitors of miR-125a and miR-125b were chemosynthesized; vascular endothelial cell H5V was transfected; expression of miR-125a and miR-125b was detected by Real-time PCR, and the efficiency of transfection was validated.
3. miR-125a and miR-125b were over-expressed by co-transfecting H5Vs with pSuper-miR-125a and pSuper-miR-125b, and miR-125a and miR-125b were downregulated by inhibitors to measure the expression of ppET-1 by Western blot. The influence of miR-125a/b on ppET-1 expression was investigated.
4. Re-combined fluorescent reporter plasmid pGL3 was constructed with ET-1 mRNA 3'UTR.293A cells were co-transfected by pSuper-miR-125a and pSuper-miR-125b. The level of luciferase activity was assayed to define the influence of miR-125a/b on ET-1 expression.
5. H5Vs and B.end3s (two kinds of vascular endothelial cells) were stimulated with ox-LDL(10μg/ml) for 6,12,24,36 and 48h. The expressions of miR-125a and miR-125b were determined by Real-time PCR, and the expression of ppET-1 was determined by Western blot
6. Expression of miR-125a, miR-125b, and ET-1 in aortas of SHR-SP rats and WKY rats aged 6 weeks was detected.
Endothelial miR-126 and its miRNA* species co-operationally control bone marrow mesenchymal stem cell migration by direct targeting of stromal-derived factor-1
1. Distribution of miR-126 and miR-126* in different tissues and cells of mice was firstly determined by Real-time PCR. The expression of miR-126 and miR-126* in H5V cells (a kind of vascular endothelial cells stimulated by treating with hypoxia and cobalt chloride) was detected in ischemic tissues of the acute cerebral ischemia model.
2. The vector pSuper-miR-126, which over-expressed miR-126 and miR-126*, was constructed by subcloning based on their same targeting gene of SDF-1 was found though bioinformatics analysing. The mimic and inhibitor of miR-126 and miR-126* were also artificially synthesized at the same time. Vascular endothelial cells were transfected with the plasmid and compounds mentioned above. The expression of miR-126 and miR-126* was detected by Real-time PCR to validate the efficiency of the over-expression and knockdown in vascular endothelial cells.
3. Vascular endothelial cells that over-expressed miR-126 and miR-126* were treated with hypoxia, and the expression of SDF-1 of the hypoxia-injured cells was detected by Western blot. Downregulation of miR-126 and miR-126* by co-transfecting with miR-126 and miR-126* inhibitors influenced the protein expression of SDF-1. The content of SDF-1 in the culture supernatant was detected by ELISA.
4. Fluorescent reporters were used for the sake of investigating the influence of...on SDF-1 expression by miR-126 and miR-126*. Re-combined plasmid pGL3 with SDF-1 mRNA 3'UTR was constructed, and the two mutations were achieved by mutating the forecasted targeting sequences on SDF-1 mRNA 3'UTR.293A cells were co-transfected with the three pGL3 carrying pSuper-miR-126, miR-126 mimic and miR-126* mimic correspondingly, and the luciferase activity was assayed after co-transfection. The functions of miR-126 and miR-126* on the targeting sites of SDF-1 mRNA 3'UTR were analysed.
5. SDF-1 expression was determined after over-expressing miR-126 and miR-126* simultaneously or respectively in the miR-126 and miR-126* mimics co-transfected vascular cells. SDF-1 expression was also determined after downregulating miR-126 and miR-126* simultaneously or respectively in the miR-126 and miR-126* inhibitors co-transfected vascular cells. The effect and relationship of SDF-1 expression by miR-126 and miR-126* were also discussed.
6. Considering the chemotactic functions of SDF-1, we observed the migration of MSCs to H5Vs, which had been under hypoxic condition after over-expressing miR-126 and miR-126* by virtue of Trans well in vitro.
Result
MicroRNA-125a/b inhibits endothlin-1 expression in vascular endothlial cells
1. miR-125a and miR-125b were mainly expressed in the lung, brain, and aorta of the mice, which are rich in vascular and vascular endothelial cells. miR-125a and miR-125b were both highly expressed in vascular endothelial cells, while miR-125b was highly expressed in vascular smooth muscle cells. ppET-1 was mainly expressed in the lung.
2. pSuper-miR-125a and pSuper-miR-125b over-expressed miR-125a and miR-125b effectively in vascular endothelial cells, while miR-125a/b inhibitors decreased the miR-125a and miR-125b expression.
3. Over-expression of miR-125a and miR-125b by co-transfecting H5Vs with pSuper-miR-125a and pSuper-miR-125b inhibited ppET-1 expression significantly, while downregulation of miR-125a and miR-125b by miR-125a/b inhibitors induced ppET-1 expression.
4. pSuper-miR-125a and pSuper-miR-125b plasmids, as well as the re-combined fluorescent reporter plasmid, pGL3, with the ET-1 mRNA 3'UTR, inhibited luciferase activity after co-transfection of 293 A cells.
5. After stimulation of ox-LDL in H5Vs, miR-125a increased significantly by more than 4 fold after 6h, and then returned to normal level. Interestingly, miR-125b decreased significantly after the stimulation, and the most obviously decrease was observed at 24h. ppET-1 increased after 6-h stimulation, and the most obviously increase was observed at 24h.
6. miR-125a and miR-125b decreased, while ET-1 increased significantly in the aorta of SHR-SP rats, compared with WKY rats.
Endothelial miR-126 and its miRNA* species co-operationally control bone marrow mesenchymal stem cell migration by direct targeting of stromal-derived factor-1
1. Both miR-126 and miR-126* were highly expressed in vascular endothelial cells. Hypoxia and stimulation with cobalt chloride did not affect the expressions of miR-126 and miR-126* in H5Vs. The expression of miR-126 and miR-126* decreased in the ischemic tissues of the acute cerebral ischemia model.
2. miR-126 and miR-126* over-expressed effectively in vascular endothelial cells co-transfected by pSuper-miR-126, miR-126 and miR-126* mimics, while they were downregulated by co-transfected. inhibitor miR-126 and miR-126*.
3. Over-expression of miR-126 and miR-126* in vascular endothelial cells prevented increase of SDF-1 expression and SDF-1 secretion significantly. However, downregulation of miR-126 and miR-126* induced SDF-1 expression and secretion significantly.
4. Over-expression of miR-126 and miR-126* with pSuper-miR-126 and miR-126/126* mimics simultaneously or respectively inhibited the expression of luciferase reporter carrying SDF-1 mRNA 3'UTR of wild type. However, the inhibitory effect diminished after mutating the targeting site of miR-126/126*.
5. Over-expression of miR-126 or miR-126* was not able to inhibit the increase of SDF-1 induced by hypoxia. Similarly, downregulation of miR-126 or miR-126* respectively induced the expression of SDF-1, neither. Over-expressing of miR-126 and miR-126* simultaneously exerted a greater inhibition on the re-combined luciferase reporter with SDF-1 mRNA 3'UTR than respectively.
6. Over-expression of miR-126 and miR-126* with pSuper-miR-126 and miR-126/126* mimics simultaneously inhibited hypoxia-induced migration of MSC to ischemic vascular endothelial cells markedly, or downregulation of miR-126 and miR-126* induced the migration.
Conclusion
1. miR-125a and miR-125b were both highly expressed in vascular endothelial cells.
2. miR-125a and miR-125b could inhibit ET-1 expression by targeting on ET-1 mRNA 3'UTR. By virtue of this, miR-125a and miR-125b may participate in the regulation of ET-1 by stimulating ox-LDL and increasing blood pressure in SHR-SP rats.
3. Both miR-126 and miR-126* were highly and specially expressed in vascular endothelial cells.
4. miR-126 and miR-126* could inhibit the expression and secretion of SDF-1 in vascular endothelial cells based on targeting to the special sequences on the SDF-1 mRNA 3'UTR in a synergistic way.
5. miR-126 and miR-126* could regulate the migration of MSC to hypoxia-injured vascular endothelial cells by inhibiting the expression of SDF-1.
血管内皮细胞既是循环和组织器官间的第一道屏障,也是许多心血管活性物质的主要来源,直接参与了心血管活动的调节,其功能是维持循环系统平衡和稳定的基础。内皮细胞的功能调节又有赖于一系列心血管活性物质,如血管内皮生长因子、内皮素、一氧化氮和血管紧张素Ⅱ等等。近年来研究发现的一种小分子调节物质microRNA,能够通过调控这些与内皮细胞功能密切相关的基因的表达而参与内皮细胞和血管功能的调节。尤其是一些血管内皮细胞特异性和高表达的microRNA,在血管的生理和病理生理过程中发挥着重要的调控功能。miR-125和miR-126是目前报道的与血管内皮细胞及血管功能密切相关的microRNA。研究表明,miR-125家族的两个成员：miR-125a和miR-125b在球囊损伤内皮引起内膜增生的血管中表达显著下降；而在单核巨噬细胞中,miR-125a能够为氧化低密度脂蛋白刺激所诱导表达增加,并能抑制单核巨噬细胞对脂质的摄取以及炎症因子的分泌,提示miR-125a/b参与了动脉粥样硬化疾病的血管炎症反应和血管内皮损伤及内膜增生等血管病理过程。miR-126是一种血管内皮细胞特异性表达的microRNA,它能够通过调控Spred-1、VCAM-1、HoxA9、v-Crk、EGFL-7和VEGF等基因的表达参与调节血管发育、新生血管形成以及血管炎症反应等血管的生理和病理生理过程。通过生物信学预测,我们发现：miR-125a和miR-125b可能调控内皮素-1 (Endothelin-1,ET-1)这一重要的心血管活性物质的基因表达,并可能藉此影响动脉粥样硬化和高血压等心血管疾病的发病过程；而由一种microRNA前体：pre-miR-126剪切生成的两种血管内皮特异性表达的microRNA:miR-126和miR-126*可能共同调控一种干细胞趋化因子：间质源性因子-1 (Stromal derived factor-1, SDF-1)的基因表达,影响组织干细胞向缺血组织和器官的迁移,进而参与心脑急性缺血损伤后的机体自身修复和重建过程。本课题利用多种microRNA的表达和功能研究方法,研究和探讨了这些科学问题。
[实验方法]
一、MicroRNA-125a/b抑制血管内皮细胞内皮素-1基因表达及机制
1、利用microRNA定量PCR技术检测miR-125a和miR-125b在小鼠不同组织和细胞的表达分布,同时利用Western blot检测ET-1基因初级翻译产物：前内皮素原(ppET-1)在这些组织细胞中的表达。
2、在H5V血管内皮细胞内转染pSuper-miR-125a和pSuper-miR-125b分别过表达miR-125a和miR-125b, Western Blot检测前内皮素原(ppET-1)的表达；利用miR-125a inhibitor和miR-125b inhibitor在血管内皮细胞分别下调miR-125a和miR-125b后,检测ppET-1表达,研究miR-125a/b对血管内皮细胞ppET-1表达的影响。
3、构建携载ET-1基因mRNA 3'非翻译区的pGL3重组萤光素酶报告基因质粒,分别与pSuper-miR-125a和pSuper-miR-125b共转染293A细胞,检测萤光素酶的活性；明确miR-125a和miR-125b调控ET-1基因表达的机制。
4、分别利用10μg/ml氧化低密度脂蛋白刺激H5V和b.END3两种血管内皮细胞6、12、24、36和48小时,定量PCR检测miR-125a和miR-125b的表达变化,同时利用Western Blot检测ppET-1的表达变化。通过转染pSuper-miR-125a和pSuper-miR-125b在H5V血管内皮细胞内分别过表达miR-125a和miR-125b后,再利用10μg／ml氧化低密度脂蛋白刺激上述转染的细胞24小时,检测细胞总蛋白中ppET-1的表达以及培养上清液中ET-1的含量,明确miR-125a/b对氧化低密度脂蛋白诱导血管内皮细胞ET-1表达的影响。
5、检测6周龄SHR-SP大鼠和WKY大鼠主动脉中miR-125a、miR-125b和ET-1的表达。
二、血管内皮miR-126／126*通过抑制SDF-1的表达调控骨髓间充值干细胞的迁移
1、利用microRNA定量PCR方法检测miR-126和miR-126*在小鼠不同组织和部分细胞中的表达分布；并检测低氧培养和氯化钴两种模拟缺氧的理化刺激条件下H5V血管内皮细胞中miR-126和miR-126*的表达变化,以及小鼠急性脑缺血模型脑组织中miR-126和miR-126*的表达变化。
2、在血管内皮细胞,通过转染pSuper-miR-126同时过表达miR-126和miR-126*随后对血管内皮细胞进行低氧培养,Western blot检测SDF-1蛋白表达变化,研究miR-126和miR-126*对低氧诱导SDF-l表达的影响；通过共转染miR-126 inhibitor和miR-126* inhibitor同时下调miR-126和miR-126*的表达,检测SDF-1蛋白的表达,研究在生理条件下下调血管内皮细胞miR-126和miR-126*对SDF-1表达的影响。同时利用ELISA检测上述实验中细胞培养上清液SDF-1的含量。
3、构建携载SDF-1基因mRNA 3'非翻译区的pGL3重组质粒,并分别对SDF-1基因mRNA 3'非翻译区上预测的miR-126和miR-126*的作用靶序列进行突变,得到两种突变型的pGL3重组质粒。通过将pSuper-miR-126、miR-126 mimic以及miR-126* mimic分别与这三种pGL3的重组质粒共转染293A细胞,检测共转染后重组萤光素酶报告基因的活性,研究miR-126和miR-126*对SDF-1基因mRNA3’非翻译区上各自预测靶位点的功能作用。
4、分别利用miR-126 mimic和miR-126* mimic转染血管内皮细胞同时或单独过表达miR-126和miR-126*,检测SDF-1的表达变化。利用miR-126 inhibitor和miR-126*inhibitor在血管内皮细胞同时或单独下调miR-126和miR-126*,检测SDF-1的表达变化。研究单独或同时过表达和下调miR-126及miR-126*对SDF-1表达的影响及二者的相互关系。
5、鉴于SDF-1对干细胞的趋化功能,在H5V血管内皮细胞过表达miR-126和miR-126*后对其进行低氧培养,利用体外Transwell迁移实验研究miR-126和miR-126*对骨髓间充质干细胞(MSC)向缺氧血管内皮细胞迁移的影响
[结果]
一、MicroRNA-125a/b抑制血管内皮细胞内皮素-1基因表达及机制
1、miR-125a和miR-125b主要表达于小鼠的肺、脑和主动脉这些富含血管和血管内皮细胞的组织。在小鼠不同细胞中,miR-125a和miR-125b均高表达血管内皮细胞,miR-125b还高表达于血管平滑肌细胞。ppET-1主要表达于肺和主动脉组织,相对高表达于成纤维细胞和血管内皮细胞。
2、在H5V血管内皮细胞内转染pSuper-miR-125a和pSuper-miR-125b分别过表达miR-125a和miR-125b后,能够显著抑制ppET-1蛋白的表达；而利用miR-125ainhibitor和miR-125b inhibitor在血管内皮细胞分别下调miR-125a和miR-125b,能够诱导ppET-1的表达。
3、将pSuper-miR-125a和pSuper-miR-125b两种microRNA表达质粒分别与携载ET-1基因mRNA 3'非翻译区的pGL3重组萤光素酶报告基因质粒共转染293A细胞,二者均能够显著抑制重组萤光素酶报告基因的活性。
4、利用氧化低密度脂蛋白刺激H5V血管内皮细胞后,miR-125a在刺激后6小时显著升高(>4倍),随后逐渐回落至正常水平；而miR-125b则在刺激后显著降低,于刺激24小时后降低最为明显；ppET-1则在刺激后6小时表达增加,于刺激后24小时升高最为明显。在H5V血管内皮细胞内分别过表达miR-125a和miR-125b均能够显著抑制氧化低密度脂蛋白诱导的ET-1表达和分泌的增加。
5、与WKY相比,miR-125a和miR-125b在SHR-SP的主动脉中表达显著降低,而ET-1的表达显著升高。
二、血管内皮miR-126／126*通过抑制SDF-1的表达调控骨髓间充值干细胞的迁移
1、验证了miR-126是一种血管内皮细胞特异性高表达的microRNA,同时也证明miR-126*也是一种血管内皮细胞特异性高表达的microRNA;并且发现低氧培养和氯化钴刺激均不影响HSV血管内皮细胞中miR-126和miR-126"的表达,而在小鼠急性脑缺血模型脑组织miR-126和miR-126'表达均下调。
2、在血管内皮细胞同时过表达miR-126和miR-126*能够显著抑制低氧所诱导的SDF-1表达增加和分泌,而同时下调miR-126和miR-126*则能够显著诱导SDF-1的表达和分泌。
3、利用pSuper-miR-126和miR-126/126* mimic同时和分别过表达miR-126和miR-126'均能够抑制携载野生型SDF-1基因mRNA 3'非翻译区的萤光素酶报告基因表达,而将SDF-1基因mRNA 3'非翻译区上miR-126和miR-126*的靶位点突变后,miR-126和miR-126"对重组萤光素酶报告基因的抑制作用消失。
4、在血管内皮细胞单独过表达miR-126和miR-126*并不能抑制低氧所诱导的SDF-1表达增加,而单独下调miR-126和miR-126"也不能诱导SDF-1的表达,只有同时过表达或下调miR-126和miR-126*才能够抑制或诱导SDF-1的表达；同时过表达miR-126和miR-126*对携载野生型SDF-1基因mRNA 3'非翻译区的重组萤光素酶报告基因的抑制作用比分别单独过表达二者更为显著。
5、在血管内皮细胞利用pSuper-miR-126和miR-126/126* mimic同时过表达miR-126和miR-126*能够显著抑制低氧诱导的MSC向缺氧血管内皮细胞的迁移；同时下调miR-126和miR-126*则能够诱导MSC向血管内皮细胞的迁移。
[结论]
1、miR-125a和miR-125b均为血管内皮细胞高表达的microRNA。
2、miR-125a和miR-125b能够通过作用于ET-1基因mRNA 3'非翻译区上特异性靶位点抑制ET-1基因的表达,并可能藉此参与ox-LDL刺激对血管内皮细胞ET-1的表达调控以及SHR-SP大鼠血压的调节。
3、miR-126和miR-126*均为血管内皮特异性高表达的microRNA。
4、miR-126和miR-126*能够作用于SDF-1基因mRNA 3'非翻译区上各自特异性靶序列抑制血管内皮细胞SDF-1的表达和分泌,二者作用具有协同效应。
5、miR-126／126*能够通过抑制SDF-1的表达调控骨髓间充质干细胞向缺氧内皮细胞的迁移。
Background
Vascular endothelial cells (VECs) were not only the barrier between the circulation and the organs, but also the resource of many cardiovascular active substrates. They directly participated in the regulation of cardiovascular activity and maintained the balance and stabilization of our circulatory system. Meanwhile, the regulation of the endothelial cell function also depended on various cardiovascular active substrates, such as vascular endothelial growth factor (VEGF), Endothelin, NO, Angiotensin II and so on. A small regulatory RNA which was called MicroRNA, was found to influence the function of endothelial cells and vessels by regulating the expression of genes which were significant to endothelial cells. Particularly, some microRNAs which were specially and highly expressed in vascular endothelial cells play great roles in the physiological and pathological processes of vessel. It was reported that miR-125 and miR-126 were critical for the function of vascular and vascular endothelial cells. Some studies show that the expression of miR-125a and miR-125b were decreased in the vessels with endometrial hyperplasia induced by saccules injury. Forthermore, the expression of miR-125a could be induced by ox-LDL stimulation in monocyte macrophages, as well as inhibited the uptake of lipid and secretion of inflammatory factors. These results suggested that miR-125a/b may participate in the inflammation of atherosclerosis, the dysfunction of vascular endothelium and the endometrial hyperplasia. miR-126 was specially expressed in vascular endothelial cells and took part in the processes of vascular development, angiogenesis and vessel inflammation by targeting genes of Spred-1、VCAM-1、HoxA9、v-Crk、EGFL-7 and VEGF. Bioinformatics analysis show that miR-125a and miR-125b may regulate the expression of endothelin-1 (ET-1), a key cardiovascular active peptide for atherosclerosis, hypertension and some other cardiovascular disease. While, vascular endothelial cell-specific microRNAs:miR-126 and miR-126* which were processed from the same precursor:pre-miR-126, have different target sites in 3'UTR of stromal derived factor-1 (SDF-1) which was a very important chemokine for stell cell, and impact on the migration of tissue stem cells to the ischemia areas in tissue repairment and rebuilding after heart infraction and stroke.
Method
MicroRNA-125a/b inhibits endothlin-1 expression in vascular endothlial cells
1. First of all, distribution of miR-125a and miR-125b in different tissues and cells of mice were determined by Real-time PCR. At the same time, the expression of the primary translated product of ET-1, ppET-1 was detected by Western blot.
2. Expressing plasmid of miR-125a and miR-125b, and pSuper-miR-125a/b was constructed; inhibitors of miR-125a and miR-125b were chemosynthesized; vascular endothelial cell H5V was transfected; expression of miR-125a and miR-125b was detected by Real-time PCR, and the efficiency of transfection was validated.
3. miR-125a and miR-125b were over-expressed by co-transfecting H5Vs with pSuper-miR-125a and pSuper-miR-125b, and miR-125a and miR-125b were downregulated by inhibitors to measure the expression of ppET-1 by Western blot. The influence of miR-125a/b on ppET-1 expression was investigated.
4. Re-combined fluorescent reporter plasmid pGL3 was constructed with ET-1 mRNA 3'UTR.293A cells were co-transfected by pSuper-miR-125a and pSuper-miR-125b. The level of luciferase activity was assayed to define the influence of miR-125a/b on ET-1 expression.
5. H5Vs and B.end3s (two kinds of vascular endothelial cells) were stimulated with ox-LDL(10μg/ml) for 6,12,24,36 and 48h. The expressions of miR-125a and miR-125b were determined by Real-time PCR, and the expression of ppET-1 was determined by Western blot
6. Expression of miR-125a, miR-125b, and ET-1 in aortas of SHR-SP rats and WKY rats aged 6 weeks was detected.
Endothelial miR-126 and its miRNA* species co-operationally control bone marrow mesenchymal stem cell migration by direct targeting of stromal-derived factor-1
1. Distribution of miR-126 and miR-126* in different tissues and cells of mice was firstly determined by Real-time PCR. The expression of miR-126 and miR-126* in H5V cells (a kind of vascular endothelial cells stimulated by treating with hypoxia and cobalt chloride) was detected in ischemic tissues of the acute cerebral ischemia model.
2. The vector pSuper-miR-126, which over-expressed miR-126 and miR-126*, was constructed by subcloning based on their same targeting gene of SDF-1 was found though bioinformatics analysing. The mimic and inhibitor of miR-126 and miR-126* were also artificially synthesized at the same time. Vascular endothelial cells were transfected with the plasmid and compounds mentioned above. The expression of miR-126 and miR-126* was detected by Real-time PCR to validate the efficiency of the over-expression and knockdown in vascular endothelial cells.
3. Vascular endothelial cells that over-expressed miR-126 and miR-126* were treated with hypoxia, and the expression of SDF-1 of the hypoxia-injured cells was detected by Western blot. Downregulation of miR-126 and miR-126* by co-transfecting with miR-126 and miR-126* inhibitors influenced the protein expression of SDF-1. The content of SDF-1 in the culture supernatant was detected by ELISA.
4. Fluorescent reporters were used for the sake of investigating the influence of...on SDF-1 expression by miR-126 and miR-126*. Re-combined plasmid pGL3 with SDF-1 mRNA 3'UTR was constructed, and the two mutations were achieved by mutating the forecasted targeting sequences on SDF-1 mRNA 3'UTR.293A cells were co-transfected with the three pGL3 carrying pSuper-miR-126, miR-126 mimic and miR-126* mimic correspondingly, and the luciferase activity was assayed after co-transfection. The functions of miR-126 and miR-126* on the targeting sites of SDF-1 mRNA 3'UTR were analysed.
5. SDF-1 expression was determined after over-expressing miR-126 and miR-126* simultaneously or respectively in the miR-126 and miR-126* mimics co-transfected vascular cells. SDF-1 expression was also determined after downregulating miR-126 and miR-126* simultaneously or respectively in the miR-126 and miR-126* inhibitors co-transfected vascular cells. The effect and relationship of SDF-1 expression by miR-126 and miR-126* were also discussed.
6. Considering the chemotactic functions of SDF-1, we observed the migration of MSCs to H5Vs, which had been under hypoxic condition after over-expressing miR-126 and miR-126* by virtue of Trans well in vitro.
Result
MicroRNA-125a/b inhibits endothlin-1 expression in vascular endothlial cells
1. miR-125a and miR-125b were mainly expressed in the lung, brain, and aorta of the mice, which are rich in vascular and vascular endothelial cells. miR-125a and miR-125b were both highly expressed in vascular endothelial cells, while miR-125b was highly expressed in vascular smooth muscle cells. ppET-1 was mainly expressed in the lung.
2. pSuper-miR-125a and pSuper-miR-125b over-expressed miR-125a and miR-125b effectively in vascular endothelial cells, while miR-125a/b inhibitors decreased the miR-125a and miR-125b expression.
3. Over-expression of miR-125a and miR-125b by co-transfecting H5Vs with pSuper-miR-125a and pSuper-miR-125b inhibited ppET-1 expression significantly, while downregulation of miR-125a and miR-125b by miR-125a/b inhibitors induced ppET-1 expression.
4. pSuper-miR-125a and pSuper-miR-125b plasmids, as well as the re-combined fluorescent reporter plasmid, pGL3, with the ET-1 mRNA 3'UTR, inhibited luciferase activity after co-transfection of 293 A cells.
5. After stimulation of ox-LDL in H5Vs, miR-125a increased significantly by more than 4 fold after 6h, and then returned to normal level. Interestingly, miR-125b decreased significantly after the stimulation, and the most obviously decrease was observed at 24h. ppET-1 increased after 6-h stimulation, and the most obviously increase was observed at 24h.
6. miR-125a and miR-125b decreased, while ET-1 increased significantly in the aorta of SHR-SP rats, compared with WKY rats.
Endothelial miR-126 and its miRNA* species co-operationally control bone marrow mesenchymal stem cell migration by direct targeting of stromal-derived factor-1
1. Both miR-126 and miR-126* were highly expressed in vascular endothelial cells. Hypoxia and stimulation with cobalt chloride did not affect the expressions of miR-126 and miR-126* in H5Vs. The expression of miR-126 and miR-126* decreased in the ischemic tissues of the acute cerebral ischemia model.
2. miR-126 and miR-126* over-expressed effectively in vascular endothelial cells co-transfected by pSuper-miR-126, miR-126 and miR-126* mimics, while they were downregulated by co-transfected. inhibitor miR-126 and miR-126*.
3. Over-expression of miR-126 and miR-126* in vascular endothelial cells prevented increase of SDF-1 expression and SDF-1 secretion significantly. However, downregulation of miR-126 and miR-126* induced SDF-1 expression and secretion significantly.
4. Over-expression of miR-126 and miR-126* with pSuper-miR-126 and miR-126/126* mimics simultaneously or respectively inhibited the expression of luciferase reporter carrying SDF-1 mRNA 3'UTR of wild type. However, the inhibitory effect diminished after mutating the targeting site of miR-126/126*.
5. Over-expression of miR-126 or miR-126* was not able to inhibit the increase of SDF-1 induced by hypoxia. Similarly, downregulation of miR-126 or miR-126* respectively induced the expression of SDF-1, neither. Over-expressing of miR-126 and miR-126* simultaneously exerted a greater inhibition on the re-combined luciferase reporter with SDF-1 mRNA 3'UTR than respectively.
6. Over-expression of miR-126 and miR-126* with pSuper-miR-126 and miR-126/126* mimics simultaneously inhibited hypoxia-induced migration of MSC to ischemic vascular endothelial cells markedly, or downregulation of miR-126 and miR-126* induced the migration.
Conclusion
1. miR-125a and miR-125b were both highly expressed in vascular endothelial cells.
2. miR-125a and miR-125b could inhibit ET-1 expression by targeting on ET-1 mRNA 3'UTR. By virtue of this, miR-125a and miR-125b may participate in the regulation of ET-1 by stimulating ox-LDL and increasing blood pressure in SHR-SP rats.
3. Both miR-126 and miR-126* were highly and specially expressed in vascular endothelial cells.
4. miR-126 and miR-126* could inhibit the expression and secretion of SDF-1 in vascular endothelial cells based on targeting to the special sequences on the SDF-1 mRNA 3'UTR in a synergistic way.
5. miR-126 and miR-126* could regulate the migration of MSC to hypoxia-injured vascular endothelial cells by inhibiting the expression of SDF-1.
引文
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