| BackgroundVascular 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. MethodMicroRNA-125a/b inhibits endothlin-1 expression in vascular endothlial cells1. 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 blot6. 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-11. 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.ResultMicroRNA-125a/b inhibits endothlin-1 expression in vascular endothlial cells1. 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-11. 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.Conclusion1. 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.
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