Function And Expression Profile Of Vascular Endothelial MicroRNAs

Background: Life science has entered post-genome era after the accomplishment of Human Genome Project. It has been revealed that the total number of protein coding genes is about 30,000, much less than 100,000 which was once anticipated. It is found that about 98% regions of human genome are non-protein coding RNA genes which are always located in the non-protein coding region. These huge regions may play pivotal roles in regulating the protein coding genes expression. The non-coding RNAs found recently include small interfering RNAs (siRNAs), microRNAs (miRNAs), repeat associated small interfering RNAs (rasiRNAs) and piwi-interacting RNAs (piRNAs). Great progresse has been made in the study of non-coding RNAs, especially in microRNAs regulatory roles in cardiovascular system, cancer, stem cells, etc. The aim of this study is to investigate the expression profile and function of microRNAs which are highly expressed in vascular endothelial cells and to explore the roles that these miRNAs play in the process of pathophysiology of cardiovascular system. The results may help to elucidate the mechanism of diseases, including atherosclerosis, tumor angiogenesis and provide new therapeutic targets to treat cardiovascular diseases using RNA interference techniques.Methods: To construct microRNA library, total RNAs of human umbilical vein endothelial cells (HUVECs) were separated on a 15% denatured PAGE gel. 19-25 nt RNAs were recovered and ligated with 5'and 3'adaptors. The products were amplified by RT-PCR and then T-A cloned into vectors. The sequences of small RNAs were determined using commercial sequencing. To detect the profile of cloned miRNAs by Northern Blot, total RNAs were separated on 15% denatured PAGE gels and then electro-transferred to nylon membranes, after being cross-linked by UV, the membranes were hybrided withγ32P-ATP labeled oligo probes then developed under -80℃with X-ray films. The relative expression level of miRNAs in tissues and cell lines was also tested by Real-time PCR. To explore the mechanism of biogenesis and maturation of miR-126, miR-126-embeded intron7 of Egfl7 along with its consecutive exons were cloned in-framely into pEGFPc1 and transfected into 293T cells. The transcription products were tested by PCR and Northern Blot. Bioinformatics include PicTar, TargetScan5.1, miRanda and miRBase were used to anticipate target genes of miRNAs. Luciferase report genes were constructed by cloning the speculated 3'UTR of target genes into pGL3-control at XbaI site. Constructs and miRNA mimics or control mimics were co-transfected into 293T to verify the target site. The regulatory relationship of miRNAs and target genes was then verified by using Western Blot. BCECF-AM labeled Jurkat T cells were used to detect the adhesion capability of HUVECs which were over-expressed of miR-155 or miR-221/222 followed by Ang II stimulating. Cell scratch assay was used to detect the role of miRNAs in the regulation of HUVECs migration. Statistic analysis: All data were reported as mean±SD. Statistical comparisons were performed between two groups using t-test and among multiple groups by ANOVA. A value of P < 0.05 was considered to be statistically significant.Results: HUVEC microRNA library was successfully constructed. In the library,the clone number of mir-26a, mir-26b, miR-126, miR-155 was among the highest. Northern Blot confirmed that miR-126, miR-155 and miR-221 were highly expressed in HUVECs. Although notably expressed in heart and lung, miR-126 could also be detected in liver, spleen and kidney tissues. Interestingly, miR-126 was strictly expressed in HUVECs but can't be detected in other cell lines, including Human Coronary Artery Smooth Muscle Cells (HCASMCs), Hela, 293T and mouse embryonic fibroblasts (MEFs). Besides, miR-155 and miR-221 was also highly expressed in VSMCs but could only be detected at trace level in Jurkat T, HEK293 and Hela cells.miR-126 precursor sequences and its genome location were highly conserved among human, rat and mouse. miR-126 gene was located in the 7th intron of egfl7, which had similar expression pattern with miR-126 . miR-126-embedded intron can be recognized, excised and processed in cells to form mature and functional miR-126. On the other hand, the exons of egfl7 could be spliced to form mRNA. Bioinformatics analysis anticipated that 3'untranslated region (UTR) of VEGF, RGS3, v-CRK, PIK3R2 and EGFL7 may contain the potential target sites which may be recognized by 5'"seed sequence"of miR-126. Luciferase report gene screening and Western Blot showed that both VEGF and PIK3R2 were the targets of miR-126. Further, over-expression of miR-126 in MCF-7 could decrease the phosphorylation level of AKT via the regulation of VEGF and PIK3R2. Moreover, the expression of miR-126 was dramatically down-regulated in human breast cancer where the phosphorylation level of AKT was enhanced while the expression of VEGF and PIK3R2 were up-regulated.Bioinformatics analysis revealed that many putative target sites of miR-155 and miR-221/222 may locate in the 3'UTR of ETS-1. Luciferase report gene assay and Western Blot both confirmed that miR-155 and miR-221/222 could simultaneously regulate the expression of ETS-1. Moreover, miR-155 could also regulate the expression of Angiotensin II type 1 receptor (AT1R) at post-transcription level. Not only can Ang II up-regulate the expression of ETS-1 and its downstream genes include VCAM1, ICAM1, MCP1 and FLT1 in HUVECs, but enhance the capability of HUVECs adhesion to Jurkat T cells and migration activity. Over-expression of miR-155 or miR-221/222 in HUVECs could partially reverse the Ang II-induced up-regulation of ETS-1 and its downstream genes include VCAM1, MCP1 and FLT1, but ICAM1 mRNA level remained unchanged, compared to control. Importantly, over-expression of miR-155 or miR-221/222 in HUVECs could effectively decrease the adhesion of Jurkat T cells to Ang II-activated HUVECs. Different from miR-221/222, miR-155 could also reverse the effect of Ang II-induced HUVECs migration.Conclusions:(1). The HUVEC miRNA library was successfully constructed, many HUVECs highly or specifically expressed miRNAs include miR-126, miR-155, miR-221/222 were confirmed.(2). miR-126-embedded intron7 of egfl7 can be recognized, excised and processed in cells to form mature and functional miR-126 without affecting its host gene splicing. Both VEGF and PIK3R2 are the target genes of miR-126. Over-expression of miR-126 in MCF-7 could decrease the phosphorylation level of AKT via the regulation of VEGF and PIK3R2. Moreover, the expression of miR-126 was dramatically down-regulated in human breast cancer where the VEGF/PI3K/AKT pathway was activated.(3). ETS-1 is a target of both miR-155 and miR-221/222. miR-155 could also regulate AT1R expression. Both miR-155 and miR-221/222 could effectively decrease the adhesion of Jurkat T cells to Ang II-activated HUVECs. Moreover, miR-155 could also reverse the effect of Ang II-induced HUVECs migration.(4). A single miRNA may have two or more targets genes while many miRNAs may co-target one genes simultaneously.(5). Endothelial highly expressed miRNAs may form a regulatory network and play important roles in the initiation and progress of diseases, including vascular inflammation, atherosclerosis, tumor angiogenesis, etc.