血管内皮细胞microRNA的表达谱分析和功能研究
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摘要
研究背景和目的
随着人类基因组计划的完成,生命科学进入了后基因组时代。研究表明,编码蛋白的基因总数不到3万个,远远少于最初预计的10万个左右。动植物基因组的非蛋白质编码区存在大量非编码RNA基因,约占整个基因组的98%,如此庞大的区域担负着基因的表达调控等重要功能。近年来发现的非编码小RNA主要包括小干扰RNA(small interfering RNAs, siRNAs)、微RNA(microRNAs, miRNAs)、重复关联小RNA(repeat associated small interfering RNAs, rasiRNAs)和piwi交互RNA(piwi - interacting RNAs, piRNAs)。目前人们对非编码RNA,尤其是microRNA在心血管、肿瘤、干细胞等领域的研究取得了长足的进步,极大的丰富和拓展了人们对这些重要生命科学领域的认知。本课题旨在选择血管内皮细胞高表达的microRNA对其进行相关表达谱分析和功能研究,探寻microRNA可能参与的心血管相关病理生理过程,进一步阐明诸如动脉粥样硬化、肿瘤血管生成等的发病机制,为今后运用RNA干扰治疗心血管疾病提供新的靶点。
研究方法
提取人脐静脉内皮细胞(Human Umbilical Vascular Endothelial Cell, HUVEC)的总RNA,变性胶富集大小约19-25个核苷酸大小的RNA后在3’和5’端分别连接合适的连接子,经过RT-PCR反应扩增后进行T-A克隆,重组质粒送商业测序,最终构建HUVEC的miRNA文库。运用Northern Blot检测文库中miRNA的表达谱:15%变性胶分离总RNA,电转移至尼龙膜,紫外线交联后运用γ32P-ATP标记的寡核苷酸探针进行杂交,然后运用X胶片-80℃显影。运用实时荧光定量RT-PCR检测miRNA在组织和细胞系中的相对表达量。将包含miR-126基因的EGFL7的第7个内含子连同相邻的外显子克隆至pEGFPc1质粒并转染293T细胞进行表达,运用PCR和Northern Blot检测转录产物以确定miR-126的生物来源和成熟机制。运用生物信息学工具PicTar、TagetScan5.1、miRanda、miRBase预测miRNA的靶基因。将预测靶基因的3’UTR克隆入pGL3-control的XbaI位点构建萤光素酶报告基因,与miRNA mimics和对照mimics共转染293T细胞,通过分析萤光素酶的表达量以初步筛选这些内皮高表达miRNA的靶基因。进一步通过Western Blot在细胞中确认miRNA对其靶基因的调控关系。干预HUVEC中miR-155或miR-221/222的表达,血管紧张素(Angiotensin, Ang)II刺激HUVEC后运用BCECF-AM标记的Jurkat T细胞检测HUVEC与单个核细胞的黏附性。采用细胞划痕法检测miRNA对AngII刺激下HUVEC迁移的影响。数据统计分析:两组间数据比较采用student t检验,多组间数据比较采用ANOVA方差分析,P<0.05代表结果存在统计学意义。
研究结果
成功建立了HUVEC的microRNA文库,发现miR-26a, miR-26b, miR-126, miR-155等在文库中的克隆数目相对较多。Northern Blot结果确认miR-126, miR-155和miR-221在HUVEC中高表达。其中miR-126在小鼠各器官组织中普遍表达并且在心脏和肺组织中高表达,然而在细胞水平,miR-126特异表达于内皮细胞而在人冠状动脉平滑肌细胞(Human Coronary Artery Smooth Muscle Cell,HCASMC)以及293T、Hela、小鼠胚胎成纤维细胞中均无表达。miR-155, miR-221和miR-222在血管平滑肌细胞也呈高表达但在Jurkat T, HEK293和Hela细胞中表达丰度很低或检测不出。miR-126前体序列和基因组位置在人、大鼠和小鼠基因组中保守。miR-126基因位于EGFL7的第7个内含子中,Real-Time PCR显示EGFL7与miR-126有着相似的表达谱。包含miR-126基因的内含子在工具细胞中能够被识别并剪切修饰形成成熟的具有功能的miR-126,而其宿主基因EGFL7也能够拼接形成mRNA,两者的成熟和表达互不影响。生物信息学预测结果表明,VEGF, RGS3, v-CRK, PIK3R2和EGFL7的3’UTR与miR-126的5’端“种子序列”有较佳的匹配位点。萤光素酶报告基因筛选和Western Blot结果显示,VEGF和PIK3R2是miR-126共同的靶基因。进一步,在MCF-7细胞中过表达miR-126可以通过对VEGF和PIK3R2的调控而降低AKT磷酸化水平。此外,miR-126在乳腺癌组织中表达明显下调而VEGF, PIK3R2的表达量以及AKT磷酸化水平均有明显升高。
生物信息学分析表明ETS-1的3’UTR区存在多个miR-155和miR-221/222可能同时调控的靶点。萤光素酶报告基因和Western Blot结果证实miR-155和miR-221/222能够同时调控靶基因ETS-1的表达。miR-155还能调控AngII的1型受体在蛋白水平的表达。AngII能够刺激HUVEC上调转录因子ETS-1及其下游基因VCAM1, ICAM1, MCP1和FLT1 mRNA等的表达,同时还能增强HUVEC与Jurkat T细胞的粘附作用以及HUVEC的迁移活性。然而在HUVEC中过表达miR-155或miR-221/222并以AngII刺激后,与对照相比,转录因子ETS-1及其下游基因VCAM1, MCP1和FLT1 mRNA等的表达均有不同程度的逆转,ICAM1 mRNA无明显变化。同时,过表达miR-155或miR-221/222的HUVEC对Jurkat T细胞的粘附作用明显降低。此外,过表达miR-155还能降低AngII诱导的HUVEC迁移活性,而miR-221/222无此作用。
研究结论
(1)成功建立了HUVEC的miRNA文库,确认miR-126在HUVEC特异表达,miR-155,miR-221/222在HUVEC高表达。
(2) miR-126来源于EGFL7基因的第7个内含子并且miR-126与其宿主基因的成熟和修饰互不干扰。VEGF和PIK3R2均为miR-126的靶基因。在MCF-7细胞系中,miR-126能够降低VEGF/PI3K/AKT通路的活性。在乳腺癌组织中,miR-126的表达降低而VEGF,PIK3R2的表达以及AKT磷酸化水平均明显升高。
(3) ETS-1是miR-155和miR-221/222共同的靶基因。miR-155还能够调控AT1R基因的表达。miR-155和miR-221/222均能通过调控ETS-1及其下游基因的表达而减轻AngII诱导的内皮细胞炎症反应。同时miR-155还能够抑制AngII诱导的内皮细胞迁移活性。
(4)一个miRNA可能具有两个或两个以上靶基因而一个基因也有可能受两个或两个以上miRNA的调控。
(5)内皮细胞高表达miRNA在血管炎症反应、动脉粥样硬化以及肿瘤血管新生等疾病的发生、发展过程中可能起重要的调控作用。miRNA有望为这些疾病提供新的治疗靶点和治疗手段。
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.
随着人类基因组计划的完成,生命科学进入了后基因组时代。研究表明,编码蛋白的基因总数不到3万个,远远少于最初预计的10万个左右。动植物基因组的非蛋白质编码区存在大量非编码RNA基因,约占整个基因组的98%,如此庞大的区域担负着基因的表达调控等重要功能。近年来发现的非编码小RNA主要包括小干扰RNA(small interfering RNAs, siRNAs)、微RNA(microRNAs, miRNAs)、重复关联小RNA(repeat associated small interfering RNAs, rasiRNAs)和piwi交互RNA(piwi - interacting RNAs, piRNAs)。目前人们对非编码RNA,尤其是microRNA在心血管、肿瘤、干细胞等领域的研究取得了长足的进步,极大的丰富和拓展了人们对这些重要生命科学领域的认知。本课题旨在选择血管内皮细胞高表达的microRNA对其进行相关表达谱分析和功能研究,探寻microRNA可能参与的心血管相关病理生理过程,进一步阐明诸如动脉粥样硬化、肿瘤血管生成等的发病机制,为今后运用RNA干扰治疗心血管疾病提供新的靶点。
研究方法
提取人脐静脉内皮细胞(Human Umbilical Vascular Endothelial Cell, HUVEC)的总RNA,变性胶富集大小约19-25个核苷酸大小的RNA后在3’和5’端分别连接合适的连接子,经过RT-PCR反应扩增后进行T-A克隆,重组质粒送商业测序,最终构建HUVEC的miRNA文库。运用Northern Blot检测文库中miRNA的表达谱:15%变性胶分离总RNA,电转移至尼龙膜,紫外线交联后运用γ32P-ATP标记的寡核苷酸探针进行杂交,然后运用X胶片-80℃显影。运用实时荧光定量RT-PCR检测miRNA在组织和细胞系中的相对表达量。将包含miR-126基因的EGFL7的第7个内含子连同相邻的外显子克隆至pEGFPc1质粒并转染293T细胞进行表达,运用PCR和Northern Blot检测转录产物以确定miR-126的生物来源和成熟机制。运用生物信息学工具PicTar、TagetScan5.1、miRanda、miRBase预测miRNA的靶基因。将预测靶基因的3’UTR克隆入pGL3-control的XbaI位点构建萤光素酶报告基因,与miRNA mimics和对照mimics共转染293T细胞,通过分析萤光素酶的表达量以初步筛选这些内皮高表达miRNA的靶基因。进一步通过Western Blot在细胞中确认miRNA对其靶基因的调控关系。干预HUVEC中miR-155或miR-221/222的表达,血管紧张素(Angiotensin, Ang)II刺激HUVEC后运用BCECF-AM标记的Jurkat T细胞检测HUVEC与单个核细胞的黏附性。采用细胞划痕法检测miRNA对AngII刺激下HUVEC迁移的影响。数据统计分析:两组间数据比较采用student t检验,多组间数据比较采用ANOVA方差分析,P<0.05代表结果存在统计学意义。
研究结果
成功建立了HUVEC的microRNA文库,发现miR-26a, miR-26b, miR-126, miR-155等在文库中的克隆数目相对较多。Northern Blot结果确认miR-126, miR-155和miR-221在HUVEC中高表达。其中miR-126在小鼠各器官组织中普遍表达并且在心脏和肺组织中高表达,然而在细胞水平,miR-126特异表达于内皮细胞而在人冠状动脉平滑肌细胞(Human Coronary Artery Smooth Muscle Cell,HCASMC)以及293T、Hela、小鼠胚胎成纤维细胞中均无表达。miR-155, miR-221和miR-222在血管平滑肌细胞也呈高表达但在Jurkat T, HEK293和Hela细胞中表达丰度很低或检测不出。miR-126前体序列和基因组位置在人、大鼠和小鼠基因组中保守。miR-126基因位于EGFL7的第7个内含子中,Real-Time PCR显示EGFL7与miR-126有着相似的表达谱。包含miR-126基因的内含子在工具细胞中能够被识别并剪切修饰形成成熟的具有功能的miR-126,而其宿主基因EGFL7也能够拼接形成mRNA,两者的成熟和表达互不影响。生物信息学预测结果表明,VEGF, RGS3, v-CRK, PIK3R2和EGFL7的3’UTR与miR-126的5’端“种子序列”有较佳的匹配位点。萤光素酶报告基因筛选和Western Blot结果显示,VEGF和PIK3R2是miR-126共同的靶基因。进一步,在MCF-7细胞中过表达miR-126可以通过对VEGF和PIK3R2的调控而降低AKT磷酸化水平。此外,miR-126在乳腺癌组织中表达明显下调而VEGF, PIK3R2的表达量以及AKT磷酸化水平均有明显升高。
生物信息学分析表明ETS-1的3’UTR区存在多个miR-155和miR-221/222可能同时调控的靶点。萤光素酶报告基因和Western Blot结果证实miR-155和miR-221/222能够同时调控靶基因ETS-1的表达。miR-155还能调控AngII的1型受体在蛋白水平的表达。AngII能够刺激HUVEC上调转录因子ETS-1及其下游基因VCAM1, ICAM1, MCP1和FLT1 mRNA等的表达,同时还能增强HUVEC与Jurkat T细胞的粘附作用以及HUVEC的迁移活性。然而在HUVEC中过表达miR-155或miR-221/222并以AngII刺激后,与对照相比,转录因子ETS-1及其下游基因VCAM1, MCP1和FLT1 mRNA等的表达均有不同程度的逆转,ICAM1 mRNA无明显变化。同时,过表达miR-155或miR-221/222的HUVEC对Jurkat T细胞的粘附作用明显降低。此外,过表达miR-155还能降低AngII诱导的HUVEC迁移活性,而miR-221/222无此作用。
研究结论
(1)成功建立了HUVEC的miRNA文库,确认miR-126在HUVEC特异表达,miR-155,miR-221/222在HUVEC高表达。
(2) miR-126来源于EGFL7基因的第7个内含子并且miR-126与其宿主基因的成熟和修饰互不干扰。VEGF和PIK3R2均为miR-126的靶基因。在MCF-7细胞系中,miR-126能够降低VEGF/PI3K/AKT通路的活性。在乳腺癌组织中,miR-126的表达降低而VEGF,PIK3R2的表达以及AKT磷酸化水平均明显升高。
(3) ETS-1是miR-155和miR-221/222共同的靶基因。miR-155还能够调控AT1R基因的表达。miR-155和miR-221/222均能通过调控ETS-1及其下游基因的表达而减轻AngII诱导的内皮细胞炎症反应。同时miR-155还能够抑制AngII诱导的内皮细胞迁移活性。
(4)一个miRNA可能具有两个或两个以上靶基因而一个基因也有可能受两个或两个以上miRNA的调控。
(5)内皮细胞高表达miRNA在血管炎症反应、动脉粥样硬化以及肿瘤血管新生等疾病的发生、发展过程中可能起重要的调控作用。miRNA有望为这些疾病提供新的治疗靶点和治疗手段。
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.
引文
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