| Introduction:Nucleolin is an important RNA binding protein involved in the regulation of transcription and transport of ribosomal RNA and ribosomal subunits assembling. Recently, increasing evidences have shown that regulation of mRNA stability of many genes is driven by nucleolin at the posttranscriptional level by protein-RNA interaction, for example:Bcl-2, P53, GADD45, IL-2and so on.Previously, our results have showed that VEGF-mediated nucleolin upregulation was involved in tube formation of the human umbilical vein endothelial cell. Moreover, myocardium targeted-expressed nucleolin promoted angiogenesis in ischemic myocardium in mice. These results suggested that nucleolin would play an important role in the regulation of angiogenesis process, and nucleolin would involve in regulating the expression and secretion of angiogenesis-related factors in cardiomyocytes then regulating angiogenesis. However, it is not well known how nucleolin regulates angiogenesis at physiological and pathological situation, and whether or not the nucleolin-mediated regulation of angiogenesis is associated with the quality of nucleolin-mRNA interaction which leads to the stability of target mRNA molecular.Methods:In this study, we used bioinformatics analyzes of human mRNA sequence of several angiogenesis-related genes to screen the potential target mRNA molecules with the specific nucleolin binding element. Subsequently, rat cardiomyocytes H9C2and human vein endothelial cells (HUVEC) were cultured as the in vitro cell models Protein-mRNA interaction was analyzed by using co-immunoprecipitation combined with quantitative PCR. Strategies of gene-gain and loss function assays, Western blotting and quantitative PCR were performed for assessing the effects of nucleolin on the target gene expression. Stability of target mRNAs was assessed by calculating the degradation velocity of target mRNAs after actinomycin D-mediated transcription block. Finally, nucleolin cardiac-targeted expression mouse was used to establish the myocardial ischemic model through ligation of left anterior descending coronary artery. This study was designed to clarify the possible regulatory mechanisms of angiogenesis process mediated by nucleolin at post-transcriptional level, hoping to provide new ideas and insight into the regulation of angiogenesis.Results:1. Bioinformatic analysis showed that the mRNA3’-untanslation region (UTR) or5’-UTR or coding region of angiogenesis-related genes MMP9, VEGF, hETS1, VE-Cadherin, KDR, Tie-2and MMP2contained nucleolin binding element "(T/G) CCCG (A/G)" sequences.2. Co-immunoprecipitation and Real-time PCR analysis showed that nucleolin interacted with MMP9and VEGF mRNA, nucleolin over-expression increased MMP9and VEGF expression, while down-regulation of nucleolin inhibited MMP9and VEGF expression in H9C2cells. Furthermore, nucleolin over-expression delayed the degradation velocity of MMP9mRNA and increased MMP9mRNA stability; in contrast, down-regulation of nucleolin accelerated the velocity and increased mRNA unstability in H9C2cells.3. In addition, we found that the mRNAs of VE-cadherin, Endoglin, hETS1, Angiopoietin were interacted with nucleolin protein in HUVECs. And enhanced nucleolin expression elevated their mRNA expression, but down-regulation of nucleolin decreased their mRNA expression in HUVECs.4. To clarify whether these interactions occurred under pathological situation, protein lysates from ischemic mouse heart tissues were analyzed by using protein-RNA immunoprecipitation and qPCR in nucleolin cardiac-targeted transgenic mice. The results showed that nucleolin did not bind to the mRNA of VEGF, VE-cadherin, endoglin, hETS1and angiopoietin but it interacted with MMP9mRNA in vivo.Conclusion:1. Nucleolin can regulate the expression of angiogenesis-related genes VEGF, MMP9, VE-cadherin, Endoglin, hETS1and Angiopoietin at the transcriptional level, and nucleolin can bind with different mRNAs in cardiomyocytes and HUVECs, which maybe contribute to regulation of angiogenesis.2. Nucleolin elevates the stability of MMP9mRNA in cardiomyocytes, which would be involved in angiogenesis in nucleolin cardiac-targeted transgenic mice. |
