microRNA-145通过靶向抑制CREB调节内皮细胞增殖和血管新生

MiRNA-145 inhibits proliferation and angiogenesis of endothelial cells by targeting inhibition of cAMP-responsive element-binding protein in vitro

目的构建微小RNA 145(microRNA-145,miR-145)过表达及干扰腺病毒载体,并探究miR-145对内皮细胞增殖和血管新生的影响及机制。方法利用T4连接酶分别将miR-145过表达和干扰目的序列插入到pHBAd-EF1-MCS-GFP和pHBAd-U6-CMV-GFP载体,构建出重组miR-145过表达和干扰腺病毒质粒。将重组miR-145过表达和干扰腺病毒质粒分别与骨架质粒pHBAd-BHG共转染人胚肾HEK293细胞进行重组腺病毒的包装与扩增,TCID_(50)法检测病毒感染滴度。miRNA qRT-PCR检测感染腺病毒的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)中miR-145的表达水平,MTT及Western blot检测PCNA蛋白表达量以检测细胞增殖情况。小管形成实验检测血管生成能力的变化。采用miRanda、miRwalk等生物信息学方法预测miR-145的靶基因及与环磷腺苷反应元件结合蛋白(cAMP response element-binding protein,CREB)3'UTR的结合位点。Western blot检测CREB及其调控的VEGF的表达情况,双荧光素酶实验验证miR-145与目的基因CREB 3'UTR相互作用。结果基因测序提示miR-145过表达及干扰质粒序列与目标序列一致,qRT-PCR结果亦提示病毒构建成功。细胞增殖和小管形成实验结果显示,与转染空载体组相比,过表达miR-145使570 nm处光密度值明显降低(P<0.05),亦显著抑制了HUVECs PCNA蛋白表达和小管样结构的形成(P<0.01),而干扰miR-145表达则使光密度值增高(P<0.01),亦促进HUVECs PCNA蛋白表达和小管样结构的形成(P<0.05)。同时,过表达miR-145可显著抑制CREB和VEGF蛋白表达(P<0.05),而干扰miR-145表达则明显促进CREB和VEGF蛋白上调(P<0.05)。双荧光素酶实验亦证实了miR-145与CREB 3'UTR的直接结合作用。结论 miR-145调节内皮细胞增殖和血管新生,其机制与miR-145直接靶向抑制CREB/VEGF信号有关。

Objective To construct recombinant adenoviruses of microRNA-145 （miRNA-145） stable expression and interference, and to investigate their effects and the underlying mechanism on the proliferation and angiogenesis of endothelial cells. Methods The shuttle plasmid of pHBAd-EF1-MCS-GFP and pHBAd-U6-CMV-GFP were used to construct miR-145 overexpression and interference adenoviruses with the aid of T4 ligase. After packaged with backbone plasmid pHBAd-BHG and amplified in HEK293 cells, the infection titers of the viruses were detected by TCID50 assay. The vectors were used to infect human umbilical vein endothelial cells （HUVECs）, and then the miRNA-145 level was detected by qRT-PCR, cell proliferation was evaluated by MIT assay and the expression level of proliferating cell nuclear antigen （PCNA） was detected by Western blotting. Angiogenesis was assessed by tube formation assay. Target genes and interaction between miR-145 and cAMP-responsive element binding protein （CREB） were predicted by bioinformatics software, miRanda and miRwalk. Then CREB and VEGF proteins after virus transfection were determined by Western blotting. And the binding site of miR-145 to CREB 3＇UTR was examined by Luciferase reporter assay. Results Gene sequencing verified that the motifs of miRNA-145 overexpression and interference plasmids were in accordance with target sequences, and the results of qRT-PCR indicated the successful construction of the vectors. In cell proliferation and angiogenesis assays, miRNA-145 overexpression inhibited cell proliferation （ P 〈 0.05 ）, suppressed the expression of PCNA protein and tube formation when compared with blank vector infection （P 〈 0.05 ）. While, miRNA-145 interference promoted cell proliferation （P 〈0.01 ）, PCNA expression and tube formation （P 〈 0.05）. MiRNA-145 overexpression inhibited the expression of CREB and VEGF proteins （P 〈 0.05）, whereas, the interference up-regulated both of them （P 〈0.05）. Luciferase reporter assay showed miR-145 directly bound to CREB 3＇ UTR. Conclusion miRNA-145 regulates the proliferation and angiogenesis of endothelial cells, which is associated with its inhibition to CREB/VEGF signaling.