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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 - 第三军医大学学报 2018年40卷第13期 页码：1213-1220
- 作者机构：
400010 重庆，重庆医科大学附属第二医院老年心血管病科
- 作者简介：
[陈庆伟，E-mail：chenqwcq@163.com CHEN Qingwei, E-mail: chenqwcq@163.com
- 基金信息：
DOI：10.16016/j.1000-5404.201712004
中图分类号： R322.12;R329.28;R394.2收稿：2017-12-14，
修回：2018-01-14，
纸质出版：2018-07-15
- 稿件说明：
移动端阅览
阳喜喜, 陈庆伟, 叶力文, 等. microRNA-145通过靶向抑制CREB调节内皮细胞增殖和血管新生[J]. 第三军医大学学报, 2018,40(13):1213-1220.
Xixi YANG, Qingwei CHEN, Liwen YE, et al. MiRNA-145 inhibits proliferation and angiogenesis of endothelial cells by targeting inhibition of cAMP-responsive element-binding protein
in vitro [J]. Journal of Third Military Medical University, 2018, 40(13): 1213-1220.阳喜喜, 陈庆伟, 叶力文, 等. microRNA-145通过靶向抑制CREB调节内皮细胞增殖和血管新生[J]. 第三军医大学学报, 2018,40(13):1213-1220. DOI： 10.16016/j.1000-5404.201712004.
Xixi YANG, Qingwei CHEN, Liwen YE, et al. MiRNA-145 inhibits proliferation and angiogenesis of endothelial cells by targeting inhibition of cAMP-responsive element-binding protein
in vitro [J]. Journal of Third Military Medical University, 2018, 40(13): 1213-1220. DOI： 10.16016/j.1000-5404.201712004.
摘要
目的
2
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
2
利用T
4
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
2
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
2
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。
Abstract
Objective
2
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
2
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 T
4
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 MTT 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
2
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 a
ssay showed miR-145 directly bound to CREB 3′UTR.
Conclusion
2
miRNA-145 regulates the proliferation and angiogenesis of endothelial cells
which is associated with its inhibition to CREB/VEGF signaling.
关键词
Keywords
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