miR-154-3p靶向Smad7对人脐静脉内皮细胞生长、运动及免疫的影响  

Effects of miR⁃154⁃3p on the growth,movement and immunity of human umbilical vein endothelial cells by targeting Smad7

作　　者：袁晓丽 江莉 黄莉莉[1] 胡斌 YUAN Xiaoli;JIANG Li;HUANG Lili;HU Bin(Department of Cardiology,Mianyang Central Hospital,Mianyang 621000,China;Department of Cardiology,Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)

机构地区：[1]绵阳市中心医院心血管内科,四川绵阳621000 [2]西南医科大学附属医院心血管内科,四川泸州646000

出　　处：《广东药科大学学报》2021年第3期123-129,共7页Journal of Guangdong Pharmaceutical University

基　　金：四川省科技厅科技计划重点研发基金资助项目(2019SZYZF015)。

摘　　要：目的探究miR-154-3p通过靶向Smad7对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)的生长、运动及免疫的影响。方法体外培养HUVEC,将HUVEC转染miR-154-3p mimic后采用RT-PCR检测miR-154-3p和Smad7的mRNA表达水平,采用生物学信息预测结合荧光素酶实验确定miR-154-3p和Smad7的靶向关系;HUVEC单独转染miR-154p、Smad7,采用Western-blot检测Smad7蛋白的表达情况,采用克隆形成检测细胞生长情况;采用Transwell实验检测细胞侵袭情况;采用微管形成实验结合蛋白质印迹法检测血管内皮生长因子C(VEGFC)表达情况;采用酶联免疫吸附试验结合RT-PCR检测诱导型一氧化氮合酶(iNOS)、白细胞介素-6(IL-6)和白细胞介素-4(IL-4)水平。结果荧光素酶实验发现,miR-154-3p上存在Smad7的结合位点。相比Control组,miR-154-3p组miR-154-3p、IL-10 mRNA和血清IL-10含量水平显著升高(P>0.05),Smad7蛋白、克隆形成率、单位面积细胞侵袭数、VEGF蛋白、微管形成节点数、iNOS mRNA、血清iNOS含量、IL-6 mRNA和血清IL-6含量水平显著降低(P>0.05);相比Control组,Smad7组miR-154-3p组miR-154-3p、IL-10 mRNA和血清IL-10含量水平显著降低(P>0.05),Smad7蛋白、克隆形成率、单位面积细胞侵袭数、VEGF蛋白、微管形成节点数、iNOS mRNA、血清iNOS含量、IL-6 mRNA和血清IL-6含量水平显著升高(P>0.05)。相比Smad7组,miR-154-3p+Smad7组miR-154-3p、IL-10 mRNA和血清IL-10含量水平显著升高(P>0.05),Smad7蛋白、克隆形成率、单位面积细胞侵袭数、VEGF蛋白、微管形成节点数、iNOS mRNA、血清iNOS含量、IL-6 mRNA和血清IL-6含量水平显著降低(P>0.05)。结论miR-154-3p过表达可以靶向下调Smad7表达,有效抑制HUVEC的增殖和运动能力。Objective To explore the effects of miR-154-3p on the growth,movement and immunity of human umbilical vein endothelial cells(HUVEC)by targeting Smad7.Methods HUVEC were cultured in vitro.After HUVEC were transfected with miR-154-3p mimic,mRNA expression levels of miR-154-3p and Smad7 were detected by RT-PCR.The targeted relationship between miR-154-3p and Smad7 was determined by bioinformatics and luciferase reporter assay.HUVEC were transfected with miR-154 and Smad7 alone.The expression of Smad7 protein was detected by western blot.The cells growth was detected by clone formation assay.The cells invasion was detected by Transwell assay.The expression of vascular endothelial growth factor C(VEGFC)was detected by microtubule formation assay and western blot.The levels of inducible nitric oxide synthase(iNOS),IL-6 and IL-4 were detected by enzyme-linked immunosorbent assay and RT-PCR.Results Luciferase assay showed that there was Smad7 binding site on miR-154-3p.Compared with the control group,levels of miR-154-3p,IL-10 mRNA and protein were significantly increased,while Smad7 protein,clone formation rate,number of invasion cells per unit area,VEGF protein,node number of microtubule formation,iNOS mRNA and protein,and IL-6 mRNA and protein were significantly decreased in miR-154-3p group.Compared with the control group,levels of miR-154-3p,IL-10 mRNA and protein were significantly decreased,while Smad7 protein,clone formation rate,number of invasion cells per unit area,VEGF protein,node number of microtubule formation,iNOS mRNA and protein,and IL-6 mRNA and protein were significantly increased in Smad7 group.Compared with Smad7 group,levels of miR-154-3p,IL-10 mRNA and protein were significantly increased,while Smad7 protein,clone formation rate,number of invasion cells per unit area,VEGF protein,node number of microtubule formation,iNOS mRNA and protein,and IL-6 mRNA and protein were significantly decreased in miR-154-3p+Smad7 group.Conclusion Overexpression of miR-154-3p can down regulate Smad7 expression and ef

关 键 词：人脐静脉内皮细胞 miR-154-3p Smad同源物7 细胞侵袭 

分 类 号：R392[医药卫生—免疫学]