摘要
目的:研究微小RNA-23b(miR-23b)是否通过靶基因Cxcl12调控肾性高血压。方法:通过miR-23b基因敲除(miR-23b KO)小鼠,运用RT-qPCR、ELISA、HE染色、PAS染色、免疫组化、彩色多普勒血流成像(CDFI)、肾皮质磁共振成像(MRI)等方法,观察分析miR-23b对肾脏的影响,通过RNA测序(RNA-Seq)技术分析miR-23b靶基因的变化,萤光素酶报告基因实验测定靶基因。结果:从临床中发现肾病综合征患者miR-23b表达水平降低(P<0.01),为了探讨miR-23b与肾病综合征的相关性,对miR-23b KO小鼠的研究发现,miR-23b基因敲除后,小鼠出现尿微量白蛋白显著升高(P<0.05)。通过CDFI和肾皮质MRI测得miR-23b基因敲除后小鼠肾脏血管阻力指数显著升高(P<0.01),肾皮质变薄(P<0.05)。通过ELISA确定miR-23 KO小鼠血清中肾素和血管紧张素蛋白表达显著增高(P<0.05)。对miR-23b KO小鼠肾脏组织的RNA-Seq发现,肾脏总体变化基因与TargetScan数据库中的miR-23b靶基因有交集,GO Term分析发现这些交集的基因主要富集于血流。RT-qPCR结果显示,与健康人群相比,肾病综合征患者Cxcl12基因表达水平显著升高(P<0.01),同时miR-23b KO小鼠Cxcl12基因表达水平也较WT小鼠显著升高(P<0.05)。体外萤光素酶报告基因实验表明,miR-23b过表达可以显著抑制Cxcl12萤光素酶活性(P<0.01),从而确定miR-23b通过调控靶基因Cxcl12发挥其生物学功能。结论:miR-23b可通过靶基因Cxcl12参与肾性高血压的发生与发展。本研究发现miR-23b在肾性高血压中的重要作用,miR-23b可能成为肾性高血压的一种崭新、有效的治疗靶点。
AIM:To investigate whether inhibition of microRNA-23b(miR-23b)induces renal hypertension by targeting Cxcl12.METHODS:Using single miR-23b gene knockout(miR-23b KO)mice,the effects of miR-23b on kidney were observed and analyzed by RT-qPCR,ELISA,HE staining,PAS staining and immunohistochemistry,color Doppler flow imaging(CDFI)and renal cortex magnetic resonance imaging(MRI).The alterations of miR-23b target genes were analyzed by the technique of RNA sequencing(RNA-Seq),and the target gene was verified by luciferase as⁃say.RESULTS:The level of miR-23b in nephrotic syndrome patients was significant decreased as compared with healthy controls(P<0.01).Furthermore,increased urine microalbumin in miR-23b KO mice was also found as compared with wild-type mice(P<0.05).Such phenotype was consistent with obvious increased renal vascular resistance index(P<0.01)and renal cortex became thinner(P<0.05)in miR-23b KO mice.To further decipher the abnormal kidney struc⁃ture,the serum renin and angiotensin in miR-23b KO mice were significantly increased as evidenced by ELISA(P<0.05).The results of RNA-Seq analysis from kidney tissue showed that significant altered genes,from the cross of global change genes and miR-23b target database,were enriched in blood circulation terms.The results of RT-qPCR showed that the expression of Cxcl12 gene in nephrotic syndrome patients(P<0.01)and miR-23b KO mice(P<0.05)was significant⁃ly increased as compared with healthy controls and wild-type mice,respectively.In vitro luciferase reporter assay showed that over-expression of miR-23b significantly inhibited Cxcl12 luciferase activity(P<0.01),which confirmed that miR-23b played its biological function by targeting Cxcl12 gene.CONCLUSION:miR-23b may participate in the occurrence of renal hypertension through targeting Cxcl12 gene.Our study may promote miR-23b as a potential therapeutic target for renal hypertension.
作者
李响
于闪闪
胡艳玲
王天贺
李洪志
赵冰海
邱德来
王会岩
LI Xiang;YU Shan-shan;HU Yan-ling;WANG Tian-he;LI Hong-zhi;ZHAO Bing-hai;QIU De-lai;WANG Hui-yan(Department of Physiology and Pathophysiology,School of Medicine,Yanbian University,Jilin 133002,China;Jilin Medical College,Jilin 132013,China;School of Basic Medicine,Beihua University,Jilin 132013,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2021年第4期626-633,共8页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.81770856,No.81970644)
吉林省教育厅“十三五”科学技术项目(No.JJKH20200058KJ)
吉林省卫生健康青年科技骨干培养计划项目(No.2019Q019)
吉林省抗体工程科技协同创新中心(No.20180623045TC)。
