miR-23b-3p调控肾间质成纤维细胞成骨样分化参与Randall斑形成的机制研究  

Study on the mechanism of miR-23b-3p regulating osteogenic differentiation of renal interstitial fibroblasts and participating in Randall′s plaque formation

作　　者：雷波 邱明星 刘健男 Lei Bo;Qiu Mingxing;Liu Jiannan(Clinical College of Southwest Medical University,Luzhou 646099;Dept of Urology,Sichuan Provincial People′s Hospital,Chengdu 610072)

机构地区：[1]西南医科大学临床学院,泸州646099 [2]四川省人民医院泌尿外科,成都610072

出　　处：《安徽医科大学学报》2023年第12期2064-2072,共9页Acta Universitatis Medicinalis Anhui

基　　金：四川省自然科学基金青年科学基金(编号:2022NSFSC 1583)。

摘　　要：目的探究miR-23b-3p对肾间质成纤维(hRIFs)细胞成骨样分化和Randall斑形成的影响及其可能机制。方法采用qRT-PCR技术检测草酸钙(CaOx)结石患者Randall斑组织(RP)和接受肾切除术的肾肿瘤患者正常乳头状组织(nRP)的miR-23b-3p和肌细胞特异性增强子因子2C(MEF2C)、成骨标志物人骨钙蛋白(OCN)、骨桥蛋白(OPN)、Runt相关转录因子2(Runx2)的mRNA表达水平;体外分离、培养hRIFs细胞,将miR-23b-3p过表达质粒pSi-miR-23b-3p及空载质粒pSi-NC和MEF2C慢病毒过表达质粒Lv-MEF2C及空载质粒Lv-NC转染至hRIFs细胞中,并诱导细胞成骨分化14 d;ELISA法检测细胞碱性磷酸酶(ALP)活性;茜素红染色观察各组细胞矿化结节形成情况;qRT-PCR检测细胞中miR-23b-3p和MEF2C、OCN、OPN、Runx2 mRNA表达水平;Western blot检测细胞中MEF2C蛋白表达水平;双荧光素酶报告基因实验验证miR-23b-3p与MEF2C的靶向关系。结果①与nRP组比较,RP组中miR-23b-3p低表达,而MEF2C、OCN、OPN和Runx2 mRNA高表达;②hRIFs成骨诱导14 d后,细胞中ALP活性升高,矿化结节形成能力增强,miR-23b-3p表达水平降低,而MEF2C、OCN、OPN和Runx2 mRNA表达水平及MEF2C蛋白表达水平升高;③miR-23b-3p过表达降低成骨诱导后hRIFs细胞中ALP活性,抑制细胞矿化结节形成能力,下调细胞中OCN、OPN、Runx2 mRNA表达水平;④MEF2C过表达可逆转miR-23b-3p过表达对hRIFs细胞成骨分化的抑制作用;⑤MEF2C是miR-23b-3p下游靶基因。结论miR-23b-3p在RP组织及hRIFs细胞成骨样分化过程中低表达,上调miR-23b-3p可抑制hRIFs细胞的成骨样分化,其作用机制可能与靶向沉默MEF2C有关。Objective To explore the effect of miR-23b-3p regulation on osteogenic differentiation of renal interstitial fibroblasts(hRIFs)on the formation of Randall plaque and its possible mechanism.Methods qRT-PCR was used to detect the expression levels of miR-23b-3p and osteogenic marker:myocyte enhancer factor 2C(MEF2C),osteocalcin(OCN),osteopontin(OPN),runt-related transcription factor 2(Runx2)mRNA in Randall plaque tissue of CaOx stone patients(RP)and normal papillary tissue of kidney tumor patients undergoing nephrectomy(nRP).Isolation and culture of human normal hRIFs were isolated and cultured in vitro.The miR-23b-3p overexpression plasmid pSi-miR-23b-3p and its negative no-load plasmid pSi-NC,the MEF2C lentivirus overexpression plasmid Lv-MEF2C and the no-load plasmid Lv-NC were transfected into hRIFs cells,and the cells were induced to osteogenic differentiation for 14 days.The activity of alkaline phosphatase(ALP)was determined by ELISA.Alizarin red staining was used to observe the formation of mineralized nodules.The expression levels of miR-23b-3p and MEF2C,OCN,OPN,Runx2 mRNA were detected by qRT-PCR.The expression level of MEF2C protein was detected by Western blot.Dual luciferase reporter gene assay verified the targeting relationship between miR-23b-3p and MEF2C.Results①Compared with the nRP group,miR-23b-3p was low expressed and MEF2C,OCN,OPN,and Runx2 were highly expressed in the RP group.②14 days after osteogenic induction of hRIFs cells,the activity of ALP in cells significantly increased,the ability of cells to form mineralized nodules was enhanced,the expression level of miR-23b-3p significantly decreased,the mRNA expression levels of MEF2C,OCN,OPN,and Runx2 significantly increased,and the expression level of MEF2C protein significantly increased.③Overexpression of miR-23b-3p decreased the activity of ALP in hRIFs cells after osteogenic induction,inhibited the formation of mineralized nodules in cells,and down-regulated the mRNA expression levels of OCN,OPN,and Runx2 in cells.④Overexpression

关 键 词：miR-23b-3p 人肾间质成纤维细胞 肌细胞增强因子2C 成骨样分化 Randall斑 草酸钙结石 

分 类 号：R691.4[医药卫生—泌尿科学]