miRNA-410-3p通过MSX2抑制人肾间质成纤维细胞的成骨样分化
Suppression of osteogenic-like differentiation in human renal interstitial fibroblasts by miRNA-410-3p through MSX2.
作者信息
Cui Yu, Zeng Feng, Zhu Zewu, Huang Fang, Chen Jinbo, He Cheng, Li Yang, Chen Zhiyong, Yang Zhongqing, Zu Xiongbing, Chen Hequn
机构信息
Department of Urology, Xiangya Hospital, Central South University, Changsha, China.
出版信息
Transl Androl Urol. 2020 Oct;9(5):2082-2093. doi: 10.21037/tau-20-607.
BACKGROUND
The aim of this stay was to determine the effect of calcium ions in promoting osteogenic-like differentiation in human renal interstitial fibroblasts (hRIFs). The role of miRNA-410-3p in upregulating Msh homeobox 2 (MSX2) level in hRIFs was also investigated.
METHODS
Quantitative polymerase chain reaction (qPCR) analysis was used to assess the expression levels of miRNA-410-3p in Randall's plaque (RP) and normal renal papillary (nRP) tissues. Furthermore, the expression levels of osteogenesis-related protein in the RP and nRP tissues were assessed with qPCR and immunohistochemistry (IHC). hRIFs were cultured from isolated human kidney papilla before treatment with calcium chloride or osteogenic medium, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed at 1, 5, 9, and 14 days post-treatment. Alizarin red staining was used to estimate the deposits of calcium aggregates. After the overexpression or knockdown of miRNA-410-3p, we evaluated the changes in the osteogenic-like differentiation and osteogenesis-related protein by alizarin red staining and qPCR, respectively. A binding relationship between miRNA-410-3p and MSX2 was established through a dual-luciferase reporter gene assay. Rescue experiments demonstrated that miRNA-410-3p regulated the osteogenic-like differentiation by targeting MSX2.
RESULTS
miRNA-410-3p levels were lower in RP tissue than in control nRP tissues. qPCR and IHC showed that the level of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN) were higher in RP tissues. The calcium deposition of hRIFs showed a time-dependent trend when treated with osteogenic medium or calcium chloride. The overexpression of miRNA-410-3p downregulated the levels of osteogenesis-related expression and attenuated mineralization. The knockdown of miRNA-410-3p yielded the opposite trend. Dual-luciferase reporter gene assay and rescue experiments indicated that miRNA-410-3p could target MSX2, while the overexpression of MSX2 reversed the effects of miRNA-410-3p on osteogenic-like differentiation.
CONCLUSIONS
The current findings suggest that calcium ions could promote the osteogenic-like differentiation of hRIFs and miRNA-410-3p regulates hRIFs osteogenic-like differentiation by inhibiting MSX2.
背景
本研究旨在确定钙离子在促进人肾间质成纤维细胞（hRIFs）向成骨样细胞分化中的作用。同时还研究了miRNA-410-3p在上调hRIFs中Msh同源盒2（MSX2）水平方面的作用。
方法
采用定量聚合酶链反应（qPCR）分析评估Randall斑（RP）和正常肾乳头（nRP）组织中miRNA-410-3p的表达水平。此外，通过qPCR和免疫组织化学（IHC）评估RP和nRP组织中成骨相关蛋白的表达水平。从分离的人肾乳头培养hRIFs，然后用氯化钙或成骨培养基处理，并在处理后1、5、9和14天进行3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）检测。茜素红染色用于评估钙聚集物的沉积。在过表达或敲低miRNA-410-3p后，我们分别通过茜素红染色和qPCR评估成骨样分化和成骨相关蛋白的变化。通过双荧光素酶报告基因检测建立miRNA-410-3p与MSX2之间的结合关系。挽救实验表明，miRNA-410-3p通过靶向MSX2调节成骨样分化。
结果
RP组织中miRNA-410-3p水平低于对照nRP组织。qPCR和IHC显示，RP组织中与 runt 相关的转录因子2（Runx2）、骨钙素（OCN）和骨桥蛋白（OPN）水平较高。用成骨培养基或氯化钙处理时，hRIFs的钙沉积呈时间依赖性趋势。miRNA-410-3p的过表达下调了成骨相关表达水平并减弱了矿化作用。miRNA-410-3p的敲低则产生相反的趋势。双荧光素酶报告基因检测和挽救实验表明，miRNA-410-3p可靶向MSX2，而MSX2的过表达逆转了miRNA-410-3p对成骨样分化的影响。
结论
目前的研究结果表明，钙离子可促进hRIFs的成骨样分化，而miRNA-410-3p通过抑制MSX2调节hRIFs的成骨样分化。
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