miR-497-5p在前成...化和矿化中的作用及机制探讨_胡熹.pdf

收稿日期 2021-11-30；修回日期 2022-01-18基金项目江西省卫生健康委科技计划（SKJP20211554）；江西省中医药管理局科技计划（2022A177）作者简介胡熹（1987-），女，硕士研究生，主治医师，E-mail:通信作者罗俊，E-mail:c2023年版权归上海口腔医学编辑部所有miR-497-5p 在前成骨细胞 MC3T3-E1分化和矿化中的作用及机制探讨胡熹1，罗俊2（1.南昌大学第二附属医院口腔医学诊疗中心，江西南昌330006；2.南昌大学附属口腔医院正畸科，江西省口腔生物医学重点实验室，江西南昌330006）摘要目的：研究微小RNA（miR）-497-5p在前成骨细胞MC3T3-E1分化和矿化中的作用，并探讨其相关机制。方法：取第3代MC3T3-E1细胞，分别转染miR-497-5p过表达质粒miR-497-5p mimics、低表达质粒miR-497-5pinhibitor和阴性对照质粒miR-497-5p NC，设为miR-497-5p mimics组、miR-497-5p inhibitor组和miR-497-5p NC组。取不做处理的细胞作为空白组。成骨诱导后14天，检测碱性磷酸酶（ALP）活性；免疫印迹法检测成骨分化相关蛋白骨钙素（OCN）、I型胶原（COL-I）蛋白表达量；茜素红染色法观察矿化情况；免疫印迹法检测Smad泛素化调节因子2（Smurf2）蛋白表达量；双荧光素酶实验验证miR-497-5p与Smurf2的靶向关系。采用SPSS 25.0软件包进行统计学分析。结果：与空白组、miR-497-5p NC组相比，miR-497-5p mimics组ALP活性显著增强，OCN、COL-I蛋白表达量及矿化结节面积构成比显著升高，Smurf2蛋白表达量显著降低（P0.05）；miR-497-5p inhibitor组ALP活性显著减弱，OCN、COL-I蛋白表达量及矿化结节面积构成比显著降低，Smurf2蛋白表达量显著升高（P0.05）；与Smurf2 3-UTR-WTmiR-497-5p NC组、Smurf2 3-UTR-MTmiR-497-5p mimics组和Smurf2 3-UTR-MTmiR-497-5p NC组相比，Smurf2 3-UTR-WTmiR-497-5p mimics组双荧光素酶活性值显著降低（P0.05）。结论：过表达miR-497-5p对前成骨细胞MC3T3-E1的分化和矿化具有促进作用，其作用机制可能与负性靶向调控Smurf2蛋白表达有关。关键词前成骨细胞；微小RNA-497-5p；分化；矿化中图分类号 R681文献标志码 ADOI:10.19439/j.sjos.2023.01.004Role and mechanism of miR-497-5p in the differentiation and mineralization of pre-osteoblast MC3T3-E1HUXi1,LUO Jun2.(1.Center of Stomatology,The Second Affiliated Hospital of Nanchang University.Nanchang 330006;2.Department of Orthodontics,Affiliated Stomatological Hospital of Nanchang University;Key Laboratory of OralBiomedicine of Jiangxi Province.Nanchang 330006,Jiangxi Province,China)Abstract PURPOSE:To study the role of microRNA(miR)-497-5p in the differentiation and mineralization of pre-osteoblasts MC3T3-E1,and to explore the related mechanisms.METHODS:The third generation MC3T3-E1 cells weretransfected into the miR-497-5p overexpression plasmid miR-497-5p mimics,the low expression plasmid miR-497-5pinhibitor,and the negative control plasmid miR-497-5p NC.They were set up as the miR-497-5p mimics group,miR-497-5p inhibitor group,and miR-497-5p NC group.The cells untreated was set up as the blank group.Fourteen daysafter osteogenic induction,alkaline phosphatase(ALP)activity was detected.The expression of osteocalcin(OCN)and typeI collagen(COL-I)proteins related to osteogenic differentiation were detected by Western blotting.Mineralization wasobserved by alizarin red staining method.The expression of Smad ubiquitination regulatory factor 2(Smurf2)protein wasdetected by Western blotting.The targeting relationship between miR-497-5p and Smurf2 was verified by dual luciferaseexperiment.Statistical analysis was performed by SPSS 25.0 software package.RESULTS:Compared with the blankgroup and miR-497-5p NC group,ALP activity of the miR-497-5p mimics group was enhanced,the expression of OCN,COL-I protein and the ratio of the area of mineralized nodules was increased,and the expression of Smurf2 protein was上海口腔医学2023年2月 第32卷第1期Shanghai Journal of Stomatology Vol32 No1 February,202317decreased(P0.05).ALP activity of the miR-497-5p inhibitor group was weakened,the expression of OCN,COL-I proteinand the ratio of the area of mineralized nodules was decreased,and the expression of Smurf2 protein was increased(P0.05).Compared with Smurf2 3-UTR-WT+miR-497-5p NC group,Smurf2 3-UTR-MT+miR-497-5p mimics group,Smurf23-UTR-MT+miR-497-5p NC group,the activity of dual luciferase in the WT+miR-497-5p mimics group was decreased(P0.05).CONCLUSIONS:Overexpression of miR-497-5p can promote the differentiation and mineralization of pre-osteoblasts MC3T3-E1,and its mechanism may be related to the negatively targeted regulation of Smurf2 protein expression.Key words Preosteoblasts;MicroRNA-497-5p;Differentiation;MineralizationShanghai J Stomatol,2023,32(1):17-22.前成骨细胞来自多潜能间充质干细胞，在成骨信号的诱导下，可向成骨细胞定向分化。因此，成骨分化过程包括间充质干细胞定向分化为前成骨细胞、前成骨细胞，进一步分化为成熟成骨细胞2个阶段。近年来研究1-2表明，miRNA在前成骨细胞的增殖与分化中起着重要作用。miR-497是miR-15/16/195簇成员，定位于染色体17p13.1上。有学者3发现，其广泛参与细胞分裂、血管生成以及机体代谢等过程。Ma等4的研究表明，miR-497-5p参与骨代谢并与骨质疏松引起的进行性骨丢失相关，且其可能作为监测抗骨质疏松疗法效果的潜在生物标志物。本研究通过体外细胞实验，观察过表达、低表达miR-497-5p对前成骨细胞MC3T3-E1分化、矿化的影响，并探讨其可能机制。1材料与方法1.1细胞系小鼠前成骨细胞MC3T3-E1（赛业生物科技有限公司）。1.2药物、主要试剂、仪器miR-497-5p过表达质粒miR-497-5p mimics、低表达质粒miR-497-5p inhibitor、阴性对照质粒miR-497-5p NC、Smurf2低 表 达 质 粒siRNA-Smurf2、Smurf2过表达质粒pcDNA3.1-Smurf2（西安淳风生物科技有限公司）；实时荧光定量聚合酶链反应（qRT-PCR）试剂盒（上海钦诚生物科技有限公司）；碱性磷酸酶（alkaline phosphatase，ALP）测定试剂盒（上海泽叶生物科技有限公司）；兔抗小鼠骨钙素（osteocalcin，OCN）、I型胶原（I collagen，COL-I）、Smad泛素化调节因子2（Smad ubiquitin regulatoryfactor2，Smurf2）一抗（美国Abcam公司）；双荧光素酶检测试剂盒（美国R&D公司）。Stepone QRT-PCR仪（美国ABI公司）；InfiniteF50酶标仪（瑞士Tecan公司）；Gene GENIUS凝胶成像系统（英国syngene公司）；Synergy-HD显微镜（日本Toshiba株式会社）。1.3前成骨细胞MC3T3-E1培养、转染及诱导将MC3T3-E1细胞培养于DMEM培养基中（含有10%胎牛血清），培养条件为标准条件（5%CO2、37、饱和湿度），培养至第3代，胰蛋白酶消化后离心，PBS冲洗，调整密度为1106个细胞/mL，接种至6孔板，按照LipofectamineTM3000试剂盒说明书，分别转染miR-497-5p mimics、miR-497-5p inhibitor和miR-497-5p NC，设为miR-497-5p mimics组、miR-497-5p inhibitor组和miR-497-5p NC组。取不做处理的细胞作为空白组。每组均设5个复孔，转染48 h后，将培养基更换为成骨诱导液（10%胎牛血清、甘油磷酸钠10 mmol/L、维生素C 50 g/L、地塞米松10 nmol/L）。1.4qRT-PCR法检测miR-497-5p表达量转染48