piR-7472影响电压调控钾离子通道促进小鼠成骨细胞分化的机制  

piR-7472 affects the mechanism by which voltage-regulated potassium channels promote osteogenic differentiation in mice

作　　者：隆宇斌 王湘斌 樊基耿 杨厚志 杨洋 李勇 Long Yubin;Wang Xiangbin;Fan Jigeng;Yang Houzhi;Yang Yang;Li Yong(Department of Spinal Surgery,The Central Hospital of Shaoyang,Shaoyang 422000,Hunan Province,China;Graduate School of Tianjin Medical University,Tianjin 300070,China;Department of Oncology,The Central Hospital of Shaoyang,Shaoyang 422000,Hunan Province,China)

机构地区：[1]邵阳市中心医院脊柱外科,湖南省邵阳市422000 [2]天津医科大学研究生院,天津市300070 [3]邵阳市中心医院肿瘤内科,湖南省邵阳市422000

出　　处：《中国组织工程研究》2025年第14期2868-2874,共7页Chinese Journal of Tissue Engineering Research

基　　金：湖南省自然科学基金(2022JJ50024),项目负责人:李勇;湖南省自然科学基金(2024JJ7474),项目负责人:隆宇斌。

摘　　要：背景:现有研究在PIWI相互作用RNA(piRNA)抗骨质疏松方面取得了重大进展,但piRNA发挥功能的具体靶点和相关机制仍有待探索。目的:探讨piR-7472对小鼠成骨细胞(MC3T3-E1细胞)分化的影响及下游机制。方法:①12只C57/BL6J小鼠随机分为假手术组和卵巢切除组,每组6只,术后8周通过Micro-CT检测小鼠的骨量,RT-qPCR检测piR-7472的表达;②将MC3T3-E1细胞分为NC mimics组、piR-7472 mimics组、NC inhibitor组与piR-7472 inhibitor组,转染后成骨诱导7 d采用RT-qPCR检测piR-7472、骨桥蛋白、胶原蛋白Ⅰ、Runt相关转录因子2、钾电压门控通道调节因子亚家族F成员1 mRNA的表达;成骨诱导7 d采用Western blot检测骨桥蛋白、Runt相关转录因子2、骨形态发生蛋白2、钾电压门控通道调节因子亚家族F成员1(KCNF1)蛋白表达;成骨诱导14 d通过碱性磷酸酶染色检测碱性磷酸酶的表达;成骨诱导21 d通过茜素红染色检测矿化结节数量;③双荧光素酶报告基因实验观察piR-7472是否能与钾电压门控通道调节因子亚家族F成员1相结合。结果与结论:①与假手术组相比,卵巢切除组小鼠骨密度、骨体积分数、骨小梁厚度、骨小梁数量明显下降,骨小梁分离度明显升高,并且骨组织中piR-7472 mRNA表达明显下调;②与NC组相比,piR-7472 mimics组骨桥蛋白、胶原蛋白Ⅰ、Runt相关转录因子2的mRNA表达明显升高,骨桥蛋白、Runt相关转录因子2、骨形态发生蛋白2的蛋白表达明显升高,矿化沉积与碱性磷酸酶表达增多;与NC inhibitor组相比,piR-7472 inhibitor组骨桥蛋白、胶原蛋白Ⅰ、Runt相关转录因子2的mRNA表达明显降低,骨桥蛋白、Runt相关转录因子2、骨形态发生蛋白2的蛋白表达明显降低,矿化沉积与碱性磷酸酶表达降低;③通过miRanda数据库预测发现piR-7472能与KCNF1相互作用;双荧光素酶报告基因实验发现piR-7472 mimcis能与钾电压门控通道调节因子亚家族F成员1结合并�BACKGROUND:Existing studies have made significant progress in PIWI-interacting RNAs(piRNAs)against osteoporosis,but the specific targets and related mechanisms by which piRNAs exert their functions remain to be explored.OBJECTIVE:To investigate the effects and downstream mechanisms of piR-7472 on the differentiation of mouse osteoblasts(MC3T3-E1 cells).METHODS:(1)Twelve C57/BL6J mice were randomly divided into a sham-operated and an ovariectomized group,with six mice in each group.Changes in bone mass and the expression of piR-7472 were detected using Micro-CT and RT-qPCR,respectively,at 8 weeks after surgery.(2)MC3T3-E1 cells were divided into NC mimics group,piR-7472 mimics group,NC inhibitor group,and piR-7472 inhibitor group.The mRNA expression of piR-7472,osteopontin,type I collagen,Runt-related transcription factor 2,and potassium voltage-gated channel modifier subfamily F member 1 were detected by RT-qPCR after 7 days of osteogenic induction.The protein expression of osteopontin,Runt-related transcription factor 2,bone morphogenetic protein 2,and potassium voltage-gated channel modifier subfamily F member 1(KCNF1)was detected using western blot assay.The expression of alkaline phosphatase was detected by alkaline phosphatase staining after 14 days of osteogenic induction,and the number of mineralized nodules was detected by alizarin red staining after 21 days of induction.Whether piR-7472 could bind to KCNF1 was observed by the dual luciferase reporter gene assay.RESULTS AND CONCLUSION:(1)Bone mineral density,bone volume fraction,bone trabecular thickness,bone trabecular number were significantly decreased and bone trabecular separation was significantly increased in ovariectomized mice,and piR-7472 in bone tissue was significantly down-regulated in osteoporotic mice.(2)Compared with the NC group,the mRNA expression of osteopontin,type I collagen,and Runt-related transcription factor 2 were significantly increased,the protein expression of osteopontin,Runt-related transcription factor 2,and bone morphogene

关 键 词：成骨细胞MC3T3-E1 PIWI相互作用RNA 成骨分化 骨质疏松症 电压调控的钾离子通道 KCNF1 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R681