miR-181a调控PINK1/Parkin通路对骨质疏松大鼠破骨细胞线粒体自噬的影响  

Effect of miR-181a regulating PINK1/Parkin pathway on mitochondrial autophagy of osteoclasts in osteoporotic rats

作　　者：祝震亚[1] 童蕾[1] 陆燕群 Zhu Zhen-Ya;Tong Lei;Lu Yan-Qun(Department of Orthopedics,Jiaxing Hospital of Traditional Chinese Medicine,Jiaxing,Zhejiang 314001,China)

机构地区：[1]嘉兴市中医医院骨科,浙江嘉兴314001

出　　处：《解放军医学杂志》2022年第6期569-578,共10页Medical Journal of Chinese People's Liberation Army

基　　金：浙江省医药卫生科技计划项目(2021KY358)。

摘　　要：目的探讨miR-181a调控PTEN诱导激酶1(PINK1)/帕金森病相关基因(Parkin)通路对骨质疏松(OP)大鼠破骨细胞线粒体自噬的影响。方法健康雌性SD大鼠20只,随机分为OP模型组(n=10)与对照组(n=10)。OP模型组大鼠制备OP模型,提取OP大鼠破骨细胞,设置OP组(未转染)、si-miR-181a组(转染si-miR-181a载体质粒)、si-NC组(转染si-NC阴性对照质粒)、ad-miR-181a组(转染ad-miR-181a载体质粒)与ad-NC组(转染阴性ad-NC对照质粒),对照组大鼠破骨细胞正常培养,设为正常对照组。采用RT-PCR检测miR-181a的表达,MTT法及流式细胞术检测破骨细胞的存活及凋亡情况,透射电镜观察线粒体自噬情况,免疫荧光共定位检测线粒体中Parkin的表达情况,Western blotting检测PINK1/Parkin及自噬、凋亡相关蛋白的表达情况。敲低OP大鼠破骨细胞miR-181a后下调Parkin的表达,验证Parkin对miR-181a的逆转作用。双荧光素酶报告实验验证miR-181a与Parkin的靶向调控关系。结果与正常对照组相比,OP组破骨细胞中miR-181a(1.59±0.15 vs.1.02±0.11)、Parkin^(+)TOMM2^(+)(2.02±0.20 vs.0.13±0.10)、Parkin(1.83±0.18 vs.1.13±0.10)、PINK1(1.93±0.19 vs.1.03±0.10)表达水平,自噬标志蛋白LC3-Ⅱ/Ⅰ比值(1.89±0.18 vs.1.15±0.10)及细胞存活率(157.06%±12.32%vs.100.09%±0.05%)升高(P<0.05)。与OP组相比,上调OP大鼠破骨细胞中miR-181a的表达后,细胞存活率(222.96%±22.15%)升高,Parkin^(+)TOMM2^(+)蛋白表达水平(1.01±0.11)、LC3-Ⅱ/Ⅰ比值(1.36±0.12)及细胞凋亡率(3.28%±0.35%)降低(P<0.05);下调OP大鼠破骨细胞中miR-181a的表达后,细胞存活率(106.96%±10.15%)降低,Parkin^(+)TOMM2^(+)蛋白表达水平(2.97±0.29)、LC3-Ⅱ/Ⅰ比值(2.47±0.24)及细胞凋亡率(19.71%±1.83%)升高(P<0.05)。双荧光素酶报告实验结果显示,Parkin为miR-181a的靶基因。下调Parkin的表达可逆转miR-181a低表达发挥的促进线粒体自噬及抑制细胞存活的作用(P<0.05)。结论上调OP大鼠破骨细胞中miR-1Objective To explore the regulatory effect of miR-181a on PTEN-induced kinase 1(PINK1)/Parkin-related genes(Parkin)pathway,and on mitochondrial autophagy of osteoclasts in osteoporosis(OP)rats.Methods Twenty healthy female SD rats were randomly divided into osteoporosis(OP)model group and control group(10 each).Rats in OP model group were employed to prepare OP model.Osteoclasts were extracted from OP rats,and set them as:OP group(not transfected),si-miR-181a group(transfected with si-miR-181a vector plasmid),si-NC group(transfected with negative control plasmid),ad-miR-181a group(transfected with ad-miR-181a vector plasmid),and ad-NC group(transfected with negative ad-NC control plasmid),and the osteoclasts of rats in control group were cultured normally and set as normal control group.The expression of miR-181a was detected by RT-PCR,and MTT and flow cytometry were performed to detect the survival and apoptosis of osteoclasts,the mitochondria autophagy was observed with transmission electron microscope,the expression of Parkin in mitochondria was detected by immunofluorescence co-localization,Western blotting was performed to detect the expression of PINK1/Parkin and autophagy,as well as apoptosis related proteins.Knockdown of miR-181a in osteoclasts of OP rats to down regulate the expression of Parkin and verify the reversal effect of Parkin on miR-181a.Double Luciferase Report experiment verified the targeted regulation between miR-181a and Parkin.Results Compared with normal control group,miR-181a(1.59±0.15 vs.1.02±0.11),Parkin^(+)TOMM2^(+)(2.02±0.20 vs.0.13±0.10),Parkin(1.83±0.18 vs.1.13±0.10)in osteoclasts of OP rats,and PINK1(1.93±0.19 vs.1.03±0.10)expression and survival rate(157.06%±12.32%vs.100.09%±0.05%),autophagy marker protein-LC3-Ⅱ/Ⅰratio(1.89±0.18 vs.1.15±0.10)increased(P<0.05).Compared with OP group,after up-regulating the expression of miR-181a in osteoclasts of OP rats,the cell survival rate(222.96%±22.15%)increased,Parkin^(+)TOMM2^(+)(1.01±0.11),LC3-Ⅱ/Ⅰratio(1.36±0.12)an

关 键 词：骨质疏松症 miR-181a TEN诱导激酶1/帕金森相关基因 自噬 破骨细胞 

分 类 号：R58[医药卫生—内分泌]