解剖学报

解剖学报 ›› 2024, Vol. 55 ›› Issue (2): 174-180.doi: 10.16098/j.issn.0529-1356.2024.02.007

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微小RNA-103a-3p通过肿瘤蛋白53调控凋亡抑制剂1/P53对骨质疏松症的影响

黄皆和1 王茜2  郏舜杰1 杨晟1*   

  1. 1.浙江省台州市立医院骨科; 2.肾内科，浙江 台州318099
  • 收稿日期:2023-03-16 修回日期:2023-06-21 出版日期:2024-04-06 发布日期:2024-04-06
  • 通讯作者: 杨晟 E-mail:13857665787@139.com
  • 基金资助:
    浙江省医药卫生科技计划

Effects of microRNA-103a-3p on osteoporosis through tumor protein 53-regulated inhibitor of apoptosis 1/P53

HUANG  Jie-he1  WANG  Qian2  JIA  Shun-jie1  YANG  Sheng1*   

  1. 1.Department of Orthopedics; 2.Department of Nephrology, Taizhou Municipal Hospital, Zhejiang Taizhou  318099, China
  • Received:2023-03-16 Revised:2023-06-21 Online:2024-04-06 Published:2024-04-06
  • Contact: YANG Sheng E-mail:13857665787@139.com

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摘要：

目的  探讨微小RNA（miR）-103a-3p调控细胞肿瘤蛋白53调控凋亡抑制剂1（TRIAP1）对成骨细胞分化以及去卵巢小鼠骨量的影响。  方法  MC3T3-E1细胞分为正常对照（NC）组、miR-103a-3p-NC组、miR-103a-3p模拟（mimc）组、miR-103a-3p mimic + TRIAP1-NC组、miR-103a-3p mimic + TRIAP1 mimic组。Real-time PCR检测细胞miR-103a-3p、TRIAP1、P53的mRNA表达，MTT法和流式细胞术检测细胞增殖及凋亡，免疫荧光染色和茜红素染色检测细胞骨架F-actin表达和矿化情况，ELISA检测细胞碱性磷酸酶（ALP）活性。24只雌性小鼠设为sham组、骨质疏松症（OP）组、miR-103a-3p antagonist-NC组和miR-103a-3p antagonist组，每组6只摘取双侧卵巢制备OP模型，sham组仅分离卵巢组织周围脂肪。测定骨组织miR-103a-3p、TRIAP1、P53、ALP、骨钙素（OCN）、骨桥蛋白（OPN）的mRNA表达，microCT测定骨密度（BMD）、骨矿物质含量（BMC），HE染色观察骨组织病理改变。  结果  细胞转染miR-103a-3p mimic后，miR-103a-3p及P53表达升高、TRIAP1表达降低，细胞增殖降低、凋亡增加，F-actin表达减弱，钙结节数量减少，ALP活性降低（P<0.01）；而在增加转染TRIAP1 mimic后，以上miR-103a-3p mimics导致的结果均得到显著逆转（P<0.01）。OP组小鼠骨组织miR-103a-3p、P53表达升高，TRIAP1、ALP、OCN、OPN基因表达降低，BMD、BMC降低，骨组织结构破坏（P<0.05）；miR-103a-3p antagonist组小鼠骨组织miR-103a-3p及P53表达降低，TRIAP1、ALP、OCN、OPN基因表达升高，BMD、BMC升高，骨组织结构改善（P<0.05）。  结论  MiR-103a-3p可介导TRIAP1/P53抑制成骨细胞增殖及矿化，而miR-103a-3p拮抗治疗可减少OP小鼠骨量丢失。

关键词: 骨质疏松症, 微小RNA-103a-3p, 肿瘤蛋白53调控细胞凋亡抑制剂1, P53, 骨分化, 骨密度, 实时定量聚合酶链反应, 小鼠

Abstract:

Objective To investigate the efeects of microRNA(miR)-103a-3p regulates tumor protein 53-regulated inhibitor of apoptosis 1(TRIAP1) on osteoblast differentiation and bone mass in ovariectomized mice.    Methods MC3T3-E1 cells were divided into normal group, miR-103a-3p-NC group, miR-103a-3p mimic group, miR-103a-3p mimic + TRIAP1-NC group, miR-103a-3p mimic + TRIAP1 mimic group. mRNA expression of miR-103a-3p, TRIAP1, P53 were detected by Real-time PCR; Cell proliferation and apoptosis were detected by MTT test and flow cytometry; cytoskeleton and mineralization of cells were detected by F-actin immunofluorescence staining and alizarin staining; alkaline phosphatase(ALP) activity was detected by ELISA. 24 female mice were divided into sham group, osteoporosis(OP) group, miR-103a-3p antagonist-NC group, miR-103a-3p antagonist group(six in each group), extract bilateral ovaries to establish an OP model, sham group mice only isolated fat around ovarian tissue. mRNA expression of miR-103a-3p, TRIAP1, P53, ALP, osteocalcin(OCN), osteopontin(OPN) of bone tissue were detected; microCT detect bone mineral density (BMD), bone mineral content (BMC); haematoxylin eosin staining was used to observe pathological changes of bone tissue.   Results After miR-103a-3p mimic was transfected into cells, the miR-103a-3p and P53 expression increased, TRIAP1 expression decreased, cell proliferation decreased, apoptosis increased, F-actin expression decreased, the number of calcium nodules decreased, and ALP enzyme activity decreased (P<0.01); however, after TRIAP1 mimic was additionally transfected into cells, the above result  caused by miR-103a-3p mimics were significantly reversed (P<0.01). In OP group, the miR-103a-3p and P53 expression in bone tissue increased, the TRIAP1, ALP, OCN and OPN expression decreased, BMD and BMC were decreased, and bone tissue construct was damaged(P<0.05); in miR-103a-3p antagonist group, the miR-103a-3p and P53 expression in bone tissue decreased, TRIAP1, ALP, OCN, OPN expression increased, BMD and BMC increased, and bone tissue construct was improved (P<0.05).   Conclusion MiRNA-103a-3p mediate TRIAP1/P53 to inhibit proliferation and mineralization of osteoblast, while miR-103a-3p antagonistic treatment reduce bone loss in OP mice.

Key words: Osteoporosis, MircroRNA-103a-3p, Tumor protein 53-regulated inhibitor of apoptosis 1, P53, Osteogenic differentiation, Bone mineral density, Real-time PCR, Mouse 

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