吉林大学学报(医学版) ›› 2023, Vol. 49 ›› Issue (4): 947-957.doi: 10.13481/j.1671-587X.20230416

• 基础研究 • 上一篇    下一篇

miR-7对激素性股骨头缺血坏死细胞模型成骨分化的影响及内质网应激介导的凋亡机制

艾克热木江·阿尔肯null,日夏提·帕尔哈提null,张峥,翟生()   

  1. 新疆医科大学第五附属医院骨科中心,新疆 乌鲁木齐 830011
  • 收稿日期:2022-08-05 出版日期:2023-07-28 发布日期:2023-07-26
  • 通讯作者: 翟生 E-mail:609409432@qq.com
  • 作者简介:艾克热木江·阿尔肯(1989-),男,新疆维吾尔自治区乌鲁木齐市人,主治医师,医学硕士,主要从事运动医学和关节创伤方面的研究。
  • 基金资助:
    新疆维吾尔自治区科技厅自然科学基金项目(2021D01C426)

Effect of miR-7 on osteogenic differentiation of steroid-induced avascular necrosis of femoral head cell model and mechanism of endoplasmic reticulum stress-mediated apoptosis

AIKEREMUJIANG·Aerken, RIXIATI·Paerhati,Zheng ZHANG,Sheng ZHAI()   

  1. Orthopedic Center,Fifth Affiliated Hospital,Xinjiang Medical University,Urumqi 830011,China
  • Received:2022-08-05 Online:2023-07-28 Published:2023-07-26
  • Contact: Sheng ZHAI E-mail:609409432@qq.com

摘要:

目的 探讨微小RNA-7(miR-7)对激素性股骨头缺血坏死(SANFH)体外细胞模型的影响,并阐明其作用机制。 方法 将慢病毒miR-7模拟物(miR-7 mimic)和miR阴性对照(miR NC)转染至成骨细胞系MC3T3-E1中,同时设对照组,采用实时荧光定量PCR(RT-qPCR)法检测各组MC3T3-E1细胞中miR-7转染情况。MC3T3-E1细胞分为对照组、SANFH组、miR-7 mimic组和miR-7 mimic+衣霉素(TM)组,甲基强的松龙刺激细胞模拟SANFH离体状态,并以TM激活内质网应激反应。采用CCK-8法检测各组细胞增殖活性,流式细胞术检测各组细胞凋亡率,TUNEL染色法检测各组细胞TUNEL阳性率,碱性磷酸酶(ALP)染色法观察各组细胞ALP染色情况,比色法检测各组细胞中ALP水平,茜素红S染色观察各组细胞矿化结节形成情况, RT-qPCR法和Western blotting法检测各组细胞中Runt相关转录因子2(Runx2)、骨钙蛋白(OCN)和骨桥蛋白(OPN)mRNA及蛋白表达水平,Western blotting法检测各组细胞中葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-12、Caspase-9和Caspase-3蛋白表达水平,免疫荧光双标记染色法检测各组细胞中GRP78和Caspase-3蛋白表达荧光强度。 结果 与对照组和miR NC组比较,miR-7 mimic组细胞中miR-7表达水平明显升高(P<0.05)。与对照组比较,SANFH组细胞增殖活性明显降低(P<0.05),细胞凋亡率和TUNEL阳性率明显升高(P<0.05),ALP染色细胞较少且ALP活性明显降低(P<0.05),细胞中矿化结节形成减少,细胞中Runx2、OCN和OPN mRNA及蛋白表达水平明显降低(P<0.05),细胞中GRP78、CHOP、Caspase-12、Caspase-9和Caspase-3蛋白表达水平明显升高(P<0.05),细胞中GRP78和Caspase-3蛋白表达荧光强度明显升高(P<0.05);与SANFH组比较,miR-7 mimic组细胞增殖活性明显升高(P<0.05),细胞凋亡率和TUNEL阳性率明显降低(P<0.05),ALP染色细胞增加且ALP活性明显升高(P<0.05),细胞中矿化结节形成明显增加,细胞中Runx2、OCN和OPN mRNA及蛋白表达水平明显升高(P<0.05),GRP78、CHOP、Caspase-12、Caspase-9和Caspase-3蛋白表达水平明显降低(P<0.05),细胞中GRP78和Caspase-3蛋白表达荧光强度明显降低(P<0.05);与miR-7 mimic组比较,miR-7 mimic+ TM组细胞增殖活性明显降低(P<0.05),细胞凋亡率和TUNEL阳性率明显升高(P<0.05),ALP染色细胞较少且ALP活性明显降低(P<0.05),细胞中矿化结节形成减少,细胞中Runx2、OCN和OPN mRNA及蛋白表达水平明显降低(P<0.05),细胞中GRP78、CHOP、Caspase-12、Caspase-9和Caspase-3蛋白表达水平明显升高(P<0.05),细胞中GRP78和Caspase-3蛋白表达荧光强度明显升高(P<0.05)。 结论 miR-7能够促进SANFH状态下MC3T3-E1细胞的成骨分化作用,该机制可能与抑制内质网应激介导的凋亡途径有关。

关键词: 激素性股骨头缺血坏死, 微小RNA-7, 内质网应激, 细胞凋亡, 成骨分化

Abstract:

Objective To discuss the effect of microRNA-7 (miR-7) on the steroid-induced avascular nectosis of femoral head (SANFH) cell model in vitro,and to clarify its mechanism. Methods The lentivirus miR-7 mimic(miR-7 mimic) and miR negative control(miR NC) were transfected into the osteoblast MC3T3-E1 cells,and control group was set up, real-time fluorescence quantitative PCR(RT-qPCR) method was used to detect the transfection of MC3T3-E1 cells in various groups.The MC3T3-E1 cells were divided into control group, SANFH group, miR-7 mimic group, and miR-7 mimic+tumicamy (TM) group.CCK- 8 method was used to detect the proliferation activities of cells in various groups; flow cytometry was used to detect the apoptotic rates of cells in various groups; alkaline phosphatase (ALP) staining method was used to detect the ALP staining of cells in various groups;colorimetric method was used to detect the levels of ALP in cells in various groups; Alizarin red S staining was used to observe the formation of mineralization nodule in the cells in various groups; RT-qPCR method and Western blotting method were used to detect the expression levels of runt-related transcription factor 2 (Runx2), osteocalcin (OCN), osteopontin (OPN) mRNA and proteins in the cells in various groups; Western blotting method was used to detect the expression levels of glucose regulated protein 78 (GRP78), CCAAT/enhancer-binding protein(CHOP), cysteinyl aspartate specific proteinase(Caspase)-12, Caspase-9, and Caspase-3 proteins in the cells in various groups; immunofluorescence double-labeling staining was used to detect the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins in the cells in various groups. Results Compared with control group and miR NC group, the expression level of miR-7 in the MC3T3-E1 cells in miR-7 mimic group was increased (P<0.05). Compared with control group, the proliferation activity of the MC3T3-E1 cells in SANFH group was decreased (P<0.05), the apoptotic rate and TUNEL positive rate were increased (P<0.05), the number of ALP staining cells was less and the ALP activity was decreased (P<0.05), the number of formation of mineralized nodules in the cells was decreased, the expression levels of Runx2, OCN, and OPN mRNA and proteins in the cells were decreased (P<0.05), the expressions levels of GRP78, CHOP, Caspase-12, Caspase-9, and Caspase-3 proteins in the cells were increased (P<0.05), and the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins were increased (P<0.05); compared with SANFH group, the proliferation activity of the MC3T3-E1 cells in miR-7 mimic group was increased (P<0.05), the apoptotic rate and TUNEL positive rate were decreased (P<0.05), the number of ALP staining cells was increased, the ALP activity was increased (P<0.05), the number of formation of mineralized nodules in the cells was significantly increased, the expressions levels of Runx2, OCN, and OPN mRNA and proteins in the cells were increased (P<0.05), while the expression levels of GRP78, CHOP, Caspase-12, Caspase-9, and Caspase-3 proteins in the cells were decreased (P<0.05),and the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins in the cells were decreased (P<0.05);compared with miR-7 mimic group, the proliferation activity of the MC3T3-E1 cells in miR-7 mimic+TM group was decreased (P<0.05), the apoptotic rate and TUNEL positive rate were increased (P<0.05), the number of ALP staining cells was less and the ALP activity was decreased (P<0.05), the number of formation of mineralized nodules in the cells was decreased, the expression levels of Runx2, OCN, and OPN mRNA and proteins in the cells were decreased (P<0.05), the expression levels of GRP78, CHOP, Caspase-12, Caspase-9, and Caspase-3 proteins in the cells were increased (P<0.05), and the fluorescence intensities of expressions of GRP78 and Caspase-3 proteins were increased (P<0.05). Conclusion miR-7 can promote the osteogenesis of the MC3T3-E1 cells in SANFH state, and the mechanism may be related to the inhibition of endoplasmic reticulum stress-mediated apoptosis pathway.

Key words: Steroid-induced avascular necrosis of femoral head, MicroRNA-7, Endoplasmic reticulum stress, Apoptosis, Osteogenesis

中图分类号: 

  • R681.8