Department of Oral and Maxillofacial Surgery, The Affiliated Hospital of Qingdao University, 1677 Wutaishan Road, Huangdao district, Qingdao, 266555, Shandong, China.
School of Stomatology, Qingdao University, Qingdao, 266003, China.
Stem Cell Res Ther. 2022 Jul 26;13(1):359. doi: 10.1186/s13287-022-03018-4.
Osteoporosis affects the mandible resulting in bone loss. Though impairments are not life threatening, they affect a person's quality-of-life particularly vulnerable elderly. MicroRNAs (miRNAs) are novel regulatory factors that play an important role in regulating bone metabolism. Autophagy is evolutionarily conserved intracellular self-degradation process and is vital in the maintenance of both miRNA and bone homeostasis. However, the role of autophagy in the pathogenesis of miRNA regulating osteoporosis remains unclear.
In the study, we established a rat osteoporosis model induced by ovariectomy (OVX) and isolated mesenchymal stem cells from mandible (MMSCs-M). Several miRNAs were identified to regulate osteoporosis in some studies. qRT-PCR was applied to examine the expression of miRNA, autophagy and osteogenic differentiation-related genes. Western blotting assays were performed to detect the expression of autophagy and osteogenic differentiation proteins. Immunofluorescence and transmission electron microscope were used to verify the autophagy activity. Transfecting technology was used to enhance or suppress the expression of miR-152-5p which enable us to observe the relationship between miR-152-5p, autophagy and osteogenic differentiation. Additionally, the measurement of reactive oxygen species was used to investigate the mechanism of autophagy affecting osteogenic differentiation.
We found an upregulated expression of miR-152-5p in MMSCs-M in OVX group. Downregulated autophagy-related gene, proteins and autophagosome were detected in vitro of OVX group compared with sham group. Moreover, downregulation of miR-152-5p promoted osteogenic differentiation of MMSCs-M as well as enhanced autophagy-related proteins in OVX group. Conversely, overexpression of miR-152-5p showed opposite effect in sham group. Meanwhile, we found Atg14 (autophagy-related protein homolog 14) was identified to be a direct target of miR-152-5p theoretically and functionally. In other words, we confirmed inhibition of miR-152-5p promoted the osteogenic differentiation via promoting ATG14-mediated autophagy. Furthermore, miR-152-5p/ATG14-mediated autophagy regulated osteogenic differentiation by reducing the endogenous ROS accumulation and maintaining cellular redox homeostasis.
Our data suggest that miR-152-5p is the first identified to regulate osteogenic differentiation by directly targeting autophagy-related protein ATG14 and regulating oxidative stress and therapeutic inhibition of miR-152-5p may be an efficient anabolic strategy for osteoporosis.
骨质疏松症会影响下颌骨，导致骨质流失。虽然这些损伤不会危及生命，但会影响到人的生活质量，尤其是脆弱的老年人。微小 RNA（miRNA）是一种新型的调节因子，在调节骨代谢中发挥着重要作用。自噬是一种进化上保守的细胞内自我降解过程，对于维持 miRNA 和骨稳态都至关重要。然而，自噬在 miRNA 调节骨质疏松症发病机制中的作用尚不清楚。
在这项研究中，我们建立了去卵巢诱导的大鼠骨质疏松模型（OVX），并从下颌骨（MMSCs-M）中分离间充质干细胞。一些 miRNA 已被确定在某些研究中调节骨质疏松症。qRT-PCR 用于检测 miRNA、自噬和成骨分化相关基因的表达。Western blot 检测自噬和成骨分化蛋白的表达。免疫荧光和透射电镜用于验证自噬活性。通过转染技术增强或抑制 miR-152-5p 的表达，观察 miR-152-5p、自噬和成骨分化之间的关系。此外，通过测量活性氧（ROS）来研究自噬影响成骨分化的机制。
我们发现 OVX 组 MMSCs-M 中 miR-152-5p 的表达上调。与 sham 组相比，体外 OVX 组的自噬相关基因、蛋白和自噬体表达下调。此外，下调 miR-152-5p 促进 OVX 组 MMSCs-M 的成骨分化，并增强 OVX 组的自噬相关蛋白。相反，在 sham 组中过表达 miR-152-5p 则表现出相反的效果。同时，我们从理论和功能上确定 Atg14（自噬相关蛋白同源物 14）是 miR-152-5p 的直接靶标。换句话说，我们证实抑制 miR-152-5p 通过促进 ATG14 介导的自噬来促进成骨分化。此外，miR-152-5p/ATG14 介导的自噬通过减少内源性 ROS 积累和维持细胞氧化还原稳态来调节成骨分化。
我们的数据表明，miR-152-5p 是第一个被发现通过直接靶向自噬相关蛋白 ATG14 来调节成骨分化的 miRNA，并调节氧化应激，治疗性抑制 miR-152-5p 可能是骨质疏松症的一种有效的合成代谢策略。
