A novel ceRNA regulatory network involving the long noncoding NEAT1, miRNA-466f-3p and its mRNA target in osteoblast autophagy and osteoporosis
A novel ceRNA regulatory network involving the long noncoding NEAT1, miRNA-466f-3p and its mRNA target in osteoblast autophagy and osteoporosis
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DOI：
10.1007/s00109-022-02255-7
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发表时间：
2022-09
期刊：
影响因子：
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通讯作者：
Xiao‐bing Zhao;Dacheng Zhao;B. Geng;Wang Yaobin;Yayi Xia
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作者：
Xiao‐bing Zhao;Dacheng Zhao;B. Geng;Wang Yaobin;Yayi Xia
骨质疏松症(OP)是一种以骨组织体积减少为特征的全身性代谢紊乱。据报道，lncRNAs是几种人类疾病的调节者。核转录因子1参与破骨细胞和骨髓间充质干细胞的增殖、分化和凋亡，调控骨质疏松症的发生发展。然而，NEAT1与成骨细胞自噬的关系及其机制尚不清楚。采用免疫印迹法检测流体剪切力(FSS)对MC3T3-E1成骨细胞自噬的影响。对加载和不加载FSS的成骨细胞进行总转录组测序和生物信息学分析。采用定量聚合酶链式反应检测NEAT1在OP骨组织和成骨细胞中的表达。应用RNA-FISH技术研究了ncRNA NEAT1和miR-466f-3p在MC3T3-E1成骨细胞的定位。体外用免疫印迹、透射电子显微镜、免疫荧光染色和定量聚合酶链式反应等方法验证NEAT1、miR-466f-3p和HK2的生物学功能。随后，我们进行了生物信息学分析和双荧光素酶报告实验，以确定NEAT1、miR-466f-3p和HK2之间的关系。此外，对成骨细胞进行了补救试验，以阐明NEAT1/miR-466f-3p/HK2信号通路的调控网络。在体内，采用OVX小鼠模型，观察si-NEAT1对OP小鼠自噬的影响。采集股骨远端和血清进行进一步的显微CT分析、血液生化、苏木精-伊红和茜素红染色(ARS)。免疫组织化学(IHC)法检测Lc3、HK2蛋白表达。在暴露于FSS的OP组织和成骨细胞系中，NEAT1表达上调。在体外和体内，NEAT1基因的敲除都能抑制自噬。进一步的研究表明，NEAT1通过竞争内源RNA作用于miR-466f-3p而正向调控HK2的表达。此外，我们还发现NEAT1/miR-466f-3p/HK2轴调控成骨细胞的自噬。通过miR-466f-3p/HK2信号通路抑制成骨细胞的自噬，为绝经后骨质疏松症的新的分子治疗靶点提供新的思路。关键信息·流体切应力(FSS)可促进成骨细胞的自噬并进行转录组测序。·NEAT1在骨质疏松症中高表达，并调节成骨细胞的自噬。·敲除ncRNA NEAT1通过海绵miRNA-466f-3p和靶向HK2在骨质疏松中抑制成骨细胞自噬。
Osteoporosis (OP) is a systemic metabolic disorder characterized by a reduction in bone tissue volume. LncRNAs have been reported to act as regulators of several human diseases. Specifically, lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in proliferation, differentiation and apoptosis in osteoclasts and bone marrow mesenchymal stem cells and regulates the occurrence and development of OP. However, the relationship between NEAT1 and osteoblast autophagy and its mechanism are still unclear. Western blotting of LC3 and P62 was used to evaluate the effect of fluid shear stress (FSS) on autophagy in MC3T3-E1 osteoblasts. Total transcriptome sequencing and bioinformatics analyses were performed on osteoblasts loaded with and without FSS. qPCR was performed to examine the expression of NEAT1 in OP bone tissues and osteoblasts. The RNA-FISH was performed to study the localization of lncRNA NEAT1 and miR-466f-3p in MC3T3-E1 osteoblasts. In vitro, western blotting, transmission electron microscopy (TEM), immunofluorescence (IF) staining and qPCR were performed to verify the biological functions of NEAT1, miR-466f-3p and HK2. Subsequently, we conducted bioinformatics analysis and dual luciferase reporter assays to identify the relationships among NEAT1, miR-466f-3p and HK2. Additionally, rescue assays were conducted on osteoblasts to clarify the regulatory network of the NEAT1/miR-466f-3p/HK2 signalling pathway. In vivo, the OVX mouse model was used to investigate the effects of si-NEAT1 on autophagy in OP mice. The distal femur and serum were collected for further micro-CT analysis, blood biochemistry, and haematoxylin–eosin and Alizarin red staining (ARS). Immunohistochemistry (IHC) was performed to assess the protein expression of LC3 and HK2. NEAT1 expression was upregulated in OP tissues and osteoblast lines exposed to FSS. Knockdown of NEAT1 inhibited autophagy in vitro and in vivo. Further studies demonstrated that NEAT1 positively regulated HK2 expression via its competing endogenous RNA effects on miR-466f-3p. Moreover, we found the NEAT1/miR-466f-3p/HK2 axis regulated autophagy in osteoblasts. Knocking down NEAT1 inhibited autophagy in osteoblasts via the miR-466f-3p/HK2 signalling pathway, which may provide new ideas for novel molecular therapeutic targets of postmenopausal OP.Key messages• Fluid shear stress (FSS) can promote autophagy of osteoblast and performed transcriptome sequencing.• NEAT1 is overexpressed in osteoporosis and could regulate osteoblast cells autophagy.• Knockdown of lncRNA NEAT1 inhibited osteoblast cells autophagy by sponging miRNA-466f-3p and targeting HK2 in osteoporosis.