上调长链非编码RNA ANRIL的表达通过miR-7-5p/IGF-1R轴促进炎症性牙周膜干细胞的成骨作用。
Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.
作者信息
Bian Minxia, Yu Yan, Li Yuzhi, Zhou Zhou, Wu Xiao, Ye Xiaying, Yu Jinhua
机构信息
Institute of Stomatology, Nanjing Medical University, Nanjing, China.
Key Laboratory of Oral Diseases of Jiangsu Province and Stomatological Institute of Nanjing Medical University, Nanjing, China.
出版信息
Front Cell Dev Biol. 2021 Feb 22;9:604400. doi: 10.3389/fcell.2021.604400. eCollection 2021.
BACKGROUND
Long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs).
METHODS
Healthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-β-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R).
RESULTS
HPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3'-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs.
CONCLUSION
This study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.
背景
INK4基因座中的长链非编码RNA（lncRNA）反义非编码RNA（ANRIL）是一种碱基长度约为3.8 kb的lncRNA，在包括细胞增殖、迁移和衰老在内的多种生物学功能中发挥重要作用。本研究确定了lncRNA ANRIL在炎症性牙周膜干细胞（iPDLSCs）衰老和成骨分化中的作用。
方法
分别从健康/炎症性牙周膜组织中分离健康牙周膜干细胞（hPDLSCs）和iPDLSCs。通过CCK-8、EdU检测和流式细胞术（FCM）测定增殖能力。采用蛋白质免疫印迹法（WB）、定量实时聚合酶链反应（qRT-PCR）、茜素红染色、碱性磷酸酶（ALP）染色、ALP活性检测和免疫荧光染色等方法，确定lncRNA ANRIL对iPDLSCs的生物学影响。进行衰老相关（SA）-β-半乳糖苷酶（gal）染色、蛋白质免疫印迹分析和qRT-PCR以确定细胞衰老。进行双荧光素酶报告基因检测以确认lncRNA ANRIL与miR-7-5-p以及miR-7-5p与胰岛素样生长因子受体（IGF-1R）的结合。
结果
成功分离并培养了hPDLSCs和iPDLSCs。与hPDLSCs相比，iPDLSCs中lncRNA ANRIL和IGF-1R表达下降，而miR-7-5p表达上调。ANRIL的过表达增强了OSX、RUNX2、ALP的成骨蛋白表达，并降低了p16、p21、p53的衰老蛋白表达。lncRNA ANRIL可通过海绵吸附miR-7-5p促进iPDLSCs的定向分化。上调miR-7-5p可抑制iPDLSCs的成骨分化。进一步分析确定IGF-1R是miR-7-5p的直接靶点。通过双荧光素酶报告基因检测验证了lncRNA ANRIL与miR-7-5p、miR-7-5p与IGF-1R的3'-UTR的直接结合。此外，拯救实验表明，敲低miR-7-5p可逆转lncRNA ANRIL缺陷对iPDLSCs成骨的抑制作用。
结论
本研究揭示lncRNA ANRIL通过调节miR-7-5p/IGF-1R轴促进iPDLSCs的成骨分化。
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