LncRNA NR2F1-AS1 is involved in osteogenic differentiation in fracture healing via miR-423-5p.

IF 2 4区生物学 Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY

Cytotechnology Pub Date : 2025-08-01 Epub Date: 2025-07-02 DOI:10.1007/s10616-025-00786-8

Yun Chen, Kun Huang, Wenjun Ji, Miao Huang, Jincheng Sima, Jin Li, Hao Song, Wei Xiong, Chao-Qun Ma

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引用次数: 0

Abstract

To investigate the function and mechanism of action of LncRNA NR2F1-AS1 involved in osteogenic differentiation process. An in vitro model was constructed by osteogenic differentiation-induced stimulation (OS) on the hFOB1.19 cell line. Real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of NR2F1-AS1, miR-423-5p and osteogenic differentiation markers (RUNX2, OCN, OPN). Cell Counting Kit-8 (CCK8) method and flow cytometry were observed cell proliferation and apoptosis, respectively. Enzyme linked immunosorbent assay (ELISA) tested alkaline phosphatase (ALP) activity. Dual-Luciferase Report (DLR) assay and RNA immunoprecipitation (RIP) verified gene interactions. Bioinformatics methods predicted downstream target genes and their pathways of action. OS increased osteogenic differentiation markers and NR2F1-AS1 expression and decreased miR-423-5p levels. Transfection of si- NR2F1-AS1 promoted OS osteoblast apoptosis, but inhibited cell proliferation, ALP activity and osteogenic differentiation marker expression. NR2F1-AS1 is mostly present in the cytoplasm and is involved in the osteogenic differentiation process by down-regulating miR-423-5p. The use of miR-423-5p inhibitor can resist apoptosis induced by silencing NR2F1-AS1, promote osteoblast proliferation, activate ALP activity, and induce osteogenic differentiation process in osteoblasts. Bioinformatics prediction identified 82 target genes that might be involved in osteogenic differentiation, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that they were mainly associated with inter-synaptic formation and signaling. NR2F1-AS1 may promote osteoblast proliferation stimulate ALP activity, and induce osteogenic differentiation by down-regulating miR-423-5p.

Supplementary information: The online version contains supplementary material available at 10.1007/s10616-025-00786-8.

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LncRNA NR2F1-AS1通过miR-423-5p参与骨折愈合中的成骨分化。

探讨LncRNA NR2F1-AS1参与成骨分化过程的功能及作用机制。采用成骨分化诱导刺激（osteogenic differentiation induced stimulation， OS）方法对hFOB1.19细胞系建立体外模型。采用实时荧光定量PCR （RT-qPCR）检测NR2F1-AS1、miR-423-5p及成骨分化标志物RUNX2、OCN、OPN的表达。细胞计数试剂盒-8 （CCK8）法和流式细胞术分别观察细胞增殖和凋亡情况。酶联免疫吸附试验（ELISA）检测碱性磷酸酶（ALP）活性。双荧光素酶报告（DLR）试验和RNA免疫沉淀（RIP）证实了基因相互作用。生物信息学方法预测下游靶基因及其作用途径。OS增加成骨分化标志物和NR2F1-AS1表达，降低miR-423-5p水平。转染si- NR2F1-AS1促进骨肉瘤成骨细胞凋亡，抑制细胞增殖、ALP活性和成骨分化标志物表达。NR2F1-AS1主要存在于细胞质中，通过下调miR-423-5p参与成骨分化过程。使用miR-423-5p抑制剂可以抵抗NR2F1-AS1沉默诱导的细胞凋亡，促进成骨细胞增殖，激活ALP活性，诱导成骨细胞的成骨分化过程。生物信息学预测鉴定出82个可能参与成骨分化的靶基因，基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析表明，它们主要与突触间形成和信号传导有关。NR2F1-AS1可能通过下调miR-423-5p，促进成骨细胞增殖，刺激ALP活性，诱导成骨分化。补充信息：在线版本包含补充资料，可在10.1007/s10616-025-00786-8获得。

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来源期刊

Cytotechnology 生物-生物工程与应用微生物

CiteScore

4.10

自引率

0.00%

发文量

审稿时长

6-12 weeks

期刊介绍： The scope of the Journal includes: 1. The derivation, genetic modification and characterization of cell lines, genetic and phenotypic regulation, control of cellular metabolism, cell physiology and biochemistry related to cell function, performance and expression of cell products. 2. Cell culture techniques, substrates, environmental requirements and optimization, cloning, hybridization and molecular biology, including genomic and proteomic tools. 3. Cell culture systems, processes, reactors, scale-up, and industrial production. Descriptions of the design or construction of equipment, media or quality control procedures, that are ancillary to cellular research. 4. The application of animal/human cells in research in the field of stem cell research including maintenance of stemness, differentiation, genetics, and senescence, cancer research, research in immunology, as well as applications in tissue engineering and gene therapy. 5. The use of cell cultures as a substrate for bioassays, biomedical applications and in particular as a replacement for animal models.