背景:骨肉瘤(OS)是一种恶性骨肿瘤,常见于20岁以下的儿童和青少年。microRNAs(miRNAs)的失调是OS发生和发展的重要因素。微小RNAmiR-744-5p在各种肿瘤中异常表达。然而,其在OS中的作用和分子靶标尚不清楚。
方法:使用基因表达Omnibus数据集GSE65071分析OS中差异表达的miRNA,并通过加权基因共表达网络分析鉴定潜在的hubmiRNA。实时定量PCR(qRT-PCR)检测miR-744-5p在OS细胞系中的表达。体外实验,包括CCK-8检测,集落形成试验,流式细胞术细胞凋亡测定,和试管形成测定,探讨miR-744-5p对OS细胞生物学行为的影响。通过生物信息学方法预测miR-744-5p的下游靶基因,结合位点通过双荧光素酶报告基因测定进行验证。
结果:低表达的miRNA,miR-744-5p,通过生物信息学分析确定为参与OS进展的hubmiRNA。核因子IX(NFIX)在OS中被证实为miR-744-5p的直接靶标。体外研究显示过表达miR-744-5p可以抑制OS细胞的生长,而miR-744-5p抑制显示出相反的效果。还观察到,用来自miR-744-5p过表达的OS细胞的条件培养基处理导致人脐静脉内皮细胞(HUVEC)中较差的增殖和血管生成。此外,NFIX过表达恢复了miR-744-5p过表达对OS细胞生长和HUVECs血管生成的抑制作用。
结论:我们的结果表明,miR-744-5p通过靶向NFIX来抑制OS细胞的生长和HUVEC中的血管生成,是OS进展中潜在的抑瘤miRNA。
BACKGROUND: Osteosarcoma (OS) is a malignant bone tumor that commonly occurs in children and adolescents under the age of 20. Dysregulation of microRNAs (miRNAs) is an important factor in the occurrence and progression of OS. MicroRNA miR-744-5p is aberrantly expressed in various tumors. However, its roles and molecular targets in OS remain unclear.
METHODS: Differentially expressed miRNAs in OS were analyzed using the Gene Expression Omnibus dataset GSE65071, and the potential hub miRNA was identified through weighted gene co-expression network analysis. Quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-744-5p in OS cell lines. In vitro experiments, including CCK-8 assays, colony formation assays, flow cytometry apoptosis assays, and tube formation assays, were performed to explore the effects of miR-744-5p on OS cell biological behaviors. The downstream target genes of miR-744-5p were predicted through bioinformatics, and the binding sites were validated by a dual-luciferase reporter assay.
RESULTS: The lowly expressed miRNA, miR-744-5p, was identified as a hub miRNA involved in OS progression through bioinformatic analysis. Nuclear factor I X (NFIX) was confirmed as a direct target for miR-744-5p in OS. In vitro studies revealed that overexpression of miR-744-5p could restrain the growth of OS cells, whereas miR-744-5p inhibition showed the opposite effect. It was also observed that treatment with the conditioned medium from miR-744-5p-overexpressed OS cells led to poorer proliferation and angiogenesis in human umbilical vein endothelial cells (HUVECs). Furthermore, NFIX overexpression restored the suppression effects of miR-744-5p overexpression on OS cell growth and HUVECs angiogenesis.
CONCLUSIONS: Our results indicated that miR-744-5p is a potential tumor-suppressive miRNA in OS progression by targeting NFIX to restrain the growth of OS cells and angiogenesis in HUVECs.