Abstract
Osteosarcoma (OS) is a primary malignant tumor of bone occurring in young adults. OS stem cells (OSCs) play an important role in the occurrence, growth, metastasis, drug resistance and recurrence of OS. CD133 is an integral membrane glycoprotein, which has been identified as an OSC marker. However, the mechanisms of metastasis, chemoresistance, and progression in CD133(+) OSCs need to be further explored. In this study, we aim to explore differences in miRNA levels between CD133(+) and CD133(−) cells from the MG-63 cell line. We found 20 differentially expressed miRNAs (DEmiRNAs) (16 upregulated and 4 downregulated) in CD133(+) cells compared with CD133(−) cells. Hsa-miR-4485-3p, hsa-miR-4284 and hsa-miR-3656 were the top three upregulated DEmiRNAs, while hsa-miR-487b-3p, hsa-miR-493-5p and hsa-miR-431-5p were the top three downregulated DEmiRNAs. In addition, RT-PCR analysis confirmed that the expression levels of hsa-miR-4284, hsa-miR-4485-3p and hsa-miR-3656 were significantly increased, while the expression levels of hsa-miR-487b-3p, hsa-miR-493-5p, and hsa-miR-431-5p were significantly decreased in CD133(+) cells compared with CD133(−) cells. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that predicted or validated target genes for all 20 DEmiRNAs or the selected 6 DEmiRNAs participated in the “PI3K-Akt signaling pathway,” “Wnt signaling pathway,” “Rap1 signaling pathway,” “Cell cycle” and “MAPK signaling pathway”. Among the selected six DEmiRNAs, miR-4284 was especially interesting. MiR-4284 knockdown significantly reduced the sphere forming capacity of CD133(+) OS cells. The number of invasive CD133(+) OS cells was markedly decreased after miR-4284 knockdown. In addition, miR-4284 knockdown increased the p-β-catenin levels in CD133(+) OS cells. In conclusion, RNA-seq analysis revealed DEmiRNAs between CD133(+) and CD133(−) cells. MiRNAs might play significant roles in the function of OSCs and could serve as targets for OS treatment. MiR-4284 prompted the self-renewal and invasion of OSCs. The function of miR-4284 might be associated with the Wnt signaling pathway.
Highlights
Osteosarcoma (OS) is a primary malignant tumor of bone occurring in young adults, with a morbidity of 4,000,000 per year (Li et al, 2016; Yin et al, 2021)
The results confirmed that the expression levels of hsa-miR-4284, hsa-miR-4485-3p, and hsa-miR-3656 were significantly increased in CD133(+) cells compared with CD133(−) cells (Figs. 4A–4C), while the expression levels of hsa-miR-487b-3p, hsa-miR-493-5p, and hsa-miR-431-5p were significantly decreased in CD133(+) cells compared with CD133(−) cells (Figs. 4B–4F)
The results showed that the number of invasive CD133(+) MG-63 and Saos-2 cells was markedly decreased after miR-4284 knockdown (Fig. 7B)
Summary
Osteosarcoma (OS) is a primary malignant tumor of bone occurring in young adults, with a morbidity of 4,000,000 per year (Li et al, 2016; Yin et al, 2021). The molecular mechanisms underlying metastasis, chemoresistance, and progression of OS need to be clarified to improve therapeutic options. Cancer stem cells (CSCs) are a subpopulation of tumor cells with capacities of selfrenewal, differentiation, and pluripotent differentiation (Lin et al, 2021; Zhang, Ma & Li, 2019a). CSCs cause disease recurrence and chemoresistance, and lead to tumor relapse and metastasis (Camuzard et al, 2020; Tornín et al, 2021; Zhang et al, 2019b). OS stem cells (OSCs) have been shown to cause recurrence and metastasis (Wang et al, 2020a). CD133 is considered as a stem cell marker for normal and cancerous tissues (Li et al, 2013). The mechanisms underlying the link between recurrence and metastasis of OS and CD133 expression in OS cells need to be further explored
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