Long non-coding RNA FOXD3-AS1 silencing exerts tumor suppressive effects in nasopharyngeal carcinoma by downregulating FOXD3 expression via microRNA-185-3p upregulation

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Abstract

Emerging evidence indicates that the incidence of nasopharyngeal carcinoma (NPC) remains high in endemic regions despite changing environmental factors, suggesting that genetic traits contribute to its development. Recently, long non-coding RNA-microRNA-messenger RNA (lncRNA-miRNA-mRNA) axis has been reported to be implicated in the pathophysiological processes of malignancies. Moreover, initial bioinformatic analysis revealed a highly expressed lncRNA Forkhead box D3 antisense RNA1 (FOXD3-AS1) for mechanistic network underlying NPC in this present study. Therefore, this study aims to delineate the ability of lncRNA FOXD3-AS1 to influence the NPC progression. The relationship among lncRNA FOXD3-AS1, miR-185-3p, and FOXD3 was identified with bioinformatics prediction, dual-luciferase reporter gene assays, RNA-binding protein immunoprecipitation, and RNA pull-down assays. Furthermore, effects of lncRNA FOXD3-AS1 on malignant phenotypes in vitro, alongside tumor formation in vivo, of transfected NPC stem-like cells were examined with gain- and loss-of-function experiments. Our findings revealed that lncRNA FOXD3-AS1 and FOXD3 exhibited increased expression levels, while miR-185-3p exhibited diminished levels in NPC. The levels of lncRNA FOXD3-AS1 and FOXD3 were further correlated with tumor node metastasis stage and pathological type of patients with NPC. LncRNA FOXD3-AS1 was also confirmed to negatively regulate the miR-185-3p expression, which further targeted the downstream gene FOXD3. In addition, lncRNA FOXD3-AS1 knockdown repressed cell stemness, colony formation, viability, invasion, migration, and in vivo tumor growth, and accelerated cell apoptosis. Moreover, FOXD3 silencing or miR-185-3p overexpression reversed the effects of lncRNA FOXD3-AS1. Our findings provide evidence indicating that lncRNA FOXD3-AS1 could bind to miR-185-3p to upregulate the FOXD3 expression, thereby promoting the development of NPC.

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Fig. 1: LncRNA FOXD3-AS1, miR-185-3p, and FOXD3 are predicted to affect NPC.
Fig. 2: LncRNA FOXD3-AS1 expression, FOXD3 mRNA expression, and miR-185-3p expression in NPC tissues and are correlated with TNM stage and pathological type.
Fig. 3: LncRNA FOXD3-AS1 knockdown or upregulation of miR-185-3p inhibits the expression of FOXD3.
Fig. 4: Overexpression of lncRNA FOXD3-AS1 upregulated protein expression of FOXD3 and stemness-related factors (SOX2, ALDH1, OCT4, CD133, and Nanog).
Fig. 5: LncRNA FOXD3-AS1 can bind to miR-185-3p to upregulate FOXD3.
Fig. 6: LncRNA FOXD3-AS1 knockdown and miR-185-3p upregulation suppress colony formation of NPC cells.
Fig. 7: LncRNA FOXD3-AS1 knockdown and miR-185-3p upregulation accelerate NPC cell apoptosis.
Fig. 8: LncRNA FOXD3-AS1 knockdown and miR-185-3p upregulation repress NPC cell invasion and migration.
Fig. 9: FOXD3 silencing reverses the effect of lncRNA FOXD3-AS1 in NPC cells.
Fig. 10: LncRNA FOXD3-AS1 knockdown and miR-185-3p upregulation repress the growth of xenograft tumors of C666-1 cells in nude mice.
Fig. 11: The mechanism graph of the regulatory network and function of lncRNA FOXD3-AS1 in NPC.

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Acknowledgements

The authors would like to acknowledge the helpful suggestions concerning this study received from their colleagues. This work was supported by Key Projects of Natural Science Foundation of Hubei Province in 2015 (No. 2015CFA076).

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Correspondence to Jiang Hu.

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Hu, J., Pan, J., Luo, Z. et al. Long non-coding RNA FOXD3-AS1 silencing exerts tumor suppressive effects in nasopharyngeal carcinoma by downregulating FOXD3 expression via microRNA-185-3p upregulation. Cancer Gene Ther (2020). https://doi.org/10.1038/s41417-020-00242-z

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