LncRNA FTX/OCT4/miR-122-5p orchestrates self-renewal and lineage commitment of human dental pulp stem cells by directly targeting FOXO3.

backgroundHuman dental pulp stem cells (hDPSCs) possess multipotent properties and play a pivotal role in tissue regeneration. Elucidating the molecular mechanisms governing hDPSC pluripotency is essential for advancing their clinical application. We previously demonstrated that long noncoding RNA five prime to Xist (lncRNA FTX) suppresses the proliferation and multilineage differentiation capacity of hDPSCs by inhibiting octamer-binding transcription factor 4 (OCT4). This study aims to explore the molecular mechanisms by which FTX regulates hDPSC proliferation/differentiation via downstream miRNAs and their biological implications.

methodsSmall RNA sequencing was utilized to identify differentially expressed miRNAs between the FTX-overexpressing and vector hDPSCs. Bioinformatic analysis, luciferase assay, and chromatin immunoprecipitation (ChIP) were employed to elucidate the regulatory network of the FTX/OCT4/miR-122-5p axis. Cell counting kit-8 (CCK-8) assay, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, alizarin red staining, and oil red O staining were performed to validate the functional roles of miR-122-5p and forkhead box O3 (FOXO3) in FTX-mediated proliferation and differentiation potential in hDPSCs. Furthermore, the effect of FTX/miR-122-5p on dentin formation was assessed using a subcutaneous implantation model.

resultsSequencing revealed 115 differentially expressed miRNAs (26 upregulated and 89 downregulated; fold change ≥ 1.5, P < 0.05). miR-122-5p displayed a significant decrease in expression in FTX-overexpressing hDPSCs. Overexpression of miR-122-5p partially alleviated FTX's inhibitory effect on hDPSC proliferation and differentiation. Cotransfection experiments demonstrated that FTX overexpression impaired the pluripotency of hDPSCs in part through miR-122-5p-mediated regulation of FOXO3 in vitro and in vivo. Mechanistically, FTX specifically suppressed OCT4 expression, which led to the transcriptional inactivation of miR-122-5p. Furthermore, upregulation of FTX resulted in enhanced expression levels of FOXO3, an effect that could be counteracted by miR-122-5p.

conclusionsLncRNA FTX overexpression exerts a suppressive effect on the transcriptional expression of miR-122-5p, which subsequently activates FOXO3 and thereby impedes cell proliferation as well as multilineage differentiation capacity of hDPSCs. Moreover, a novel lncRNA FTX-OCT4-miR-122-5p interaction pathway was discovered. These findings shed light on two novel regulatory mechanisms - lncRNA FTX/OCT4/miR-122-5p regulation and lncRNA FTX/miR-122-5p/FOXO3 regulation - that unravel crucial signaling pathways governing the pluripotency of hDPSCs, potentially offering promising RNA-based therapeutic strategies and gene-editing targets for dentin regeneration.