The lncRNA CYTOR Regulates SOX11 to Promote Osteogenic Differentiation of Human Periodontal Ligament Stem Cells, Potentially a Novel Target for Period

Periodontitis is a highly prevalent chronic inflammatory disease associated with the loss of periodontal tissues (including gums, periodontal ligaments, alveolar bone, particularly around teeth), ultimately resulting in tooth loss. Periodontal ligament stem cells (PDLSCs) are considered an ideal cell source for periodontal and alveolar bone tissue regeneration [1-2], holding promise for periodontitis-related alveolar bone regeneration therapy. Thus, gaining deeper insights into the mechanisms that control PDLSC osteogenic differentiation would greatly advance the development of novel therapeutic approaches for tissue regeneration [3].

In March 2023, Professor Ai Hong's research team at the Third Affiliated Hospital of Sun Yat-sen University published an article titled 'lncRNA CYTOR Facilitates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating SOX11 via Sponging miR-6512-3p' in the journal 'Stem Cells International'. The study demonstrated that CYTOR acts as a positive regulator of PDLSC osteogenic differentiation and revealed its role as a competitive endogenous RNA (ceRNA) that modulates SOX11 through the sponging of miR-6512-3p. These research findings offer a novel perspective on the fundamental mechanism underlying PDLSC osteogenic differentiation and hold the potential to become a new therapeutic approach for periodontal tissue regeneration [3].

In this study, specific siRNA targeting SOX11 (siSOX11) designed and synthesized by GencefeBiotech was employed alongside its control (siNC). Through qRT-PCR and Western Blot validation, it was confirmed that siSOX11 effectively silenced the mRNA and protein levels of SOX11.

Research Findings:

1.Long non-coding RNA CYTOR primarily expresses in the cytoplasm of PDLSCs and promotes the osteogenic differentiation of PDLSCs.

2.CYTOR directly binds to miR-6512-3p and promotes the osteogenic differentiation of PDLSCs through competitive binding to miR-6512-3p.

Through bioinformatics analysis predicting potential miRNAs with binding sequences to CYTOR, eight candidate miRNAs were identified. Subsequent experimental validation revealed that only the expression of hsa-miR-6512-3p decreased, and it could be suppressed by the overexpression of CYTOR during the osteogenic differentiation of PDLSCs. To further assess whether the binding between CYTOR and miR-6512-3p affects the osteogenic differentiation of PDLSCs, researchers applied miR-6512-3p mimics and miR-6512-3p inhibitors to modulate the expression of miR-6512-3p in PDLSCs. Experimental results demonstrated that miR-6512-3p mimics/inhibitors effectively controlled the levels of miR-6512-3p in PDLSCs.

3.CYTOR counteracts the inhibition of SOX11 by miR-6512-3p; CYTOR promotes the osteogenic differentiation of PDLSCs by regulating SOX11.

Bioinformatics analysis predicted potential targets of miR-6512-3p, identifying SOX11 as a common target. Series of experiments validated the mutual influence between CYTOR and miR-6512-3p on the level of SOX11. Overall, these data indicate that CYTOR reduces the inhibitory effect of miR-6512-3p on SOX11.

Researchers further confirmed the functional role of SOX11 in osteogenic differentiation through RNA interference (RNAi). Three specific small interfering RNAs (siSOX11) targeting human SOX11 or control (siNC) were transfected into PDLSCs. Both mRNA and protein levels of SOX11 were effectively silenced by siSOX11. Further experiments demonstrated that silencing SOX11 attenuated the increased levels of osteogenic marker proteins induced by CYTOR overexpression in PDLSCs. This illustrates that CYTOR enhances the osteogenic differentiation of PDLSCs by modulating SOX11.

Discussion:

This study demonstrates the pivotal role of CYTOR in the osteogenic differentiation of PDLSCs. CYTOR acts as a ceRNA, upregulating SOX11 levels by sponging miR-6512-3p, thereby promoting the osteogenic differentiation of PDLSCs (see Figure 4). The CYTOR/miR-6512-3p/SOX11 axis holds promise as a novel therapeutic target in periodontal regenerative medicine.

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Reference：[1]?B.-M. Seo, M. Miura, S. Gronthos et al., “Investigation of multipotent postnatal stem cells from human periodontal ligament,” The Lancet, vol. 364, no. 9429, pp. 149–155, 2004.

[2] P. M. Bartold and S. Gronthos, “Standardization of criteria defining periodontal ligament stem cells,” Journal of Dental Research, vol. 96, no. 5, pp. 487–490, 2017.

[3] Tu, Shaoqin et al. “lncRNA CYTOR Facilitates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating SOX11 via Sponging miR-6512-3p.” Stem cells international vol. 2023 5671809. 3 Mar. 2023.