WITHDRAWN: LncRNA NHS-AS1 regulates osteo/odontogenic differentiation of stem cells from apical papilla via miR-5011-5p/SMAD7 axis

Abstract

Background: Elucidating the osteo/odontogenic differentiation mechanisms in human stem cells from apical papilla (SCAPs) is the key to promoting dentin regeneration. A growing number of studies have identified long non-coding RNAs (lncRNAs) as key roles in osteogenesis. Nonetheless, their functions and mechanisms in osteo/odontogenic differentiation of SCAPs have yet to be clarified. Methods: We analyzed the expression profiles of lncRNAs between the osteo/odontogenic differentiation group and control group by lncRNAs microarray and found NHS-AS1 was a key factor during the osteo/odontogenic differentiation of SCAPs, as evidenced by quantitative real-time PCR (qRT-PCR). Explored the function of NHS-AS1 by transfecting NHS-AS1 overexpression plasmid vectors into SCAP cells. qRT-PCR, Western blot, alkaline phosphatase (ALP) staining and alizarin red staining (ARS) were used to detect changes in osteo/odontogenesis-related indicators. Bioinformatics analysis indicated that NHS-AS1 could sponge miR-5011-5p and miR-5011-5p could target SMAD7, which were further affirmed by dual luciferase reporter assay. The role of miR-5011-5p and SMAD7 in the osteo/odontogenic differentiation of SCAP were also clarified by cell transfection techniques. We performed co-transfection experiments to confirm the involvement of miR-5011-5p and SMAD7 in NHS-AS1-mediated osteo/odontogenic differentiation of SCAP.Results: The expression of NHS-AS1 was down-regulated after osteo/odontogenesis of SCAP. Over-expression of NHS-AS1 significantly inhibited the osteo/odontogenesis of SCAPs. Dual luciferase reporter assay demonstrated NHS-AS1 could sponge miR-5011-5p. Further study showed that miR-5011-5p mimics promoted osteo/odontogenic differentiation of SCAPs and reversed NHS-AS1’s inhibitory impact on the process. Meanwhile, as the target gene of miR-5011-5p, SAMD7 depressed the osteo/odontogenic differentiation of SCAPs and could overcome the positive influence of miR-5011-5p mimics on the process. NHS-AS1 competitively sponged miR-5011-5p and alleviated the suppressive action of miR-5011-5p against SMAD7 expression, thereby significantly inhibiting the osteo/odontogenic differentiation of SCAPs. Conclusion: NHS-AS1 inhibits the osteo/odontogenic differentiation of SCAPs via miR-5011-5p/SMAD7 axis.