Li et al. Cell Death and Disease (2020) 11:795 https://doi.org/10.1038/s41419-020-03006-7 Cell Death & Disease ARTICLE Open Access A novel lncRNA LNC_000052 leads to the dysfunction of osteoporotic BMSCs via the miR-96- 5p–PIK3R1 axis Mingyang Li1, Rong Cong2,LiyuYang1, Lei Yang1,YiqiZhang1 and Qin Fu1 Abstract Bone marrow-derived mesenchymal stem cells (BMSCs) in postmenopausal osteoporosis models exhibit loss of viability and multipotency. Identification of the differentially expressed RNAs in osteoporotic BMSCs could reveal the mechanisms underlying BMSC dysfunction under physiological conditions, which might improve stem cell therapy and tissue regeneration. In this study, we performed high-throughput RNA sequencing and showed that the novel long non-coding RNA (lncRNA) LNC_000052 and its co-expressed mRNA PIK3R1 were upregulated in osteoporotic BMSCs. Knockdown of LNC_000052 could promote BMSC proliferation, migration, osteogenesis, and inhibit apoptosis via the PI3K/Akt signaling pathway. We found that both LNC_000052 and PIK3R1 shared a miRNA target, miR-96-5p, which was downregulated in osteoporotic BMSCs. Their binding sites were confirmed by dual-luciferase assays. Downregulation of miR-96-5p could restrain the effects of LNC_000052 knockdown while upregulation of miR-96-5p together with LNC_000052 knockdown could improve the therapeutic effects of BMSCs. In summary, the LNC_000052–miR-96-5p–PIK3R1 axis led to dysfunction of osteoporotic BMSCs and might be a novel therapeutic target for stem cell therapy and tissue regeneration. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Introduction are widely employed in the treatment of various diseases, Bone marrow-derived mesenchymal stem cells (BMSCs) such as acute pulmonary injury and myocardial injury5,6. are common multipotent stem cells with self-renewal However, due to the limited amounts of cells that can be ability and are regarded as an ideal type of seed cells for collected from one donor and the unavoidable differ- regenerative cell therapy1. They are easy to harvest from entiation during in vitro cell culture, a continuous supply bone marrow and exhibit low immunogenicity due to the of standardized clinical-grade stem cells is urgently negligible expression of immune antigens on their surface required for clinical translation7. Some groups attempted as well as their inhibitory effects on major immune to regulate the gene expression of BMSCs to improve cells2,3. Moreover, their homing ability, together with their functions. Notably, the mechanism of competing directed differentiation and secretion in injured sites, endogenous RNAs (ceRNAs), in which long non-coding makes them become an attractive cell source in tissue RNAs (lncRNAs) act as microRNA (miRNA) sponges to regeneration4. Owing to these unique properties, BMSCs regulate target gene expression, has become a hot topic in the search for novel practical targets8. Shang et al.9 investigated differentially expressed lncRNAs in BMSCs with or without glucocorticoid treatment by lncRNA 10 11 Correspondence: Qin Fu ([email protected]) microarray analysis. Tang et al. and Sun et al. studied 1Department of Orthopedics, Shengjing Hospital of China Medical University, the osteogenesis-associated lncRNAs by treating BMSCs Shenyang, China with osteogenic reagents. 2Department of Obstetrics and Gynecology, Shengjing Hospital of China Medical University, Shenyang, China Edited by Y. Shi © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Li et al. Cell Death and Disease (2020) 11:795 Page 2 of 13 Nevertheless, all previous studies used inducers to treat BMSCs were prominently decreased (Fig. 2a, b). Trans- BMSCs in vitro to search for differentially expressed well assays showed that after AD-lnc52-EGFP transfec- lncRNAs, which may be insufficient to simulate the actual tion, the migration ability was significantly decreased (Fig. changes in BMSCs in vivo. During studying the patho- 2c). Moreover, significantly increased percentages of genesis of postmenopausal osteoporosis, our team found apoptotic cells and cells in the G0–G1 phase in the viability and differentiation properties of BMSCs LNC_000052 overexpressing BMSCs were detected by collected from ovariectomy (OVX)-induced osteoporotic flow cytometry (Fig. 2d, e). To analyze BMSC multi- rats were remarkably reduced compared with sham- potency, we took osteogenic differentiation as a reference operated (SHAM) rats. Accordingly, we hypothesized that and found calcium deposition was decreased upon the changes in gene expression during postmenopausal LNC_000052 overexpression (Fig. 2f). osteoporosis may be of paramount importance for the biological behavior of BMSCs. Therefore, we explored the MiR-96-5p directly bound to PIK3R1 but was sponged by transcriptional profiles of OVX BMSCs and SHAM LNC_000052 BMSCs by high-throughput RNA sequencing (RNA-seq), In order to identify miRNAs with binding sites with both and identified that the novel lncRNA, LNC_000052, and LNC_000052 and PIK3R1 that are lowly expressed in its co-expressed mRNA phosphoinositide-3-kinase reg- osteoporotic BMSCs, we screened the bioinformatics ulatory subunit 1 (PIK3R1), a negative regulatory subunit databases TargetScan (http://www.targetscan.org/vert_72/) of PI3K12 as well as an inhibitor of the Akt signaling and Bibiserv (https://bibiserv.cebitec.uni-bielefeld.de/)and pathway13, were upregulated in OVX BMSCs compared identified miR-96-5p as the only eligible miRNA (Fig. with SHAM BMSCs. Then we conducted a series of 3a–c). Luciferase activity significantly decreased upon co- experiments to characterize the interaction between transfection of miR-96-5p mimics and wild-type LNC_000052 and PIK3R1 and their roles in BMSCs. LNC_000052 compared with the other three groups. Meanwhile, the co-transfection group with miR-96-5p Results mimics and wild-type PIK3R1 exhibited prominently The novel lncRNA LNC_000052 and its co-expressed mRNA decreased luciferase activity compared with the other PIK3R1 were upregulated in osteoporotic BMSCs groups. We further altered LNC_000052 expression and The RNA-seq analysis provided an overview of the found miR-96-5p expression was negatively correlated with lncRNAs that were differentially expressed in OVX LNC_000052 levels (Fig. 3a-iii). Then we transfected BMSCs compared with SHAM BMSCs. In total, 2541 BMSCs with miR-96-5p agomir or antagomir, and found lncRNAs were markedly differentially expressed in that PIK3R1 expression was downregulated in agomir-96- osteoporotic BMSCs, i.e., 849 lncRNAs were upregulated 5p cells and upregulated in antagomir-96-5p cells, while p- in osteoporotic BMSCs while 1692 were downregulated Akt/Akt changed oppositely (Fig. 3b-iii-e). Collectively, (Fig. 1a). We aimed to identify some hyper-activated miR-96-5p interacts with LNC_000052 and PIK3R1 via targets that could be inhibited to improve the function of these specific binding sites. BMSCs for stem cell therapy. Therefore, a novel lncRNA, LNC_000052, and its co-expressed mRNA, PIK3R1, Effects of miR-96-5p on BMSC proliferation, migration, caught our attention (Fig. 1b). We first validated the apoptosis, cell cycle, and differentiation upregulation of LNC_000052 in OVX BMSCs by qRT- CCK-8 and EdU assays demonstrated that decreases in PCR (Fig. 1c). Then we confirmed PIK3R1 was upregu- miR-96-5p expression significantly inhibited cell pro- lated in OVX BMSCs at both mRNA and protein level liferation (Fig. 4a, b). The migration of BMSCs was (Fig. 1d, e). To prove PIK3R1 was co-expressed with inhibited in response to miR-96-5p knockdown (Fig. 4c). LNC_000052, BMSCs were transfected with an Insufficient miR-96-5p also remarkably promoted the LNC_000052 silencing plasmid (si-LNC_000052) and apoptosis of BMSCs and increased the percentage of cells LNC_000052 overexpression adenovirus (AD-lnc52- in the G0–G1 phase (Fig. 4c, d). Furthermore, antagomir- EGFP). qRT-PCR, western blot, and immunofluorescence 96-5p significantly delayed BMSC osteogenesis (Fig. 4e). analyses confirmed that the changes in PIK3R1 levels By contrast, miR-96-5p overexpression significantly pro- were consistent with those of LNC_000052 (Fig. 1f–h). moted cell proliferation, migration, osteogenesis, and And the downstream signaling p-Akt/Akt showed an inhibited apoptosis. opposite trend against PIK3R1 (Fig. 1g). Knockdown of miR-96-5p restrained the positive effects of Effects of LNC_000052 on BMSC proliferation, migration, LNC_000052 downregulation on BMSCs apoptosis, cell cycle, and differentiation We co-transfected BMSCs with si-LNC_000052 and Cell Counting Kit-8 (CCK8) and EdU assays demon- antagomir-96-5p, and the cells were divided into the fol- strated