Aberrantly expressed long noncoding RNAs in adipose-derived mesenchymal stem cells differentiation to nucleus pulposus-like cells

Aberrantly expressed long noncoding RNAs in adipose-derived mesenchymal stem cells differentiation to nucleus pulposus-like cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Aberrantly expressed long noncoding RNAs in adipose-derived mesenchymal stem cells differentiation to nucleus pulposus-like cells Jian Zhu, Libin Jin, Kaipeng Jin, Yongping Wu, Lingling Sun, Yuluan Huang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7130762/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 18 You are reading this latest preprint version Abstract Background: Stem cells were often used for intervertebral disc degeneration (IDD) regeneration. The underlying mechanisms remain to be explored. LncRNAs were found to be related to the physiological process such as apoptosis and differentiation. Many studies focus on the messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) between normal nucleus pulposus and degeneration nucleus pulposus. However, few studies have shed light on the different expression of lncRNA and mRNA in the differentiation. In the present study, we aimed to determine mRNAs and lncRNAs, which are differentially expressed during in human adipose-derived mesenchymal stem cells (hADSCs) differentiation process into np-like cell types and to explore the related signaling pathways and the regulatory networks. Methods: hADSCs were induced to differentiation into np-like cell under the cytokine circumstance. The mark genes of np-like cell were determined by PCR and immunology staining. Then RNA-seq was used to analysis the expression of lncRNA and mRNA in the differentiation of hADSCs into np-like cell types. The significant genes were confirmed by Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Results: We found 14 lncRNAs and 601 mRNAs were significantly differentially expressed in hADSCs differentiation. The RNA-seq data were confirmed by real-time PCR. Furthermore, we found Gene Ontology terms were upregulated, and downregulated and significantly enriched pathways. Moreover, gene network shows significant differentially expressed genes. Meanwhile, the relationship of significantly changed mRNAs and lncRNAs were revealed by mRNA-lncRNA co-expression network. Conclusion: Our results first explore differentially expressed lncRNAs and mRNAs in the differentiation of hADSCs into np-like cell types. These may supply useful information for better understanding of stem cell therapy and IDD regeneration. Biological sciences/Cell biology Biological sciences/Computational biology and bioinformatics Biological sciences/Developmental biology Biological sciences/Genetics Biological sciences/Molecular biology Biological sciences/Stem cells Intervertebral disc degeneration Human adipose-derived mesenchymal stem cells Nucleus pulposus cells RNA-seq Long non-coding RNAs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Low back pain caused by degenerative disc disease (DDD) is very common in people of all ages and is the main cause of limited working ability in people under 45 years of age[1]. The incidence of low back pain associated with DDD is 60 to 90%, and the United States costs up to $50 billion to $100 billion in DDD medical costs each year [2], and the prevalence rate of chronic diseases was 9.5‰, both ranking sixth among all diseases. The social burden caused by DDD in China is also very huge. At present, symptomatic surgery is often used in clinical treatment of DDD[3], but surgery has a high recurrence rate and more complications, and brings great physical and mental pain and economic burden to patients. Studies at home and abroad have shown that the decrease of nucleus pulposus cells is the main initiating factor of DDD. Nucleus pulposus cells are the main cells in intervertebral disc, and many animal model studies have shown that intervertebral disc degeneration is often accompanied by a decrease in cell density[4, 5]. Similarly, in juvenile patients with neuromuscular scoliosis, the number of cells in the intervertebral disc on the curved convex side (degenerative side) is significantly lower than that on the concave side (normal side), and the water content is also reduced[6]. In addition, with the increase of age, the number of senescent cells in the nucleus pulposus increased from about 2% in the fetal period to 80% in the old age[7]. In addition, nucleus pulposus cells play an important role in the metabolic balance of intervertebral disc stroma. Nucleus pulposus cells are twice as capable of synthesizing nucleus pulposus stroma as chondroid cells, and can enhance chondroid cell synthesis by secreting cytokines such as connective tissue growth factor (CTGF) [8]. Therefore, restoring the number of nucleus pulposus cells in the degenerated intervertebral disc is expected to repair the degenerated intervertebral disc, which also provides a theoretical basis for stem cell transplantation treatment of DDD. Numerous studies have confirmed that stem cell transplantation can not only induce differentiation into nucleus pulposus cells, but also promote the synthesis of matrix proteins such as type II collagen and proteoglycan in the degraded intervertebral disc, significantly restore the water content of nucleus pulposus, and significantly increase the intervertebral disc height index[9-11]. The adverse microenvironment of the degenerated intervertebral disc (low pH, low oxygen, changes in osmotic pressure and accumulation of inflammatory factors) may be an important reason for the low survival rate, limited differentiation and low matrix synthesis of the transplanted stem cells in vivo[12-14]. However, the exact mechanism by which stem cell differentiation is limited in the intervertebral disc microenvironment remains poorly understood. In-depth research on the methods and mechanisms of how to improve the directional differentiation of ADSCs into nucleus pulposus cells in the intervertebral disc microenvironment has important guiding value for the rational application of stem cell transplantation in the treatment of DDD and the formulation of more effective treatment plans. Intervertebral disc degeneration (IDD) is the main cause to low back pain which induces serious burden to the society. Numerous studies have been done to seek the methods to intervertebral disc regeneration[15, 16]. Stem-cell based therapy is one of the most promising methods to regenerate intervertebral disc[17, 18]. Mesenchymal stem cell cultured with cytokines could differentiation into np-like cell type[19]. But the underlying mechanism remains to be explored. Recently, lncRNAs are emerged as an important regulator in the differentiation process of stem cell[20]. RNA-seq technology can measure differences in the expression level of thousands of genes and allows highly effective evaluation of genome-wide expression changes. With the help of RNA-seq, we could find the significant different related lncRNA, miRNA and mRNA. Most studies focus on the different lncRNAs between normal and degeneration intervertebral disc[21-23]. As stem cell based therapy act an important role in the process of intervertebral disc regeneration[24, 25]. It is necessary to find the underlying mechanism in the differentiation process of hADSCs. Yet, there was no study focusing on the different expression of lncRNA/miRNA/mRNA in the differentiation of hADSCs into np-like cell type. Our study aimed to use RNA-seq analysis to investigate the lncRNAs, miRNA and mRNAs which related to differentiation. We also use bioinformatics methods to reveal the promising signaling pathways through KEGG analysis, and gene regulation networks between lncRNAs, miRNAs and mRNAs. We hope that our study will shed new sight into the cell based therapy and supply new target for intervertebral disc regeneration. Methods Cells and Regents hADSCs were obtained from Cyagen Biosciences (HUXMD-01001; Guangzhou, China). hADSCs were culture in medium from Cyagen Biosciences (HUXMD-90011; Guangzhou, China) in a humidified incubator at 37 °C with 5% CO2. The culture medium was replaced every 3 days. hADSCs at passages 2–4 was used for subsequent experiments. hADSC Differentiation Culture medium The NP differentiation medium was composed of 1× ITS, 0.1 μM dexamethasone, 1 mM sodium pyruvate, 0.35 mM proline, 0.17 mM ascorbic acid–2-phosphate, 1.25 mg/ml BSA, 10 ng/ml TGF-β1 and 100 ng/ml GDF5；1× Anti, 10ng/ml BMP2[26]. Cell pellet culture For preparation of 3D cell culture, 3 × 10 5 cells were centrifuged at 1500 rpm for 5 min in 15 ml polypropylene conical tubes and incubated at 37°C overnight. Pellets were formed after 24 h culture. Then pellets were cultured with different differentiation medium. The NP group was cultured with NPM differentiation medium. The control group was cultured with hADSCs culture medium. Medium was changed every three days. Pellets at 7, 14 and 21 days were selected for light microscopic analysis. After 7, 14 and 21 days culture, pellets were fixed with 4% paraformaldehyde for 24 h and then dehydrated with 30% sucrose water. Histology analysis Pellets at 7, 14 and 21 days were selected to cut into 7 um frozen section. These sections were stained by H&E. Immunofluorescence staining Pellets at 21 days were selected to be fixed in 4% paraformaldehyde and then cut into 7 um frozen sections. The frozen sections were incubated in primary rabbit collagen II (1:500 dilutions), aggrecan (1:500 dilutions) and anti-SOX-9 (1:500 dilutions) overnight at 4°C. Afterward, the cells were washed and incubated for 1-hour with fluorescence-conjugated secondary antibody. Finally, cells were washed by PBS and stained by DAPI for 5 min (BOSTER Biological Technology). Then, the cells were observed and imaged by the fluorescence microscope. RNA-seq analysis mRNA and lncRNA expression profiles were compared in hADSCs cultured with NPM and control medium. To clarify the changes in the signaling pathways of hADSCs differentiation, we further performed Gene Ontology (GO) analysis, KEGG analysis, pathway analysis, and signal-net analysis. RNA-seq analysis was performed by Lc-Bio Technologies (Hangzhou) Co., Ltd. Random variance model (RVM) t test was used to identify the different expression of mRNAs and lncRNAs. Cluster map was created after hierarchical clustering was performed. qRT-PCR analysis To validate the NP-differentiation and the RNA-seq results, these mRNAs were selected Total RNA was tracted from cells with RNAiso reagent (TaKaRa Bio, Japan). A PrimeScript RT reagent kit (TaKaRa, Bio, Japan) was used to reverse transcription. qRT-PCR was performed using the StepOnePlus Real-time PCR System (Applied Biosystems, CA, USA) and a SYBR® Premix Ex Taq™ kit (TaKaRa Bio, Japan). 18s rRNA was used as a housekeeping gene. The results were calculated using the 2-ΔΔCt method. All reactions were performed in triplicate and the sequences of used primers are shown in Table 1 were synthesized by Sangon Biotech (Shanghai, China). GO analysis Two-sided Fisher’s exact test and chi-square test are used to analyze the main function of significant differentially expressed in the GO analysis based on the GO database (http://www.geneontology.org). P < 0.01 were defined as significant regulated. Pathway analysis Base on Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/), we import data and select human species for calculation. Fisher’s exact test and the chi-square test were used to analyse the significance score of differential gene enrichment in each pathway， P < 0.05 were defined as significant regulated. Different gene expression pathways were analyzed on the basis of KEGG database. Signal-net analysis Significant crossover genes in GO analysis and pathway analysis were selected to analyze gene-gene interactions and construct network maps. Based on KEGG database, gene-gene network maps are constructed with differentially expressed gene data. The network is presented as a graph, the nodes are genes, and the edges between the nodes may indicate activation or phosphorylation. The network function of each gene is presented related to the number of upstream and downstream genes, which are expressed in the form of internal and external degrees. The mesocentricity of each gene was calculated according to the inner and outer degrees of genes. The higher the mesocentricity, the more important it is in gene-network regulation. Competing endogenous RNA (ceRNA) analysis lncRNAs can regulate miRNA abundance by competitively binding miRNAs, thus regulating post-transcriptional regulatory processes. In our experiment, we used miRNAs with significant differences to map competitive RNA networks. During the drawing process, we use relevant targetscan (http://www.targets can.org/), miRDB (https://www.mirdb.org) and starbase (starBase or ENCORI: Decoding the Encyclopedia of RNA Interactomes (rnasysu.com) to process target predictions. Competing RNA networks were mapped with interacting miRNAs and lncRNAs. Statistical analysis All the state were reported as average ± standarded. The difference was analysed by Student’s t analysis by SPSS 20.0 software (Chicago IL, USA). P < 0.05 was defined as different significance. Results The specific gene expression of np-like cell type hADSCs cells were culture in 3D model. The result shows the microscopic morphology of pellet. The histological image shows the result that NP group had more np-like cell type than control groups (Figure1). The specific gene of PAX1, FoxF1, IBSP, FBLN1, SOX9, ACAN, CA12 and COL2 were measured by PCR. The np-like cell type specific gene PAX1, FoxF1 and CA12 were significant upregulated in Induced (NP) group. The cartilage specific gene IBSP and FBLN1 show no significant difference between Induce (NP) and Non-Induced group (Figure2). Immunofluorescence of sox-9, col-2 and aggrecan show that hADSCs cultured with growth factor could differentiate into np-like cell type (Figure3). Identification of differentially expressed lncRNAs and mRNAs To determine that whether lncRNA and mRNA were involved in the differentiation process, we used RNA-seq to explore the expression of hADSCs cultured with growth factors. 12092 lnRNAs and 20256 mRNAs were detected with regulation in the differentiation process. As shown in figure 4, 500 lncRNA and 601 mRNAs were significantly differently expressed in the differentiation process of hADSCs into np-like cell type. The top 10 differential expressed lncRNAs were AL355075.4, MALAT1，AC022966.2, AC006064.4, AC145207.3, AC125611.3, DDIT4-AS1, AL662797.3，AL121748.2 and AC008914.1. The top 10 differential expressed mRNA were KCTD11，ALOX15B，KANK4，RNF139，TLR2，CHMP1B，CA9，PFN1P2，LEP，RNU1-27P. The np cell specific gene such as sox-9, col2a1, and CD24 were upregulated expressed in NP group. The results were consistent with PCR result. mRNAs listed are the top ten regulated mRNAs in the differentiation. And lncRNAs listed are the top ten regulated lncRNA. Go analysis To determine the biological function of the genes, GO enrichment analysis were performed. Different expressed lncRNAs and mRNAs were used to make GO analysis. There are 51 GO terms show significant regulated in NP and Non-Induced group. The top twenty regulated GO terms were listed in figure 5. The most abundant genes are concentrated on this GO term extracellular matrix structural constituent (GO:0005201). Genes also enriched on GO term cadherin binding，glycosaminoglycan binding and kinase regulator activity. Pathway analysis Based on the latest version of the KEGG database, we made KEGG pathway analysis with significant enrichment of differentially expressed mRNAs. The top twenty significant regulated pathways in NP and Non-Induced group were listed in figure 6. The pathway PI3K-Akt and Cytoskeleton in muscle cells show highly related with the differentiation process. The significance of the corresponding pathway was denoted by P-value. We also calculated the enrichment score value, which represents the enrichment importance of pathway ID. These values are equal to - log10 (p-value). Regulation of ceRNA network A competing endogenous RNA network was constructed by fourteen differentially expressed lncRNAs, 51 differentially expressed miRNAs and 601 differentially expressed mRNAs based on the degree of correlation. RPL41, RNU4-2, U2, ZNF331, JARID2, CLVS1, GAS5, EMX2OS, MALAT1, MEG3, CYP1B1-AS1, PRICKLE2-AS1, VCAN-AS1, PAPPA-AS1 were most closed related in the competing endogenous RNA network. While ZNF331, JARID2, MEG3 show upregulated in NP group compared to control group, PRICKLE2-AS1, CYP1B1-AS1, MALAT1, MEG3 and GAS5 show both upregulated and downregulated. VCAN-AS1, PAPPA-AS1, RPL41, RNU4-2 and CLVS1 show downregulated (Figure 7). Discussion Intervertebral disc degeneration is the result of many factors, and the decrease of nucleus pulposus cells is the main initiating factor[11, 27]. Existing studies have confirmed that intervertebral disc contains stem cells[28]. Therefore, stimulating the growth of endogenous intervertebral disc stem cells and their active differentiation into nucleus pulposus cells can be an effective therapeutic strategy. However, whether stem cells can effectively differentiate into nucleus pulposus cells, and then effectively improve the pathological state caused by insufficient number of endogenous stem cells and aging, there is still a big realistic bottleneck[26]. Earlier results of our research group's induction of Human adipose stem cells (hADSCs) into nucleus pulpocyte differentiation showed that the proportion of nucleus pulpocyte only reached 40%, far from meeting the needs of clinical treatment of DDD[6]. Therefore, it is particularly critical to further study and effectively improve the mechanism and method of hADSCs nucleus pulposus differentiation efficiency, and further exploration in this direction will provide important scientific research guidance and clinical transformation value for clinical stem cell transplantation programs. Many research[29-31] have reported that long noncoding RNA (lncRNA) plays an important regulatory role in the maintenance of pluripotency and directed differentiation of stem cells. A large number of existing studies have also proved that lncRNA is more tissue-specific than protein-coding gene expression in human tissue cells, and is closely related to transcription factors, indicating that lncRNA plays an important regulatory role in cell differentiation[32-34]. Numerous studies have shown that 1ncRNA is involved in the directed differentiation of adult stem cells: for example, LncMyoD is involved in the differentiation of stem cells into skeletal muscle cells[35]. LncRNA DANCR promotes stem cell differentiation into bone cell [36]. LncRNA ADINR promotes stem cell differentiation into adipocytes[37]. Although the above studies found changes in the expression profile of LncRNAs in stem cell differentiation, little is known about their production mechanism and physiological function. Therefore, it is of great significance to study the specific expression of lncRNA in stem cell differentiation and its role and mechanism of action. Our studies on lncRNAs involved in nucleus pulposus differentiation of hADSCs found that there were 12092 lncRNAs and 20256 genes specifically expressed in hADSCs (nucleus pulposus group) and hADSCs (normal group) cultured in normal medium, respectively. At present, we further analyzed the data from the current major lncRNA databases. The summary results of various test data suggest that RPL41, RNU4-2, U2, ZNF331, JARID2, CLVS1, GAS5, EMX2OS, MALAT1, MEG3, CYP1B1-AS1, PRICKLE2-AS1, VCAN-AS1 and PAPPA-AS1 may be important regulatory lncRNAs in hADSCs nucleus pulposus differentiation. In previous studies，lncRNA GAS5, MALAT1 and MEG3, was related to nucleus pulposus cell degeneration process[38-40]. Upregulate ZNF331 and JARID2 may stimulate the differentiation process of hADSCs into nucleus pulposus cell. The role of lncRNA RPL41, RNU4-2, U2, CLVS1, EMX2OS, CYP1B1-AS1, PRICKLE2-AS1, VCAN-AS1 and PAPPA-AS1 is needed further exploration in the differentiation. Regarding the regulatory function of lncRNAs, we investigate the relation between the mRNA and lncRNA. Many gene were rich in extracelluar matrix structural constitute, collagen binding, glycosaminoglycan binding. In our study，LOX, CDKN1A, PLOD2, INSIG1, EIF1, IGFBP5, HMGCS1, DDIT4, PTP4A1, ATP5B and DHCR24 are the top ten regulated genes in the differentiation process. LOX was reported as a regulator in osteoblast differentiation[41]. Lysyl oxidase modulates the osteoblast differentiation of primary mouse calvaria cells and governs osteogenic and adipogenic cell fate by MSCs[42, 43]. Cdkn1a expression significantly contributed to osteoclast differentiation[44]. CDKN1A regulated chondrogenic differentiation of human chondrocytes in osteoarthritis[45]. KDM1A binded with PLOD2, and finally resulted in the inhibited function for the osteo/dentinogenesis in stem cells of the apical papilla[46]. IGFBP5 promotes angiogenic and neurogenic differentiation potential of dental pulp stem cells[47]. IGFBP5 enhances osteogenic differentiation potential of periodontal ligament stem cells[48]. ATP5B make a contribution to osteoclast differentiation and joint destruction[49]. DHCR24 play a role in neuronal differentiation[50]. Little studies show INSIG1, EIF1, IGFBP5, HMGCS1, DDIT4, PTP4A1 in the differentiation process. Studies may be taken to explore the mechanism of these gene in cell differentiation. During the differentiation process, PI3K/Akt signaling pathway and cytoskeleton in muscle cell play an important role. Previouslly, PI3K/Akt signaling pathway were found in the osteogenic, endothelial and epithelial, myoblast differentiation process[51-54]. Study have highlighted the importance of this pathway to development and cellular differentiation. Our study showed the PI3K/Akt signaling pathway were upregulated in the nucleus pulposus differentiation process. The top regulated lncRNAs and mRNAs listed may show a great possibility in playing a role in the differentiation process. Such a considerable divergence needed to be further in-depth investigations into the sophisticated underlying mechanism. More in vitro and in vivo studies should be done to explore the mechanism of cell differentiation. Conclusions The mechanism of the differentiation of stem cells is very complex, and it was initially thought that the differentiation of stem cells is mainly by regulation of tissue-specific transcription factors. LncRNAs play an important regulatory role in the maintenance of pluripotency and directed differentiation of stem cells. We found 14 lncRNAs and 601 mRNAs were significantly differentially expressed in hADSCs differentiation. Our results first explore differentially expressed lncRNAs and mRNAs in the differentiation of hADSCs into np-like cell types. These may supply useful information for better understanding of stem cell therapy and IDD regeneration. List of abbreviations LncRNA: Long noncoding RNA; IDD: intervertebral disc degeneration; mRNAs: messenger RNAs; hADSCs: human adipose-derived mesenchymal stem cells; DDD: degenerative disc disease; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes ; ceRNA: Competing endogenous RNA Declarations Ethics Approval and consent to participate： All procedures performed in studies involving human participants were in accordance with the ethical standards of the Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine, China. Consent for publication: Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Availability of data and material： Sequence data that support the findings of this study have been deposited in the ArrayExpress accession with the primary accession code E-MTAB-15403. Competing interests： The authors declare no conflicts of interests. Funding: This project was supported by this study by the Natural Science Foundation of Zhejiang Province (Project No. LQ22H060005, LY21H060003 and LY21H060004), the National Natural Science Foundation of China (Project No. 82102592), and the Scientific Research Project of the Department of Education of Zhejiang Province (Project No. Y202352378). These fundings were used in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Authors' contributions: All authors contributed to the study conception and design. Data collection and analysis were performed by Jian Zhu, Libin Jin, Kaipeng Jin, Yongping Wu, Lingling Sun，Yuluan Huang and Chengchun Shen. The first draft of the manuscript was written by Jian Zhu and all authors commented on previous versions of the manuscript. Weixu Li and Zengfeng Xin were responsible for the design and guidance of the article. All authors read and approved the final manuscript. Acknowledgements: Not applicable References Hoffeld K, Lenz M, Egenolf P, Weber M, Heck V, Eysel P and Scheyerer MJ (2023) Patient-related risk factors and lifestyle factors for lumbar degenerative disc disease: a systematic review. 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Stem Cell Reports 16:1006-1008. doi: 10.1016/j.stemcr.2021.03.024 Tan L, Xie Y, Yuan Y and Hu K (2021) LncRNA GAS5 as miR-26a-5p Sponge Regulates the PTEN/PI3K/Akt Axis and Affects Extracellular Matrix Synthesis in Degenerative Nucleus Pulposus Cells in vitro. Front Neurol 12:653341. doi: 10.3389/fneur.2021.653341 Zheng H, Wang T, Li X, He W, Gong Z, Lou Z, Wang B and Li X (2020) LncRNA MALAT1 exhibits positive effects on nucleus pulposus cell biology in vivo and in vitro by sponging miR-503. BMC Mol Cell Biol 21:23. doi: 10.1186/s12860-020-00265-2 Zhang C, Qiu Y and Yuan F (2023) The long non-coding RNA maternally expressed 3-micorRNA-15a-5p axis is modulated by melatonin and prevents nucleus pulposus cell inflammation and apoptosis. Basic Clin Pharmacol Toxicol 133:603-619. doi: 10.1111/bcpt.13939 Zhang J, Ye F, Ye A and He B (2023) Lysyl oxidase inhibits BMP9-induced osteoblastic differentiation through reducing Wnt/beta-catenin via HIF-1a repression in 3T3-L1 cells. 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Heliyon 10:e27466. doi: 10.1016/j.heliyon.2024.e27466 Wang L, Yang H, Lin X, Cao Y, Gao P, Zheng Y and Fan Z (2018) KDM1A regulated the osteo/dentinogenic differentiation process of the stem cells of the apical papilla via binding with PLOD2. Cell Prolif 51:e12459. doi: 10.1111/cpr.12459 Li J, Diao S, Yang H, Cao Y, Du J and Yang D (2019) IGFBP5 promotes angiogenic and neurogenic differentiation potential of dental pulp stem cells. Dev Growth Differ 61:457-465. doi: 10.1111/dgd.12632 Wang Y, Jia Z, Diao S, Lin X, Lian X, Wang L, Dong R, Liu D and Fan Z (2016) IGFBP5 enhances osteogenic differentiation potential of periodontal ligament stem cells and Wharton's jelly umbilical cord stem cells, via the JNK and MEK/Erk signalling pathways. Cell Prolif 49:618-27. doi: 10.1111/cpr.12284 Xu Y, Tan H, Liu K, Wen C, Pang C, Liu H, Xu R, Li Q, He C, Nandakumar KS and Zhou C (2021) Targeted inhibition of ATP5B gene prevents bone erosion in collagen-induced arthritis by inhibiting osteoclastogenesis. Pharmacol Res 165:105458. doi: 10.1016/j.phrs.2021.105458 Benvenuti S, Saccardi R, Luciani P, Urbani S, Deledda C, Cellai I, Francini F, Squecco R, Rosati F, Danza G, Gelmini S, Greeve I, Rossi M, Maggi R, Serio M and Peri A (2006) Neuronal differentiation of human mesenchymal stem cells: changes in the expression of the Alzheimer's disease-related gene seladin-1. Exp Cell Res 312:2592-604. doi: 10.1016/j.yexcr.2006.04.016 Yu JS and Cui W (2016) Proliferation, survival and metabolism: the role of PI3K/AKT/mTOR signalling in pluripotency and cell fate determination. Development 143:3050-60. doi: 10.1242/dev.137075 Gao S, Chen B, Zhu Z, Du C, Zou J, Yang Y, Huang W and Liao J (2023) PI3K-Akt signaling regulates BMP2-induced osteogenic differentiation of mesenchymal stem cells (MSCs): A transcriptomic landscape analysis. Stem Cell Res 66:103010. doi: 10.1016/j.scr.2022.103010 Gao W, Yuan L, Zhang Y, Si Y, Wang X, Lv T and Wang YS (2023) miR-221/222 Promote Endothelial Differentiation of Adipose-Derived Stem Cells by Regulation of PTEN/PI3K/AKT/mTOR Pathway. Appl Biochem Biotechnol 195:4196-4214. doi: 10.1007/s12010-023-04335-x Ling M, Quan L, Lai X, Lang L, Li F, Yang X, Fu Y, Feng S, Yi X, Zhu C, Gao P, Zhu X, Wang L, Shu G, Jiang Q and Wang S (2021) VEGFB Promotes Myoblasts Proliferation and Differentiation through VEGFR1-PI3K/Akt Signaling Pathway. Int J Mol Sci 22. doi: 10.3390/ijms222413352 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 10 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 28 Oct, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers agreed at journal 26 Oct, 2025 Reviewers agreed at journal 25 Oct, 2025 Reviewers agreed at journal 25 Oct, 2025 Reviewers agreed at journal 25 Oct, 2025 Reviews received at journal 24 Oct, 2025 Reviews received at journal 24 Oct, 2025 Reviewers agreed at journal 24 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers agreed at journal 23 Oct, 2025 Reviewers invited by journal 20 Aug, 2025 Editor assigned by journal 11 Aug, 2025 Editor invited by journal 06 Aug, 2025 Submission checks completed at journal 04 Aug, 2025 First submitted to journal 03 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7130762","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":502871854,"identity":"eebfc739-71c2-4a38-8a06-2cba7f1934e6","order_by":0,"name":"Jian Zhu","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jian","middleName":"","lastName":"Zhu","suffix":""},{"id":502871855,"identity":"7e7e4d19-aaae-4854-a51f-4cf3920c18e4","order_by":1,"name":"Libin Jin","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Libin","middleName":"","lastName":"Jin","suffix":""},{"id":502871857,"identity":"d25fc2b9-80a5-4053-8756-f9ea0fe4b44f","order_by":2,"name":"Kaipeng Jin","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kaipeng","middleName":"","lastName":"Jin","suffix":""},{"id":502871858,"identity":"40dc5b1c-454c-4b68-8f31-97cf33ac2cbf","order_by":3,"name":"Yongping Wu","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yongping","middleName":"","lastName":"Wu","suffix":""},{"id":502871860,"identity":"541f6fe9-33a8-4487-9067-8bd65f6d0038","order_by":4,"name":"Lingling Sun","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Lingling","middleName":"","lastName":"Sun","suffix":""},{"id":502871862,"identity":"7417b3b6-2341-4a17-8230-bcddfe2acb8d","order_by":5,"name":"Yuluan Huang","email":"","orcid":"","institution":"Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Yuluan","middleName":"","lastName":"Huang","suffix":""},{"id":502871865,"identity":"7b371e15-3e69-408b-82be-452478c9c014","order_by":6,"name":"Chengchun Shen","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chengchun","middleName":"","lastName":"Shen","suffix":""},{"id":502871866,"identity":"dce44b48-e2cf-4dd1-bfaa-4b97d61f4a2b","order_by":7,"name":"Weixu Li","email":"","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Weixu","middleName":"","lastName":"Li","suffix":""},{"id":502871868,"identity":"bf0cc3bc-b6f1-4612-8f97-0b6e5fd7ab8c","order_by":8,"name":"Zengfeng Xin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIie3RMQrCMBSA4VcKnSJZ49Je4ZWMXiYi2KnQqXOK4OQBAg6ewRukBJxKXTs4eATBxaGCbRe3Nt0E8y9vyccjCYDL9aNpgDAcBgT2hPN5pIuv5TBtCKVVbLIWk1NRITxyA/Qox8lSbYVRBNNCVuip2gC76XGCja8NYW26gwr9xd4AMjFBrqU0BDEJevK2InrTbREoSE88GzLchWiMFVyy8lAnhDUTpHsx/iQtRpEy5/srX4VUTZBvTA+fSWzP9/vkjMMul8v1V30A+vZCKBwUinQAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Zengfeng","middleName":"","lastName":"Xin","suffix":""}],"badges":[],"createdAt":"2025-07-15 12:53:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7130762/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7130762/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-026-36219-5","type":"published","date":"2026-02-10T15:57:10+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":90078525,"identity":"299afa73-d38f-4032-b0f4-06109233968b","added_by":"auto","created_at":"2025-08-28 08:24:06","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2392018,"visible":true,"origin":"","legend":"\u003cp\u003e(A) hADSCs cells were culture in 3D model. (B)The result shows the microscopic morphology of pellet. The histological image shows microscopic morphology of pellet.\u003c/p\u003e","description":"","filename":"Figure1qiuhe.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/a4b762c5ff84cace7da8b218.jpg"},{"id":90078517,"identity":"9516036d-d9da-431b-a86e-0a0a935a54f8","added_by":"auto","created_at":"2025-08-28 08:24:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":402675,"visible":true,"origin":"","legend":"\u003cp\u003eThe specific gene of PAX1, FoxF1, IBSP, FBLN1, SOX9, ACAN, CA12 and COL2 were measured by PCR. * indicate p\u0026lt;0.05, **indicate p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"Figure2PCR.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/1e2f567567b5b4ecaf34be16.jpg"},{"id":90078544,"identity":"c20a0738-efb6-4981-8d71-491acf071908","added_by":"auto","created_at":"2025-08-28 08:24:07","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1260768,"visible":true,"origin":"","legend":"\u003cp\u003eImmunofluorescence of sox-9, col-2 and aggrecan in Induce (NP) and Non-Induced group.\u003c/p\u003e\n\u003cp\u003eIdentification of differentially expressed lncRNAs and mRNAs\u003c/p\u003e\n\u003cp\u003eTo determine that whether lncRNA and mRNA were involved in the differentiation process, we used RNA-seq to explore the expression of hADSCs cultured with growth factors. 12092 lnRNAs and 20256 mRNAs were detected with regulation in the differentiation process. As shown in figure 4, 500 lncRNA and 601 mRNAs were significantly differently expressed in the differentiation process of hADSCs into np-like cell type. The top 10 differential expressed lncRNAs were AL355075.4, MALAT1，AC022966.2, AC006064.4, AC145207.3, AC125611.3, DDIT4-AS1, AL662797.3，AL121748.2 and AC008914.1. The top 10 differential expressed mRNA were KCTD11，ALOX15B，KANK4，RNF139，TLR2，CHMP1B，CA9，PFN1P2，LEP，RNU1-27P. The np cell specific gene such as sox-9, col2a1, and CD24 were upregulated expressed in NP group. The results were consistent with PCR result. mRNAs listed are the top ten regulated mRNAs in the differentiation. And lncRNAs listed are the top ten regulated lncRNA.\u003c/p\u003e","description":"","filename":"Figure3IF.tif.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/120275c8bfda854a6ac25a91.jpg"},{"id":90078520,"identity":"7ba1a4ae-9e72-4c55-a3e7-a4110fdeb915","added_by":"auto","created_at":"2025-08-28 08:24:06","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":207926,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map shows differentially expressed long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs). X-axis represent the sample groups and y-axis represent the different probes. NP: Induce (NP)group. Con: Non-Induced group.\u003c/p\u003e\n\u003cp\u003eGo analysis\u003c/p\u003e\n\u003cp\u003eTo determine the biological function of the genes, GO enrichment analysis were performed. Different expressed lncRNAs and mRNAs were used to make GO analysis. There are 51 GO terms show significant regulated in NP and Non-Induced group. The top twenty regulated GO terms were listed in figure 5. The most abundant genes are concentrated on this GO term extracellular matrix structural constituent (GO:0005201). Genes also enriched on GO term cadherin binding，glycosaminoglycan binding and kinase regulator activity.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/ceb606b7d87817ea73d6910e.jpg"},{"id":90078509,"identity":"10804126-1d11-4c1b-a951-f72ea7268674","added_by":"auto","created_at":"2025-08-28 08:24:02","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276887,"visible":true,"origin":"","legend":"\u003cp\u003eThe result of GO enrichment analysis. (A)The barplot show the top 20 most significantly upregulated Gene Ontology (GO) terms. (B)The dotplot show the top 20 most significantly upregulated Gene Ontology (GO) terms.\u003c/p\u003e\n\u003cp\u003ePathway analysis\u003c/p\u003e\n\u003cp\u003eBased on the latest version of the KEGG database, we made KEGG pathway analysis with significant enrichment of differentially expressed mRNAs. The top twenty significant regulated pathways in NP and Non-Induced group were listed in figure 6. The pathway PI3K-Akt and Cytoskeleton in muscle cells show highly related with the differentiation process. The significance of the corresponding pathway was denoted by P-value. We also calculated the enrichment score value, which represents the enrichment importance of pathway ID. These values are equal to - log10 (p-value).\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/1040dc3e6e9137e5a71c8eaa.jpg"},{"id":90078518,"identity":"2c63a4c2-b407-4df1-ab9e-559e6c6137b7","added_by":"auto","created_at":"2025-08-28 08:24:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":262369,"visible":true,"origin":"","legend":"\u003cp\u003eResults of the Kyoto Encylopedia of Genes and Genomes (KEGG) pathway analysis.\u003c/p\u003e\n\u003cp\u003eRegulation of ceRNA network\u003c/p\u003e\n\u003cp\u003eA competing endogenous RNA network was constructed by fourteen differentially expressed lncRNAs, 51 differentially expressed miRNAs and 601 differentially expressed mRNAs based on the degree of correlation. RPL41, RNU4-2, U2, ZNF331, JARID2, CLVS1, GAS5, EMX2OS, MALAT1, MEG3, CYP1B1-AS1, PRICKLE2-AS1, VCAN-AS1, PAPPA-AS1 were most closed related in the competing endogenous RNA network. While ZNF331, JARID2, MEG3 show upregulated in NP group compared to control group, PRICKLE2-AS1, CYP1B1-AS1, MALAT1, MEG3 and GAS5 show both upregulated and downregulated. VCAN-AS1, PAPPA-AS1, RPL41, RNU4-2 and CLVS1 show downregulated (Figure 7).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/9e4c54fe143932b5059c970b.jpg"},{"id":90078519,"identity":"610b2f0e-ee0e-4fd4-9c38-0d507a8887e7","added_by":"auto","created_at":"2025-08-28 08:24:05","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":568639,"visible":true,"origin":"","legend":"\u003cp\u003eThe long noncoding RNA (lncRNA)-messenger RNA (mRNA)-micro RNA (miRNA) competing endogenous RNA (ceRNA) network. Lozenge with red represent lncRNAs; nodes without blue represent mRNAs; triangle with yellow represent miRNAs.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/407ca3aa2312e506fece2961.jpg"},{"id":102786030,"identity":"40a4af6e-c8c9-43b4-86ca-5037fe65778a","added_by":"auto","created_at":"2026-02-16 16:11:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5769054,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7130762/v1/58e950d5-4108-4552-bdbc-9b3d968ce560.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Aberrantly expressed long noncoding RNAs in adipose-derived mesenchymal stem cells differentiation to nucleus pulposus-like cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLow back pain caused by degenerative disc disease (DDD) is very common in people of all ages and is the main cause of limited working ability in people under 45 years of age[1]. The incidence of low back pain associated with DDD is 60 to 90%, and the United States costs up to $50 billion to $100 billion in DDD medical costs each year [2], and the prevalence rate of chronic diseases was 9.5\u0026permil;, both ranking sixth among all diseases. The social burden caused by DDD in China is also very huge. At present, symptomatic surgery is often used in clinical treatment of DDD[3], but surgery has a high recurrence rate and more complications, and brings great physical and mental pain and economic burden to patients.\u003c/p\u003e\n\u003cp\u003eStudies at home and abroad have shown that the decrease of nucleus pulposus cells is the main initiating factor of DDD. Nucleus pulposus cells are the main cells in intervertebral disc, and many animal model studies have shown that intervertebral disc degeneration is often accompanied by a decrease in cell density[4, 5]. Similarly, in juvenile patients with neuromuscular scoliosis, the number of cells in the intervertebral disc on the curved convex side (degenerative side) is significantly lower than that on the concave side (normal side), and the water content is also reduced[6]. In addition, with the increase of age, the number of senescent cells in the nucleus pulposus increased from about 2% in the fetal period to 80% in the old age[7]. In addition, nucleus pulposus cells play an important role in the metabolic balance of intervertebral disc stroma. Nucleus pulposus cells are twice as capable of synthesizing nucleus pulposus stroma as chondroid cells, and can enhance chondroid cell synthesis by secreting cytokines such as connective tissue growth factor (CTGF) [8]. Therefore, restoring the number of nucleus pulposus cells in the degenerated intervertebral disc is expected to repair the degenerated intervertebral disc, which also provides a theoretical basis for stem cell transplantation treatment of DDD.\u003c/p\u003e\n\u003cp\u003eNumerous studies have confirmed that stem cell transplantation can not only induce differentiation into nucleus pulposus cells, but also promote the synthesis of matrix proteins such as type II collagen and proteoglycan in the degraded intervertebral disc, significantly restore the water content of nucleus pulposus, and significantly increase the intervertebral disc height index[9-11]. The adverse microenvironment of the degenerated intervertebral disc (low pH, low oxygen, changes in osmotic pressure and accumulation of inflammatory factors) may be an important reason for the low survival rate, limited differentiation and low matrix synthesis of the transplanted stem cells in vivo[12-14]. However, the exact mechanism by which stem cell differentiation is limited in the intervertebral disc microenvironment remains poorly understood. In-depth research on the methods and mechanisms of how to improve the directional differentiation of ADSCs into nucleus pulposus cells in the intervertebral disc microenvironment has important guiding value for the rational application of stem cell transplantation in the treatment of DDD and the formulation of more effective treatment plans.\u003c/p\u003e\n\u003cp\u003eIntervertebral disc degeneration (IDD) is the main cause to low back pain which induces serious burden to the society. Numerous studies have been done to seek the methods to intervertebral disc regeneration[15, 16]. Stem-cell based therapy is one of the most promising methods to regenerate intervertebral disc[17, 18]. Mesenchymal stem cell cultured with cytokines could differentiation into np-like cell type[19]. But the underlying mechanism remains to be explored.\u003c/p\u003e\n\u003cp\u003eRecently, lncRNAs are emerged as an important regulator in the differentiation process of stem cell[20]. RNA-seq technology can measure differences in the expression level of thousands of genes and allows highly effective evaluation of genome-wide expression changes. With the help of RNA-seq, we could find the significant different related lncRNA, miRNA and mRNA. Most studies focus on the different lncRNAs between normal and degeneration intervertebral disc[21-23]. As stem cell based therapy act an important role in the process of intervertebral disc regeneration[24, 25]. It is necessary to find the underlying mechanism in the differentiation process of hADSCs.\u003c/p\u003e\n\u003cp\u003eYet, there was no study focusing on the different expression of lncRNA/miRNA/mRNA in the differentiation of hADSCs into np-like cell type. Our study aimed to use RNA-seq analysis to investigate the lncRNAs, miRNA and mRNAs which related to differentiation. We also use bioinformatics methods to reveal the promising signaling pathways through KEGG analysis, and gene regulation networks between lncRNAs, miRNAs and mRNAs. We hope that our study will shed new sight into the cell based therapy and supply new target for intervertebral disc regeneration.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eCells and Regents\u003c/p\u003e\n\u003cp\u003ehADSCs were obtained from Cyagen Biosciences (HUXMD-01001; Guangzhou, China). hADSCs were culture in medium from Cyagen Biosciences (HUXMD-90011; Guangzhou, China) in a humidified incubator at 37 °C with 5% CO2. The culture medium was replaced every 3 days. hADSCs at passages 2–4 was used for subsequent experiments.\u003c/p\u003e\n\u003cp\u003ehADSC Differentiation Culture medium\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe NP differentiation medium was composed of 1× ITS, 0.1 μM dexamethasone, 1 mM sodium pyruvate, 0.35 mM proline, 0.17 mM ascorbic acid–2-phosphate, 1.25 mg/ml BSA, 10 ng/ml TGF-β1 and 100 ng/ml GDF5；1× Anti, 10ng/ml BMP2[26].\u003c/p\u003e\n\u003cp\u003eCell pellet culture\u003c/p\u003e\n\u003cp\u003eFor preparation of 3D cell culture, 3 × 10\u003csup\u003e5\u003c/sup\u003e cells were centrifuged at 1500 rpm for 5 min in 15 ml polypropylene conical tubes and incubated at 37°C overnight. Pellets were formed after 24 h culture. Then pellets were cultured with different differentiation medium. The NP group was cultured with NPM differentiation medium. The control group was cultured with hADSCs culture medium. Medium was changed every three days. Pellets at 7, 14 and 21 days were selected for light microscopic analysis. After 7, 14 and 21 days culture, pellets were fixed with 4% paraformaldehyde for 24 h and then dehydrated with 30% sucrose water.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHistology analysis\u003c/p\u003e\n\u003cp\u003ePellets at 7, 14 and 21 days were selected to cut into 7 um frozen section. These sections were stained by H\u0026amp;E.\u003c/p\u003e\n\u003cp\u003eImmunofluorescence staining\u003c/p\u003e\n\u003cp\u003ePellets at 21 days were selected to be fixed in 4% paraformaldehyde and then cut into 7 um frozen sections. The frozen sections were incubated in primary rabbit collagen II (1:500 dilutions), aggrecan (1:500 dilutions) and anti-SOX-9 (1:500 dilutions) overnight at 4°C. Afterward, the cells were washed and incubated for 1-hour with fluorescence-conjugated secondary antibody. Finally, cells were washed by PBS and stained by DAPI for 5 min (BOSTER Biological Technology). Then, the cells were observed and imaged by the fluorescence microscope.\u003c/p\u003e\n\u003cp\u003eRNA-seq analysis\u003c/p\u003e\n\u003cp\u003emRNA and lncRNA expression profiles were compared in hADSCs cultured with NPM and control medium. To clarify the changes in the signaling pathways of hADSCs differentiation, we further performed Gene Ontology (GO) analysis, KEGG analysis, pathway analysis, and signal-net analysis. RNA-seq analysis was performed by Lc-Bio Technologies (Hangzhou) Co., Ltd. Random variance model (RVM) t test was used to identify the different expression of mRNAs and lncRNAs. Cluster map was created after hierarchical clustering was performed.\u003c/p\u003e\n\u003cp\u003eqRT-PCR analysis\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate the NP-differentiation and the RNA-seq results, these mRNAs were selected Total RNA was tracted from cells with RNAiso reagent (TaKaRa Bio, Japan). A PrimeScript RT reagent kit (TaKaRa, Bio, Japan) was used to reverse transcription. qRT-PCR was performed using the StepOnePlus Real-time PCR System (Applied Biosystems, CA, USA) and a SYBR® Premix Ex Taq™ kit (TaKaRa Bio, Japan). 18s rRNA was used as a housekeeping gene. The results were calculated using the 2-ΔΔCt method. All reactions were performed in triplicate and the sequences of used primers are shown in Table 1 were synthesized by Sangon Biotech (Shanghai, China).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGO analysis\u003c/p\u003e\n\u003cp\u003eTwo-sided Fisher’s exact test and chi-square test are used to analyze the main function of significant differentially expressed in the GO analysis based on the GO database (http://www.geneontology.org). P \u0026lt; 0.01 were defined as significant regulated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePathway analysis\u003c/p\u003e\n\u003cp\u003eBase on Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/), we import data and select human species for calculation. Fisher’s exact test and the chi-square test were used to analyse the significance score of differential gene enrichment in each pathway，\u0026nbsp;P \u0026lt; 0.05 were defined as significant regulated. Different gene expression pathways were analyzed on the basis of KEGG database.\u003c/p\u003e\n\u003cp\u003eSignal-net analysis\u003c/p\u003e\n\u003cp\u003eSignificant crossover genes in GO analysis and pathway analysis were selected to analyze gene-gene interactions and construct network maps. Based on KEGG database, gene-gene network maps are constructed with differentially expressed gene data. The network is presented as a graph, the nodes are genes, and the edges between the nodes may indicate activation or phosphorylation. The network function of each gene is presented related to the number of upstream and downstream genes, which are expressed in the form of internal and external degrees. The mesocentricity of each gene was calculated according to the inner and outer degrees of genes. The higher the mesocentricity, the more important it is in gene-network regulation.\u003c/p\u003e\n\u003cp\u003eCompeting endogenous RNA (ceRNA) analysis\u003c/p\u003e\n\u003cp\u003elncRNAs can regulate miRNA abundance by competitively binding miRNAs, thus regulating post-transcriptional regulatory processes. In our experiment, we used miRNAs with significant differences to map competitive RNA networks. During the drawing process, we use relevant targetscan (http://www.targets can.org/), miRDB (https://www.mirdb.org) and starbase (starBase or ENCORI: Decoding the Encyclopedia of RNA Interactomes (rnasysu.com) to process target predictions. Competing RNA networks were mapped with interacting miRNAs and lncRNAs.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eAll the state were reported as average ± standarded. The difference was analysed by Student’s t analysis by SPSS 20.0 software (Chicago IL, USA). P \u0026lt; 0.05 was defined as different significance.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThe specific gene expression of np-like cell type\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ehADSCs cells were culture in 3D model. The result shows the microscopic morphology of pellet. The histological image shows the result that NP group had more np-like cell type than control groups (Figure1). The specific gene of PAX1, FoxF1, IBSP, FBLN1, SOX9, ACAN, CA12 and COL2 were measured by PCR. The np-like cell type specific gene PAX1, FoxF1 and CA12 were significant upregulated in Induced (NP) group. The cartilage specific gene IBSP and FBLN1 show no significant difference between Induce (NP) and Non-Induced group (Figure2). Immunofluorescence of sox-9, col-2 and aggrecan show that hADSCs cultured with growth factor could differentiate into np-like cell type (Figure3).\u003c/p\u003e\n\u003cp\u003eIdentification of differentially expressed lncRNAs and mRNAs\u003c/p\u003e\n\u003cp\u003eTo determine that whether lncRNA and mRNA were involved in the differentiation process, we used RNA-seq to explore the expression of hADSCs cultured with growth factors. 12092 lnRNAs and 20256 mRNAs were detected with regulation in the differentiation process. As shown in figure 4, 500 lncRNA and 601 mRNAs were significantly differently expressed in the differentiation process of hADSCs into np-like cell type. The top 10 differential expressed lncRNAs were AL355075.4, MALAT1，AC022966.2, AC006064.4, AC145207.3, AC125611.3, DDIT4-AS1, AL662797.3，AL121748.2 and AC008914.1. The top 10 differential expressed mRNA were KCTD11，ALOX15B，KANK4，RNF139，TLR2，CHMP1B，CA9，PFN1P2，LEP，RNU1-27P. The np cell specific gene such as sox-9, col2a1, and CD24 were upregulated expressed in NP group. The results were consistent with PCR result. mRNAs listed are the top ten regulated mRNAs in the differentiation. And lncRNAs listed are the top ten regulated lncRNA.\u003c/p\u003e\n\u003cp\u003eGo analysis\u003c/p\u003e\n\u003cp\u003eTo determine the biological function of the genes, GO enrichment analysis were performed. Different expressed lncRNAs and mRNAs were used to make GO analysis. There are 51 GO terms show significant regulated in NP and Non-Induced group. The top twenty regulated GO terms were listed in figure 5. The most abundant genes are concentrated on this GO term extracellular matrix structural constituent (GO:0005201). Genes also enriched on GO term cadherin binding，glycosaminoglycan binding and kinase regulator activity.\u003c/p\u003e\n\u003cp\u003ePathway analysis\u003c/p\u003e\n\u003cp\u003eBased on the latest version of the KEGG database, we made KEGG pathway analysis with significant enrichment of differentially expressed mRNAs. The top twenty significant regulated pathways in NP and Non-Induced group were listed in figure 6. The pathway PI3K-Akt and Cytoskeleton in muscle cells show highly related with the differentiation process. The significance of the corresponding pathway was denoted by P-value. We also calculated the enrichment score value, which represents the enrichment importance of pathway ID. These values are equal to - log10 (p-value).\u003c/p\u003e\n\u003cp\u003eRegulation of ceRNA network\u003c/p\u003e\n\u003cp\u003eA competing endogenous RNA network was constructed by fourteen differentially expressed lncRNAs, 51 differentially expressed miRNAs and 601 differentially expressed mRNAs based on the degree of correlation. RPL41, RNU4-2, U2, ZNF331, JARID2, CLVS1, GAS5, EMX2OS, MALAT1, MEG3, CYP1B1-AS1, PRICKLE2-AS1, VCAN-AS1, PAPPA-AS1 were most closed related in the competing endogenous RNA network. While ZNF331, JARID2, MEG3 show upregulated in NP group compared to control group, PRICKLE2-AS1, CYP1B1-AS1, MALAT1, MEG3 and GAS5 show both upregulated and downregulated. VCAN-AS1, PAPPA-AS1, RPL41, RNU4-2 and CLVS1 show downregulated (Figure 7).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIntervertebral disc degeneration is the result of many factors, and the decrease of nucleus pulposus cells is the main initiating factor[11, 27]. Existing studies have confirmed that intervertebral disc contains stem cells[28]. Therefore, stimulating the growth of endogenous intervertebral disc stem cells and their active differentiation into nucleus pulposus cells can be an effective therapeutic strategy. However, whether stem cells can effectively differentiate into nucleus pulposus cells, and then effectively improve the pathological state caused by insufficient number of endogenous stem cells and aging, there is still a big realistic bottleneck[26]. Earlier results of our research group\u0026apos;s induction of Human adipose stem cells (hADSCs) into nucleus pulpocyte differentiation showed that the proportion of nucleus pulpocyte only reached 40%, far from meeting the needs of clinical treatment of DDD[6]. Therefore, it is particularly critical to further study and effectively improve the mechanism and method of hADSCs nucleus pulposus differentiation efficiency, and further exploration in this direction will provide important scientific research guidance and clinical transformation value for clinical stem cell transplantation programs.\u003c/p\u003e\n\u003cp\u003eMany research[29-31] have reported that long noncoding RNA (lncRNA) plays an important regulatory role in the maintenance of pluripotency and directed differentiation of stem cells. A large number of existing studies have also proved that lncRNA is more tissue-specific than protein-coding gene expression in human tissue cells, and is closely related to transcription factors, indicating that lncRNA plays an important regulatory role in cell differentiation[32-34]. Numerous studies have shown that 1ncRNA is involved in the directed differentiation of adult stem cells: for example, LncMyoD is involved in the differentiation of stem cells into skeletal muscle cells[35]. LncRNA DANCR promotes stem cell differentiation into bone cell [36]. LncRNA ADINR promotes stem cell differentiation into adipocytes[37]. Although the above studies found changes in the expression profile of LncRNAs in stem cell differentiation, little is known about their production mechanism and physiological function. Therefore, it is of great significance to study the specific expression of lncRNA in stem cell differentiation and its role and mechanism of action. Our studies on lncRNAs involved in nucleus pulposus differentiation of hADSCs found that there were 12092 lncRNAs and 20256 genes specifically expressed in hADSCs (nucleus pulposus group) and hADSCs (normal group) cultured in normal medium, respectively. At present, we further analyzed the data from the current major lncRNA databases. The summary results of various test data suggest that RPL41, RNU4-2, U2, ZNF331, JARID2, CLVS1, GAS5, EMX2OS, MALAT1, MEG3, CYP1B1-AS1, PRICKLE2-AS1, VCAN-AS1 and PAPPA-AS1 may be important regulatory lncRNAs in hADSCs nucleus pulposus differentiation. In previous studies，lncRNA GAS5, MALAT1 and MEG3, was related to nucleus pulposus cell degeneration process[38-40]. Upregulate ZNF331 and JARID2 may stimulate the differentiation process of hADSCs into nucleus pulposus cell. The role of lncRNA RPL41, RNU4-2, U2, CLVS1, EMX2OS, CYP1B1-AS1, PRICKLE2-AS1, VCAN-AS1 and PAPPA-AS1 is needed further exploration in the differentiation.\u003c/p\u003e\n\u003cp\u003eRegarding the regulatory function of lncRNAs, we investigate the relation between the mRNA and lncRNA. Many gene were rich in extracelluar matrix structural constitute, collagen binding, glycosaminoglycan binding. In our study，LOX, CDKN1A, PLOD2, INSIG1, EIF1, IGFBP5, HMGCS1, DDIT4, PTP4A1, ATP5B and DHCR24 are the top ten regulated genes in the differentiation process. LOX was reported as a regulator in osteoblast differentiation[41]. Lysyl oxidase modulates the osteoblast differentiation of primary mouse calvaria cells and governs osteogenic and adipogenic cell fate by MSCs[42, 43]. Cdkn1a expression significantly contributed to osteoclast differentiation[44]. CDKN1A regulated chondrogenic differentiation of human chondrocytes in osteoarthritis[45]. KDM1A binded with PLOD2, and finally resulted in the inhibited function for the osteo/dentinogenesis in stem cells of the apical papilla[46]. IGFBP5 promotes angiogenic and neurogenic differentiation potential of dental pulp stem cells[47]. IGFBP5 enhances osteogenic differentiation potential of periodontal ligament stem cells[48]. ATP5B make a contribution to osteoclast differentiation and joint destruction[49]. DHCR24 play a role in neuronal differentiation[50]. Little studies show INSIG1, EIF1, IGFBP5, HMGCS1, DDIT4, PTP4A1 in the differentiation process. Studies may be taken to explore the mechanism of these gene in cell differentiation.\u003c/p\u003e\n\u003cp\u003eDuring the differentiation process, PI3K/Akt signaling pathway and cytoskeleton in muscle cell play an important role. Previouslly, PI3K/Akt signaling pathway were found in the osteogenic, endothelial and epithelial, myoblast differentiation process[51-54]. Study have highlighted the importance of this pathway to development and cellular differentiation. Our study showed the PI3K/Akt signaling pathway were upregulated in the nucleus pulposus differentiation process.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp;The top regulated lncRNAs and mRNAs listed may show a great possibility in playing a role in the differentiation process. Such a considerable divergence needed to be further in-depth investigations into the sophisticated underlying mechanism. More in vitro and in vivo studies should be done to explore the mechanism of cell differentiation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe mechanism of the differentiation of stem cells is very complex, and it was initially thought that the differentiation of stem cells is mainly by regulation of tissue-specific transcription factors. LncRNAs play an important regulatory role in the maintenance of pluripotency and directed differentiation of stem cells. We found 14 lncRNAs and 601 mRNAs were significantly differentially expressed in hADSCs differentiation. Our results first explore differentially expressed lncRNAs and mRNAs in the differentiation of hADSCs into np-like cell types. These may supply useful information for better understanding of stem cell therapy and IDD regeneration.\u003c/p\u003e"},{"header":"List of abbreviations","content":"\u003cp\u003eLncRNA: Long noncoding RNA; IDD: intervertebral disc degeneration; mRNAs: messenger RNAs; hADSCs: human adipose-derived mesenchymal stem cells; DDD: degenerative disc disease; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes ; ceRNA: Competing endogenous RNA\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics Approval and consent to participate：\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of the Research Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine, China.\u003c/p\u003e\n\u003cp\u003eConsent for publication:\u003c/p\u003e\n\u003cp\u003eWritten informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material：\u003c/p\u003e\n\u003cp\u003eSequence data that support the findings of this study have been deposited in the ArrayExpress accession with the primary accession code E-MTAB-15403.\u003c/p\u003e\n\u003cp\u003eCompeting interests：\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interests.\u003c/p\u003e\n\u003cp\u003eFunding:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis project was supported by this study by the Natural Science Foundation of Zhejiang Province (Project No. LQ22H060005, LY21H060003 and LY21H060004), the National Natural Science Foundation of China (Project No. 82102592), and the Scientific Research Project of the Department of Education of Zhejiang Province (Project No. Y202352378). These fundings were used in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003eAuthors' contributions:\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Data collection and analysis were performed by Jian Zhu,\u0026nbsp;Libin Jin, Kaipeng Jin, Yongping Wu, Lingling Sun，Yuluan Huang\u0026nbsp;and Chengchun Shen. The first draft of the manuscript was written by Jian Zhu and all authors commented on previous versions of the manuscript.\u0026nbsp;Weixu\u0026nbsp;Li\u0026nbsp;and\u0026nbsp;Zengfeng\u0026nbsp;Xin were responsible\u0026nbsp;for the design and guidance of the article. All authors read and approved the final manuscript. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAcknowledgements:\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHoffeld K, Lenz M, Egenolf P, Weber M, Heck V, Eysel P and Scheyerer MJ (2023) Patient-related risk factors and lifestyle factors for lumbar degenerative disc disease: a systematic review. Neurochirurgie 69:101482. doi: 10.1016/j.neuchi.2023.101482\u003c/li\u003e\n\u003cli\u003eNguyen C, Boutron I, Baron G, Sanchez K, Palazzo C, Benchimol R, Paris G, James-Belin E, Lefevre-Colau MM, Beaudreuil J, Laredo JD, Bera-Louville A, Cotten A, Drape JL, Feydy A, Ravaud P, Rannou F and Poiraudeau S (2017) Intradiscal Glucocorticoid Injection for Patients With Chronic Low Back Pain Associated With Active Discopathy: A Randomized Trial. 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Int J Mol Sci 22. doi: 10.3390/ijms222413352\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Intervertebral disc degeneration, Human adipose-derived mesenchymal stem cells, Nucleus pulposus cells, RNA-seq, Long non-coding RNAs","lastPublishedDoi":"10.21203/rs.3.rs-7130762/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7130762/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Stem cells were often used for intervertebral disc degeneration (IDD) regeneration. The underlying mechanisms remain to be explored. LncRNAs were found to be related to the physiological process such as apoptosis and differentiation. Many studies focus on the messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) between normal nucleus pulposus and degeneration nucleus pulposus. However, few studies have shed light on the different expression of lncRNA and mRNA in the differentiation. In the present study, we aimed to determine mRNAs and lncRNAs, which are differentially expressed during in human adipose-derived mesenchymal stem cells (hADSCs) differentiation process into np-like cell types and to explore the related signaling pathways and the regulatory networks.\u003c/p\u003e\n\u003cp\u003eMethods: hADSCs were induced to differentiation into np-like cell under the cytokine circumstance. The mark genes of np-like cell were determined by PCR and immunology staining. Then RNA-seq was used to analysis the expression of lncRNA and mRNA in the differentiation of hADSCs into np-like cell types. The significant genes were confirmed by Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database.\u003c/p\u003e\n\u003cp\u003eResults: We found 14 lncRNAs and 601 mRNAs were significantly differentially expressed in hADSCs differentiation. The RNA-seq data were confirmed by real-time PCR. Furthermore, we found Gene Ontology terms were upregulated, and downregulated and significantly enriched pathways. Moreover, gene network shows significant differentially expressed genes. Meanwhile, the relationship of significantly changed mRNAs and lncRNAs were revealed by mRNA-lncRNA co-expression network.\u003c/p\u003e\n\u003cp\u003eConclusion: Our results first explore differentially expressed lncRNAs and mRNAs in the differentiation of hADSCs into np-like cell types. These may supply useful information for better understanding of stem cell therapy and IDD regeneration.\u003c/p\u003e","manuscriptTitle":"Aberrantly expressed long noncoding RNAs in adipose-derived mesenchymal stem cells differentiation to nucleus pulposus-like cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-28 08:23:28","doi":"10.21203/rs.3.rs-7130762/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-28T21:05:45+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"331177547163986021872924773274935316187","date":"2025-10-28T00:19:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326837948556464293241928790023039250065","date":"2025-10-26T12:04:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"128579709114283834327321148194269719748","date":"2025-10-25T22:40:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293716318614374886000362733654049641283","date":"2025-10-25T18:20:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272258128269144127809165971065616195684","date":"2025-10-25T06:34:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T13:56:25+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-24T07:21:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292491787863661376813744754094596100539","date":"2025-10-24T07:06:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224323402327349580458156416682153550039","date":"2025-10-24T03:15:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113212274471772218188831388811914929759","date":"2025-10-23T23:28:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294126806621344934082915988770833720987","date":"2025-10-23T16:14:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27345339882164661378896139324033997914","date":"2025-10-23T16:01:22+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-20T04:22:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-11T19:59:48+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-06T18:11:26+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-04T06:22:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-03T10:39:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d6fb50ef-2419-43e2-94a9-0ffb70c76222","owner":[],"postedDate":"August 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":53417092,"name":"Biological sciences/Cell biology"},{"id":53417093,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":53417094,"name":"Biological sciences/Developmental biology"},{"id":53417095,"name":"Biological sciences/Genetics"},{"id":53417096,"name":"Biological sciences/Molecular biology"},{"id":53417097,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2026-02-16T16:08:16+00:00","versionOfRecord":{"articleIdentity":"rs-7130762","link":"https://doi.org/10.1038/s41598-026-36219-5","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2026-02-10 15:57:10","publishedOnDateReadable":"February 10th, 2026"},"versionCreatedAt":"2025-08-28 08:23:28","video":"","vorDoi":"10.1038/s41598-026-36219-5","vorDoiUrl":"https://doi.org/10.1038/s41598-026-36219-5","workflowStages":[]},"version":"v1","identity":"rs-7130762","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7130762","identity":"rs-7130762","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}