biostudies-arrayexpressJian Zhuhuman oral metagenomeACGT101-microRNAFastQChttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15403Background: 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.biostudies-arrayexpressSequencing - LncRNA identification: First of all, transcripts that overlapped with known mRNAs and transcripts shorter than 200 bp were discarded. Then we utilized CPC [6], CNCI [7] and Pfam [8] to predict transcripts with coding potential. All transcripts with CPC score <-1 and CNCI score <0 were removed. The remaining transcripts with class code (I, j, o, u, x) were considered as lncRNAs. Different expression analysis of mRNAs and lncRNAs: StringTie [4] was used to perform expression level for mRNAs and lncRNAs by calculating FPKM. The differentially expressed mRNAs and lncRNAs were selected with log2 (fold change) >1 or log2 (fold change) <-1 and with statistical significance (p value < 0.05) by R package Ballgown [5].Library Construction - Firstly, Cutadapt [1] was used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases. Then sequence quality was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). We used Bowtie2 [2] and Tophat2 [3] to map reads to the genome of human. The mapped reads of each sample were assembled using StringTie [4]. Then, all transcriptomes from human Samples were merged to reconstruct a comprehensive transcriptome using perl scripts. After the final transcriptome was generated, StringTie [4] and Ballgown [5] was used to estimate the expression levels of all transcriptsNucleic Acid Extraction - Total RNA was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0. Approximately 1 ug of total RNA were used to prepare small RNA library according to protocol of TruSeq Small RNA Sample Prep Kits(Illumina, San Diego, USA). And then we performed the single-end sequencing (36bp or 50bp ) on an Illumina Hiseq 2500 at the LC-BIO (Hangzhou, China) following the vendor’s recommended protocolSample Collection - 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 × 105 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.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Two diagrams were generated to show the localization and abundance of lncRNAs in genome using the program Circos [12]. One diagram showed lncRNA expression level in different samples. The lncRNAs in the other diagram were subdivided into five categories according to their class code generated by StringTie: (i) a transfrag falling entirely within a reference intron (intronic); (j) potentially novel isoform or fragment at least one splice junction is shared with a reference transcript; (o) generic exonic overlap with a reference transcript; (u) unknown, intergenic transcript (intergenic); (x) Exonic overlap with reference on the opposite strand (antisense). LncRNA conservation in different species The full length of all identified xxx lncRNAs of human were used to blast against the genome of human, with the word-size=5, and E-value<10E-5UnknownTranscriptomicsGenomicsProteomicscomputerIllumina HiSeq 1500RNA-seq of coding RNAhuman oral metagenomeJian ZhufalseNP Cell line vs control cellBackground: 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.2025-08-18T00:00:00Z2025-07-25T12:16:53.97Z2025-07-25T12:16:53.97ZE-MTAB-15403ERP177269EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184