Comprehensive analysis of lncRNA-miRNA-mRNA networks during osteogenic differentiation of bone marrow mesenchymal stem cells.

Long non-coding RNA (lncRNA) plays crucial role in osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs), involving in regulation of competing endogenous RNA (ceRNA) mechanisms and conduction of signaling pathways. However, its mechanisms are poorly understood. This study aimed to investigate lncRNAs, miRNAs and mRNAs expression profiles in rat BMMSCs (rBMMSCs) osteogenic differentiation, screen the potential key lncRNA-miRNA-mRNA networks, explore the putative functions and identify the key molecules, as the basis of studying potential mechanism of rBMMSCs osteogenic differentiation driven by lncRNA, providing molecular targets for the management of bone defect. High-throughput RNA sequencing (RNA-seq) was used to determine lncRNAs, miRNAs, and mRNAs expression profiles at 14-day rBMMSCs osteogenesis. The pivotal lncRNA-miRNA and miRNA-mRNA networks were predicted from sequencing data and bioinformatic analysis, and the results were exported by Cytoscape 3.9.0 software. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were used for functional exploration. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to validate lncRNAs, miRNAs and mRNAs. rBMMSCs were identified, and the osteogenic and adipogenic differentiation ability were detected. A total of 8634 lncRNAs were detected by RNA-seq, and 1524 differential expressed lncRNAs, of which 812 up-regulated and 712 down-regulated in osteo-inductive groups compared with control groups. 30 up-regulated and 61 down-regulated miRNAs, 91 miRNAs were differentially expressed in total. 2453 differentially expressed mRNAs including 1272 up-expressed and 1181 down-expressed were detected. 10 up-regulated lncRNAs were chosen to predict 21 down-regulated miRNAs and 650 up-regulated mRNAs. 49 lncRNA-miRNA and 1515 miRNA-mRNA interactive networks were constructed. GO analysis showed the most important enrichment in cell component and molecular function were "cytoplasm" and "protein binding", respectively. Biological process related to osteogenic differentiation such as "cell proliferation", "wound healing", "cell migration", "osteoblast differentiation", "extracellular matrix organization" and "response to hypoxia" were enriched. KEGG analysis showed differentially expressed genes were mainly enriched in "PI3K-Akt signaling pathway", "Signaling pathway regulating pluripotency of stem cells", "cGMP-PKG signaling pathway", "Axon guidance" and "Calcium signaling pathway". qRT-PCR verified that lncRNA Tug1, lncRNA AABR07011996.1, rno-miR-93-5p, rno-miR-322-5p, Sgk1 and Fzd4 were consistent with the sequencing results, and 4 lncRNA-miRNA-mRNA networks based on validations were constructed, and enrichment pathways were closely related to "PI3K-Akt signaling pathway", "Signaling pathway regulating pluripotency of stem cells" and "Wnt signaling pathway". lncRNAs, miRNAs and mRNAs expression profiles provide clues for future studies on their roles for BMMSCs osteogenic differentiation. Furthermore, lncRNA-miRNA-mRNA networks give more information on potential new mechanisms and targets for management on bone defect.

Liu J ，Yao Y ，Huang J ，Sun H ，Pu Y ，Tian M ，Zheng M ，He H ，Li Z ... -  《BMC GENOMICS》

microRNA expression profiles and the potential competing endogenous RNA networks in NELL-1-induced human adipose-derived stem cell osteogenic differentiation.

Studies have indicated that Nel-like molecule-1 (NELL-1) was an osteoblast-specific cytokine and some specific microRNAs (miRNAs) could serve as competing endogenous RNA (ceRNA) to partake in osteogenic differentiation of human adipose-derived stem cells (hASCs). The aim of this study was to explore the potential functional mechanisms of recombinant human NELL-1 protein (rhNELL-1) during hASCs osteogenic differentiation. rhNELL-1 was added to osteogenic medium to activate osteogenic differentiation of hASCs. High-throughput RNA sequencing (RNA-Seq) was performed and validated by real-time quantitative polymerase chain reaction. Gene ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed to detect the functions of differentially expressed miRNAs and genes. Coding-noncoding gene co-expression network and ceRNA networks were constructed to predict the potential regulatory role of miRNAs. A total of 1010 differentially expressed miRNAs and 1762 differentially expressed messenger RNAs (mRNAs) were detected. miRNA-370-3p, bone morphogenetic protein 2 (BMP2), and parathyroid hormone like hormone (PTHLH) were differentially expressed during NELL-1-induced osteogenesis. Bioinformatic analyses demonstrated that these differentially expressed miRNAs and mRNAs enriched in Rap1 signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, Glucagon signaling pathway, and hypoxia-inducible factor-1 signaling pathway, which were important pathways related to osteogenic differentiation. In addition, miRNA-370-3p and has-miR-485-5p were predicted to interact with circ0001543, circ0002405, and ENST00000570267 in ceRNA networks. Based on the gain or loss of functional experiments by transfection, the results showed that miR-370-3p was a key regulator in osteogenic differentiation by targeting BMP2 and disturbing the expression of PTHLH, and participated in NELL-1-stimulated osteogenesis. The present study provided the primary data and evidence for further exploration on the roles of miRNAs and ceRNAs during NELL-1-induced ossification of hASCs.

Yu L ，Cen X ，Xia K ，Huang X ，Sun W ，Zhao Z ，Liu J ... -  《-》

The long non-coding RNA, miRNA and mRNA landscapes of cementoblasts during cementogenesis.

Stimulation of cementogenesis is essential to cementum regeneration and root restoration. Long non-coding RNAs (lncRNAs) participate in the regulatory networks of periodontal regeneration processes. We identified and analysed differentially expressed lncRNAs, miRNAs and mRNAs associated with cementogenic differentiation of cementoblasts. OCCM-30 immortalized mouse cementoblast cells were induced in cementogenic medium for 7 and 14 days. Total RNA was extracted and subjected to RNA sequencing to screen for differentially expressed lncRNAs, miRNAs and mRNAs. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of RNAs. Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to clarify the potential functions of differentially expressed genes in biological processes and pathways. lncRNA-miRNA-mRNA networks were constructed based on correlation and algorithmic analyses. In all, 461 lncRNAs, 89 miRNAs and 2157 mRNAs showed differential expression in OCCM-30 cells after cementoblast differentiation. At day 7, upregulation of 248 lncRNAs, 30 miRNAs and 905 mRNAs was observed, along with downregulation of 127 lncRNAs, 34 miRNAs and 960 mRNAs. At day 14, 197 lncRNAs, 13 miRNAs and 847 mRNAs were upregulated, while 74 lncRNAs, 12 miRNAs and 760 mRNAs were downregulated. The results of qRT-PCR showed that four candidate lncRNAs, H19, Gdap10, Foxo6os and Ipw, were significantly upregulated after 7 and 14 days of cementogenic induction. The lncRNA-miRNA-mRNA network illustrated a possible competitive endogenous RNA regulatory mechanism. GO analysis showed that consistently differentially expressed mRNAs were involved in blood vessel morphogenesis, cell-substrate adhesion, cell adhesion, ossification and extracellular matrix organization. KEGG analysis indicated that extracellular matrix-receptor interaction, focal adhesion, and the PI3K-Akt, Rap1, mitogen-activated protein kinase, and Ras signalling pathways varied significantly during cementogenesis. The expressions of lncRNA, miRNA and mRNA were significantly altered in cementoblasts after cementogenesis. This study highlighted the effect of lncRNAs in the process of cementogenesis and revealed their potential for the discovery of novel biomarkers and therapeutic targets for cementum regeneration.

Zhang Y ，Huang Y 《-》

Comprehensive Analysis of lncRNA Expression Pattern and lncRNA-miRNA-mRNA Network in a Rat Model With Cavernous Nerve Injury Erectile Dysfunction.

Long noncoding RNAs (lncRNAs) are differentially expressed in erectile dysfunction (ED) associated with aging and diabetes mellitus; however, the lncRNA expression profile in cavernous nerve (CN) injury-related ED (CNI-ED) is unknown. To investigate the dysregulated lncRNAs, microRNAs (miRNAs), and mRNA expression in CNI-ED and construct a potential lncRNA-miRNA-mRNA network. 22 male Sprague-Dawley (SD) rats were divided into bilateral CN crush (BCNC) and Sham groups. Using second-generation high-throughput sequencing technology, we analyzed the expression profiles of lncRNA, miRNA, and mRNA of the 2 groups. 17 differentially expressed lncRNAs were selected and further validated by quantitative real-time polymerase chain reaction (RT-qPCR). The lncRNA-miRNA-mRNA network, Gene Ontology (GO) term enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using Cytoscape. Intra-cavernosal pressure, mean arterial pressure, smooth muscle content, and the expression of miRNA, mRNA, and lncRNA were measured. The BCNC group showed decreased intra-cavernosal/mean arterial pressure as well as decreased smooth muscle/collagen ratios compared with the Sham group. The RNA sequencing results revealed dysregulated expressions of 65 lncRNA, 14 miRNA, and 750 mRNA in the BCNC group based on the following criteria: fold change >2 and P < .05. Among the 17 lncRNAs further selected based on mean count number >4 in both groups, 3 lncRNAs (TCONS_00028173, TCONS_00049985, and TCONS_00058429) were further validated for differential expression by RT-qPCR. GO analysis suggests that these 3 lncRNAs could regulate various processes such as myotube differentiation and muscle cell differentiation. Furthermore, the KEGG pathway analysis showed that the mRNAs in the competing endogenous RNA (ceRNA) network are involved in pathways, including axon guidance and vascular endothelial growth factor signaling pathway. Our findings may provide new information on molecular pathophysiology of CNI-ED and suggest further research to find a more effective therapy for CNI-ED. This study is the first to identify the lncRNA expression pattern and propose a ceRNA network in a rat model with cavernous nerve injury-related erectile dysfunction. However, analogous studies are needed to confirm these findings in humans. In addition, we constructed the network by only confirming the lncRNA. Our study reveals differential expression profiles of lncRNAs, miRNAs, and mRNAs between the BCNC and Sham groups and suggests that these differentially expressed lncRNAs may play critical roles in CNI-ED by regulating apoptosis and fibrosis in the corpus cavernosum via targeting mRNAs or miRNAs. Cong R, Wang Y, Wang Y. Comprehensive Analysis of lncRNA Expression Pattern and lncRNA-miRNA-mRNA Network in a Rat Model With Cavernous Nerve Injury Erectile Dysfunction. J Sex Med 2020;17:1603-1617.

Cong R ，Wang Y ，Wang Y ，Zhang Q ，Zhou X ，Ji C ，Yao L ，Song N ，Meng X ... -  《-》

Differential long noncoding RNA/mRNA expression profiling and functional network analysis during osteogenic differentiation of human bone marrow mesenchymal stem cells.

Zhang W ，Dong R ，Diao S ，Du J ，Fan Z ，Wang F ... -  《Stem Cell Research & Therapy》