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 ... -  《-》

Comparative Transcriptome Analyses of Geriatric Rats Associate Age-Related Erectile Dysfunction With a lncRNA-miRNA-mRNA Regulatory Network.

The key regulatory roles of long non-coding RNAs (lncRNAs) in age-related erectile dysfunction (A-ED) are unknown. This study aimed to identify putative lncRNAs that regulate age-related erectile dysfunction via transcriptome analyses, and to predict their specific regulatory routes via bioinformatics methods. 22 geriatric male SD rats were divided into age-related erectile dysfunction (A-ED) and negative control (NC) groups after evaluations of intracavernous pressure (ICP). By comparative analysis of transcriptomes of cavernosal tissues from both groups, we identified differentially expressed lncRNAs, miRNAs, and mRNAs. Seven differentially expressed lncRNAs were selected and further verified by quantitative real-time polymerase chain reactions (RT-qPCR). The construction of the lncRNA-miRNA-mRNA network, the Gene Ontology (GO) term enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed in Cytoscape. From comparative transcriptome analyses of A-ED and NC groups, 69, 29, and 364 differentially expressed lncRNAs, miRNAs, and mRNAs were identified respectively. Differentially expressed lncRNAs were culled to seven, which were all verified by qPCR. Three of these lncRNAs (ENSRNOT00000090050, ENSRNOT00000076482, and ENSRNOT00000029245) were used to build regulatory networks, of which only ENSRNOT00000029245 was successful. Moreover, GO and KEGG analyses demonstrated that these lncRNAs possibly regulated muscle myosin complex, muscle cell cellular homeostasis, and ultimately erectile function in rats through PI3K-Akt, fluid shear stress, and atherosclerosis pathways. Our study identified differentially expressed lncRNAs, miRNAs, and mRNAs through comparisons of transcriptomes of geriatric rats. An identified lncRNA verified by qPCR, was used to construct a lncRNA-miRNA-mRNA regulatory network. LncRNA ENSRNOT00000029245 possibly regulated downstream mRNAs through this regulatory network, leading to apoptosis in the cavernous tissue, fibrosis, and endothelial dysfunction, which ultimately caused ED. These findings provide seminal insights into the molecular biology of aging-related ED, which could spur the development of effective therapeutics.

Zhou X ，Cong R ，Yao L ，Zhou X ，Luan J ，Zhang Q ，Zhang X ，Ren X ，Zhang T ，Meng X ，Song N ... -  《Frontiers in Endocrinology》

The Changes of MicroRNA Expression in the Corpus Cavernosum of a Rat Model With Cavernous Nerve Injury.

MicroRNAs (miRs) were found to be dysregulated in erectile dysfunction (ED) related to aging, type 2 diabetes mellitus, and vasculogenic abnormalities. However, miR expression in ED after radical prostatectomy (RP) is not known. To detect abnormal miR expression in post-RP ED and analyze target genes and pathways. 16 Sprague Dawley rats were divided into bilateral cavernous nerve crush (BCNC) and control groups. 4 weeks after surgery, erectile function and histological change in the corpus cavernosum were evaluated. Total RNA from 3 rats from each group was isolated and processed to analyze the miR expression profiling by RNA sequencing. The top 10 up-regulated miR profiles were chosen directly and further validated in another 5 rats per each group by quantitative real-time polymerase chain (PCR) reaction. The target genes were predicted by online databases, including: TargetScan, mirwalk, miRanda, miRDB, and DIANA. The enrichment analysis of gene ontology-term analysis and Kyoto Encyclopedia of Genes and Genomes were performed by DAVID database. Intra-cavernosal pressure, mean arterial pressure, smooth muscle content, and miR expression were measured. Compared to the control group, the BCNC group had decreased intra-cavernosal/mean arterial pressure ratio and smooth muscle marker (α-smooth muscle actin). The sequence results showed that 124 miR expression dysregulated in the BCNC group, in which 122 miR expression were up-regulated. Of the 122 miRs, 21 miR expressions were increased above 2-fold. Among the top 10 up-regulated miRs, 4 miRs (miR-101a, miR-138, miR-338, and miR-142) levels were finally validated for over-expression by quantitative (PCR) reaction. The gene ontology analysis results showed that these 4 miRs could regulate the processes of cell apoptosis, fibrosis, endothelium, and smooth muscle cells function. The Kyoto Encyclopedia of Genes and Genomes pathway analysis showed the target genes were involved in 7 pathways related to ED. Our findings provide novel insights into post-RP ED that may stimulate further studies to develop miR targeted therapy or damage detection for ED. To our knowledge, this is the first study to identify the miR profiling and function in the BCNC rat model. The rat model might not represent the human condition and the miR was only detected at 1 period. Besides that, there is a high probability of false positives for RNA sequence results. 4 dysregulated miRs were found in the BCNC rat model, which may be related to post-RP ED by regulating apoptosis, fibrosis, endothelial, and smooth muscle cells. Liu C, Cao Y, Ko TC, et al. The Changes of MicroRNA Expression in the Corpus Cavernosum of a Rat Model With Cavernous Nerve Injury. J Sex Med 2018;15:958-965.

Liu C ，Cao Y ，Ko TC ，Chen M ，Zhou X ，Wang R ... -  《-》

Whole-transcriptome analysis of rat cavernosum and identification of circRNA-miRNA-mRNA networks to investigate nerve injury erectile dysfunction pathogenesis.

Huang J ，Ma J ，Wang J ，Ma K ，Zhou K ，Huang W ，Zhao F ，Lv B ，Hu Q ... -  《-》

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》