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Exploring Relevant mRNAs and miRNAs in Injured Urethral Tissues of Rats with High-Throughput Sequencing [miRNA-seq]

ABSTRACT: Acute urethral injuries caused by urethral endoscopy and other mechanical injuries are the main reasons for secondary infection and late urethral stricture. However, there are no studies to explore the transcriptomic changes in urethral injury and the molecular mechanism of urethral injury, which is important for the treatment and cure of urethral injury. Therefore, we used RNA-seq and sRNA-seq profiles from normal and injured urethral tissues to identify and characterize differentially expressed mRNAs and miRNAs. In total, we found 166 differentially expressed mRNAs, of which 69 were upregulated, and 97 were downregulated in injured urethral tissues. The differentially expressed mRNAs were mainly involved in the positive regulation of epithelial cell differentiation, focal adhesion, cell adhesion molecules, protein activation cascade, complement activation, complement and coagulation cascades, and chemokine-mediated signaling pathway. Additionally, we found six upregulated and four downregulated miRNAs, respectively, in the injured urethral tissues. Notably, their target genes were involved in the vascular endothelial growth factor receptor 2 binding, PI3k-Akt signaling pathway, and Notch signaling pathway. In summary, our results suggest that the cell damage response induced by mechanical injury activates the pathological immune response in a variety of ways in injured urethral tissues.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE182947 | GEO | 2022/05/31

REPOSITORIES: GEO

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Project description:Background: Urethral stricture is related to scar tissue fibrosis, but its pathogenesis is still unclear. This study aims to explore the mechanism of circular RNA (circRNA) in the occurrence and development of urethral stricture. Methods: CircRNA microarray was employed to analyze circRNA expression profiles between human urethral scar tissue and normal urethral tissue. The first nine circRNA differentially expressed were selected for RT-qPCR detection. Urethral scar fibroblasts were isolated from human urethral scar tissue and cultured in vitro. The cells were transfected with si-circ_0047339, LV-circ_0047339, and miR-4691-5p mimic and their corresponding negative controls. Related gene and protein expression was measured by RT-qPCR, Western Blot and immunofluorescence staining, and the cell function was detected by CCK-8 and EDU assay. The targeting relationship between miR-4691-5p and circ_0047339 or TSP-1 was analyzed by dual-luciferase reporter assay. Results: The results of circRNA microarray showed that there were 268 differential genes between urethral scar tissue and normal urethral tissue (circRNA expression difference multiple ≥2 and p value≤0.05). The enrichment analysis of KEGG (Kyoto encyclopedia of genes and genomes) showed that these circRNAs were significantly correlated with ECM-receptor interaction. The first nine differentially expressed circRNA were selected to predict the circRNA-miRNA network. RT-qPCR results showed that circ_0047339 was upregulated considerably in urethral scar tissue. After silencing circ_0047339, the proliferation of urethral scar cells decreased significantly, and the expression of collagen 1 (COL-1) and α-smooth muscle actin (α-SMA) also reduced. Circ_0047339 as a competing endogenous RNA (ceRNA) could increase the expression of TSP-1 by competitively binding miR-4691-5p. In addition, miR-4691-5p mimic transfection could inhibit the proliferation of urethral scar fibroblasts and the presentation of thrombospondin-1 (TSP-1), α-SMA and COL-1, while circ_0047339 overexpression eliminated this inhibition. Conclusion: Our results showed that the dysfunctional circ_0047339 might promote the growth and fibrosis of urethral scar fibroblasts through the ceRNA mechanism, thus promoting the development of urethral stricture via miR-4691-5p/TSP-1 axis.

Project description:Periurethral human mesenchymal stem cell (hMSC) injections are associated with functional improvement in animal models of post-partum stress urinary incontinence (SUI). However, limited data exists on the role of hMSCs in modulating gene expression in tissue repair after urethral injury. We quantified temporal gene expression modulation in hMSCs, and injured rat urethral tissue, using RNA seq in an animal model of SUI, over a 3-day time period following urethral injury, and local hMSC therapy. We injected PKH fluorescent-labeled human hMSC into the periurethral space of rats, following a 4h vaginal distention (3 rats per time point). Control rats underwent vaginal distention injury only, and were sacrificed at 12h, 24h, 36h, 72h post injury. Rat urethral and vaginal tissues were frozen and sectioned. Fluorescent labeled hMSCs were distinguished from adjacent, unlabeled rat urethral tissue. RNA was prepared from urethral tissue obtained by laser dissection of frozen tissue sections and sequenced on an Illumina HiSeq 2500. Differentially expressed genes (DEGs) over 72h were evaluated using a 2-group t-test (p<0.05). Our transcriptional analyses identified candidate genes involved in tissue injury, that were broadly sorted by injury and exposure to hMSC, throughout the first 72h of acute phase of injury. DEGs in treated urethra, compared with untreated urethra, were functionally associated with tissue repair, angiogenesis, neurogenesis, and oxidative stress suppression. DEGs included a variety of cytokines, extracellular matrix stabilization and regeneration genes, cytokine signaling modification, cell cycle regulation, muscle differentiation and stabilization. Moreover, our results revealed DEGs changes in the hMSCs (PKH-labelled), which were harvested from injured urethra. The expressions are related to DNA damage repair, transcription activation, stem cell regulation, cell survival, apoptosis, self-renewal, cell proliferation, migration and injury response.

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Exploring Relevant mRNAs and miRNAs in Injured Urethral Tissues of Rats with High-Throughput Sequencing [miRNA-seq]

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