MiR-145-modulated SOX9-mediated hypospadias through acting on mitogen-activated protein kinase signaling pathway
Yafeng Shang1, Yanjie Kang1, Jiantao Sun1
1Department of Urology, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, China.
Abstract:
This study primarily explored how miR-145, mitogen-activated protein kinase (MAPK) signaling and a downstream transcription factor (i.e., SOX9) mediated development of hypospadias. The hypospadias tissues and preputial tissues were isolated from pediatric inpatients postoperatively. Simultaneously, the rat models of hypospadias were established, and spermatogonial stem cells were separated. The expressions of proteins that symbolized cell apoptosis and oxidative stress were quantified via western blot analysis. Furthermore, the apoptosis, proliferation, and viability of cells were evaluated by means of flow cytometry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays. The results of microarray indicated miR-145 as a differentially expressed biomarker between hypospadias tissues and normal tissues (p < 0.05). Moreover, rat models of hypospadias were observed with markedly lower vitamins A and E levels, reduced expressions of proteins relevant to oxidative stress (i.e., Nrf2, HO-1, Gpx, and SOD-1), as well as enhanced Bax and cleaved caspase-3 expressions ( p < 0.05). Furthermore, SOX9 was found to be targeted by miR-145, and it was also modified by phosphorylated extracellular-regulated kinase (p-ERK), a portion of MAPK signaling ( p < 0.05). The p-ERK was significantly regulated after altering the expression of miR-145 ( p < 0.05). Moreover, activation of p-ERK and transfection of pcDNA-SOX9 could cause higher expression of apoptins and larger apoptotic proportion of cells ( p < 0.05), yet transfection of miR-145 mimic led to improved cell apoptosis and depressed cell viability ( p < 0.05). In conclusion, SOX9, which was regulated by both miR-145 and miR-145/MAPK signaling, could be involved in the pathogenesis of hypospadias.
Insights
This study reveals that miR-145 and mitogen-activated protein kinase (MAPK) signaling regulate SOX9, impacting hypospadias development. These findings offer insights into the molecular mechanisms underlying hypospadias pathogenesis.
Area of Science:
- Urology
- Molecular Biology
- Developmental Biology
Background:
- Hypospadias is a congenital condition affecting male urinary tract development.
- The molecular mechanisms underlying hypospadias remain incompletely understood.
- MicroRNAs (miRNAs) have emerged as critical regulators in various biological processes, including development.
Purpose of the Study:
- To investigate the role of miR-145, mitogen-activated protein kinase (MAPK) signaling, and SOX9 in hypospadias development.
- To elucidate the molecular interactions between these factors in the pathogenesis of hypospadias.
Main Methods:
- Analysis of hypospadias and normal tissues from pediatric patients.
- Establishment of rat models for hypospadias and separation of spermatogonial stem cells.
- Western blot, flow cytometry, MTT assays, and colony formation assays to assess cell apoptosis, proliferation, and viability.
- Microarray analysis to identify differentially expressed miRNAs.
Main Results:
- Microarray analysis identified miR-145 as a differentially expressed biomarker in hypospadias tissues.
- Rat models exhibited reduced vitamin levels, decreased oxidative stress markers (Nrf2, HO-1, Gpx, SOD-1), and increased apoptosis markers (Bax, cleaved caspase-3).
- SOX9 was identified as a target of miR-145 and was also modulated by phosphorylated extracellular-regulated kinase (p-ERK) within the MAPK signaling pathway. Altering miR-145 expression significantly affected p-ERK levels, while p-ERK activation and SOX9 transfection increased apoptosis. Conversely, miR-145 mimic transfection improved apoptosis and reduced cell viability.
Conclusions:
- miR-145 plays a crucial role in hypospadias pathogenesis by targeting SOX9.
- The miR-145/MAPK signaling pathway modulates SOX9 expression and influences cellular apoptosis and viability.
- These findings highlight a novel molecular mechanism potentially involved in hypospadias development.
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