miR-4270 regulates cell proliferation and apoptosis in patients with Sertoli cell-only syndrome by targeting GADD45A and inactivating the NOTCH signaling pathway

Hai Wang; Zhongming Huang; Xin Zhao; Boda Guo; Zhigang Ji

miR-4270 regulates cell proliferation and apoptosis in patients with Sertoli cell-only syndrome by targeting GADD45A and inactivating the NOTCH signaling pathway

Am J Transl Res. 2020 Sep 15;12(9):5730-5740. eCollection 2020.

Authors

Hai Wang¹, Zhongming Huang¹, Xin Zhao¹, Boda Guo², Zhigang Ji¹

Affiliations

¹ Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences Beijing 100730, P. R. China.
² Department of Urology, Beijing Hospital, National Center of Gerontology Beijing 100730, P. R. China.

PMID: 33042452
PMCID: PMC7540124

Abstract

In recent decades, growing data has suggested that microRNAs (miRNAs, miRs) play a critical role in the development of Sertoli cells (SC), including regulating SC maturation, synthesis, proliferation, and apoptosis. Previous reports of miRNA microarray have identified aberrant miR-4270 expression in patients with Sertoli-cell-only syndrome (SCOS). However, it is not known whether miR-4270 is associated with the pathogenesis of SCOS. In this study, we aimed to further investigate the roles and potential mechanisms of miR-4270 on SC proliferation and apoptosis. Our data confirmed that miR-4270 was significantly upregulated in SC of SCOS patients compared with healthy controls. EdU and CCK-8 assays showed silencing of miR-4270 by specific inhibitor significantly enhanced human SC and TM4 cells proliferation. ELISA and flow cytometry assays indicated that miR-4270 knockdown prominently suppressed the apoptosis of human SC and TM4 cells. Furthermore, expression of cell cycle genes, including CCNE1 (cyclin E1), CCND1 (cyclin D1) and CDK4 (cyclin dependent kinase 4), were obviously upregulated in human SC and TM4 cells by qRT-PCR assay after knockdown of miR-4270, while expression of cell apoptotic factors, including CASP3 (caspase 3), CASP6 (caspase 6) and CASP7 (caspase 7), were all markedly decreased. Notably, GADD45A (growth arrest and DNA damage inducible alpha) mRNA was downregulated in SC of SCOS patients, and negatively corrected with miR-4270 expression. Moreover, bioinformatics tools and dual-luciferase reporter assay identified that miR-4270 directly bound the 3'-UTR of GADD45A mRNA to inhibit GADD45A expression. Meanwhile, Western blots analysis validated that the protein expression levels of NOTCH1 (notch receptor 1) and HES1 (hes family bHLH transcription factor 1) were significantly increased in SC and TM4 cells after miR-4270 silencing or GADD45A overexpression. Taken together, our data demonstrated that miR-4270 regulates proliferation and apoptosis in SC of SCOS patients by inactivating NOTCH signaling pathway via GADD45A gene, which may offer a new insight into the development of human SC and provide a promising biomarker for the treatment of SCOS.

Keywords: GADD45A; Sertoli cells; apoptosis; miR-4270; proliferation.