Wang Hai, Huang Zhongming, Zhao Xin, Guo Boda, Ji Zhigang
Department of Urology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences Beijing 100730, P. R. China.
Department of Urology, Beijing Hospital, National Center of Gerontology Beijing 100730, P. R. China.
Am J Transl Res. 2020 Sep 15;12(9):5730-5740. eCollection 2020.
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.
近几十年来，越来越多的数据表明，微小RNA（miRNA，miR）在支持细胞（SC）的发育中起关键作用，包括调节SC的成熟、合成、增殖和凋亡。先前关于miRNA微阵列的报道已确定在唯支持细胞综合征（SCOS）患者中miR-4270表达异常。然而，尚不清楚miR-4270是否与SCOS的发病机制相关。在本研究中，我们旨在进一步研究miR-4270对SC增殖和凋亡的作用及潜在机制。我们的数据证实，与健康对照相比，SCOS患者的SC中miR-4270显著上调。EdU和CCK-8检测表明，特异性抑制剂沉默miR-4270可显著增强人SC和TM4细胞的增殖。ELISA和流式细胞术检测表明，敲低miR-4270可显著抑制人SC和TM4细胞的凋亡。此外，qRT-PCR检测显示，敲低miR-4270后人SC和TM4细胞中细胞周期基因的表达，包括CCNE1（细胞周期蛋白E1）、CCND1（细胞周期蛋白D1）和CDK4（细胞周期蛋白依赖性激酶4）明显上调，而细胞凋亡因子的表达，包括CASP3（半胱天冬酶3）、CASP6（半胱天冬酶6）和CASP7（半胱天冬酶7）均显著降低。值得注意的是，SCOS患者的SC中GADD45A（生长停滞和DNA损伤诱导α）mRNA下调，且与miR-4270表达呈负相关。此外，生物信息学工具和双荧光素酶报告基因检测确定miR-4270直接结合GADD45A mRNA的3'-UTR以抑制GADD45A表达。同时，蛋白质印迹分析证实，沉默miR-4270或过表达GADD45A后，SC和TM4细胞中NOTCH1（Notch受体1）和HES1（Hes家族bHLH转录因子1）的蛋白表达水平显著升高。综上所述，我们的数据表明，miR-4270通过GADD45A基因使NOTCH信号通路失活来调节SCOS患者SC的增殖和凋亡，这可能为人类SC的发育提供新的见解，并为SCOS的治疗提供有前景的生物标志物。
