miR-326 down-regulate CYP19A1 expression and estradiol-17b production in buffalo granulosa cells through CREB and C/EBP-β.,The Journal of Steroid Biochemistry and Molecular Biology

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miR-326 down-regulate CYP19A1 expression and estradiol-17b production in buffalo granulosa cells through CREB and C/EBP-β.
The Journal of Steroid Biochemistry and Molecular Biology ( IF 2.7 ) Pub Date : 2020-01-26 , DOI: 10.1016/j.jsbmb.2020.105608
Vaishali Chaurasiya ₁ , Suman Kumari ₁ , Suneel Kumar Onteru ₁ , Dheer Singh ₁

Affiliation

Ovarian granulosa cells, known to be endocrine cells, have well active TLR4-/NFKB signalling mediated innate immune capabilities. We have previously shown that endotoxin not only transiently regulates proinflammatory cytokines but cells become tolerant on repeated exposure to endotoxin and impaired granulosa cells functions, which includes downregulation of CYP19A1 gene. To understand further endotoxin tolerance and impaired granulosa cells function, genome-wide transcriptomic profiling in endotoxin tolerant buffalo granulosa cells (bGCs) identified miR-326 as upregulated amongst top 5 DE miRNAs [unpublished data] and qPCR validation confirmed its upregulation during endotoxin tolerance. In silico analyses showed that miR-326 targets CYP19A1 gene. Therefore, in the present study, we elucidated the role of miR-326 in buffalo granulosa cells (bGCs). We first validated its expression vis-à-vis CYP19A1 gene expression in bGCs, both in vivo and in vitro. Results showed an inverse relationship between miR-326 and CYP19A1 expression. Similarly, transcription factors, known to be involved in CYP19A1 gene regulation, CREB and C/EBP-β expression was also found to be decreased in granulosa cells mimicking pre-ovulatory follicular stage. Further, miR-326 mimic was transfected to bGCs in culture and expression of CYP19A1 and CREB & C/EBP-β and genes encoding other enzymes of steroidogenesis pathway were also analyzed. The present study results showed that miR-326 significantly inhibits the expression of CYP19A1 gene while expression of transcription factors CREB and C/EBP-β was found to be upregulated. The expression of STAR and CYP11A1 was found to be unaffected. To elucidate the molecular mechanism of miR-326 mediated downregulation of CYP19A1, binding analyses of RNA polymerase II and CEBP-β to CYP19A1 gene promoter II was analyzed. The result also showed decreased binding of RNA polymerase II with increased binding of CEBP-β to CYP19A1 gene promoter II in bGCs, transfected with miR-326 as compared to control. In summary, our results suggest that miR-326 upregulate CREB and CREB may activate C/EBP-β and later inhibited the transcription of CYP19A1 and decreased estradiol-17b production. The miR-326 mediated down-regulation of the CYP19A1 gene involving CREB-C/EBP-β can be exploited in developing strategies to attenuate endotoxin-mediated tolerance induced impaired granulosa cells function to ensure proper fertility in females.

中文翻译：

miR-326通过CREB和C /EBP-β下调水牛颗粒细胞中CYP19A1的表达和雌二醇-17b的产生。

已知为内分泌细胞的卵巢颗粒细胞具有活跃的TLR4- / NFKB信号传导介导的先天免疫能力。先前我们已经表明，内毒素不仅可以瞬时调节促炎细胞因子，而且细胞在反复暴露于内毒素和颗粒细胞功能受损（包括CYP19A1基因下调）时变得耐受。为了进一步了解内毒素耐受性和受损的颗粒细胞功能，内毒素耐受性水牛颗粒细胞（bGC）中的全基因组转录组谱分析鉴定出miR-326在前5种DE miRNA中均被上调[未公开数据]，qPCR验证证实了内毒素耐受性期间其上调。计算机分析表明，miR-326靶向CYP19A1基因。因此，在本研究中，我们阐明了miR-326在水牛颗粒细胞（bGC）中的作用。我们首先在体内和体外验证了其相对于CYP19A1基因在bGC中的表达。结果显示miR-326与CYP19A1表达呈反比关系。同样，在模仿排卵前卵泡期的颗粒细胞中，已知参与CYP19A1基因调节，CREB和C /EBP-β表达的转录因子也被降低。此外，将miR-326模拟物转染到培养的bGC中，并分析了CYP19A1和CREB和C /EBP-β的表达以及编码类固醇生成途径其他酶的基因。本研究结果显示，miR-326显着抑制CYP19A1基因的表达，而转录因子CREB和C /EBP-β的表达被上调。发现STAR和CYP11A1的表达不受影响。为了阐明miR-326介导的CYP19A1下调的分子机制，分析了RNA聚合酶II和CEBP-β与CYP19A1基因启动子II的结合分析。结果还显示，与对照组相比，转染了miR-326的bGC中RNA聚合酶II的结合减少，而CEBP-β与CYP19A1基因启动子II的结合增加。总之，我们的结果表明，miR-326上调CREB，而CREB可能激活C /EBP-β，随后抑制CYP19A1的转录并降低雌二醇-17b的产生。可以利用miR-326介导的涉及CREB-C /EBP-β的CYP19A1基因下调来开发减弱内毒素介导的耐受性诱导的颗粒细胞功能受损的策略，以确保女性的正常生育能力。分析了RNA聚合酶II和CEBP-β与CYP19A1基因启动子II的结合分析。结果还显示，与对照组相比，转染了miR-326的bGC中RNA聚合酶II的结合减少，而CEBP-β与CYP19A1基因启动子II的结合增加。总之，我们的结果表明，miR-326上调CREB，而CREB可能激活C /EBP-β，随后抑制CYP19A1的转录并降低雌二醇-17b的产生。可以利用miR-326介导的涉及CREB-C /EBP-β的CYP19A1基因下调来开发减弱内毒素介导的耐受性诱导的颗粒细胞功能受损的策略，以确保女性的正常生育能力。分析了RNA聚合酶II和CEBP-β与CYP19A1基因启动子II的结合分析。结果还显示，与对照组相比，转染了miR-326的bGC中RNA聚合酶II的结合减少，而CEBP-β与CYP19A1基因启动子II的结合增加。总之，我们的结果表明，miR-326上调CREB，而CREB可能激活C /EBP-β，随后抑制CYP19A1的转录并降低雌二醇-17b的产生。可以利用miR-326介导的涉及CREB-C /EBP-β的CYP19A1基因下调来开发减弱内毒素介导的耐受性诱导的颗粒细胞功能受损的策略，以确保女性的正常生育能力。结果还显示，与对照组相比，转染了miR-326的bGC中RNA聚合酶II的结合减少，而CEBP-β与CYP19A1基因启动子II的结合增加。总之，我们的结果表明，miR-326上调CREB，而CREB可能激活C /EBP-β，随后抑制CYP19A1的转录并降低雌二醇-17b的产生。可以利用miR-326介导的涉及CREB-C /EBP-β的CYP19A1基因下调来开发减弱内毒素介导的耐受性诱导的颗粒细胞功能受损的策略，以确保女性的正常生育能力。结果还显示，与对照组相比，转染了miR-326的bGC中RNA聚合酶II的结合减少，而CEBP-β与CYP19A1基因启动子II的结合增加。总之，我们的结果表明，miR-326上调CREB，而CREB可能激活C /EBP-β，随后抑制CYP19A1的转录并降低雌二醇-17b的产生。可以利用miR-326介导的涉及CREB-C /EBP-β的CYP19A1基因下调来开发减弱内毒素介导的耐受性诱导的颗粒细胞功能受损的策略，以确保女性的正常生育能力。我们的研究结果提示miR-326上调CREB，而CREB可能激活C /EBP-β，随后抑制CYP19A1的转录并降低雌二醇17b的产生。可以利用miR-326介导的涉及CREB-C /EBP-β的CYP19A1基因下调来开发减弱内毒素介导的耐受性诱导的颗粒细胞功能受损的策略，以确保女性的正常生育能力。我们的研究结果提示miR-326上调CREB，而CREB可能激活C /EBP-β，随后抑制CYP19A1的转录并降低雌二醇17b的产生。可以利用miR-326介导的涉及CREB-C /EBP-β的CYP19A1基因下调来开发减弱内毒素介导的耐受性诱导的颗粒细胞功能受损的策略，以确保女性的正常生育能力。

更新日期：2020-01-27

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