MicroRNA-4279通过靶向核心启动子区域上调Notch-1基因表达进而抑制细胞凋亡

Micro RNA-4279 Inhibits Cell Apoptosis through Upregulating Notch-1 Expression by Targeting the Core Promoter Region

【目的】寻找与Notch-1基因核心启动子结合的micro RNA(miRNA)并阐明其调控功能,研究免疫细胞肿瘤的发生发展机制。【方法】首先使用生物信息学手段筛选与Notch-1基因核心启动子区域结合的miRNA,进而利用荧光素酶报告系统对其调控作用进行实验验证;通过突变核心启动子上的结合位点进一步确定该miRNA特异性靶向基因核心启动子区域。检测转染该miRNA后H9细胞中Notch-1的mRNA与内源蛋白表达水平。另外通过转染该miRNA并用H2O2诱导凋亡,研究该miRNA对H9细胞凋亡的影响。进一步在上述实验条件下,使用siRNA降低靶基因(Notch-1)的表达,然后检测miRNA对H9细胞系的凋亡的影响。【结果】首先通过软件预测,我们发现了miR-4279可以与Notch-1的核心启动子序列结合。荧光素酶报告实验表明miR-4279可以增强Notch-1启动子的转录活性;而在miR-4279的靶位点引入突变后,该增强作用消失。RT-qPCR和Western Blot实验表明miR-4279能够显著增强内源Notch-1 mRNA与蛋白的表达。H2O2诱导凋亡实验表明,miR-4279可以抑制H9细胞的凋亡。siRNA干扰实验表明,当Notch-1通路被抑制后,miR-4279导致的细胞凋亡抑制的效果消失。【结论】miR-4279通过靶向Notch-1基因核心启动子区域,特异性增加Notch-1基因的表达,进而增强H9细胞对H2O2诱导的凋亡的抗性。

【Objective】 To investigate the regulation of Notch-1 expression by miRNAs through targeting the core promoter region and get insight into the role of miRNA mediated transcriptional regulation in the carcinogenesis of the lymphocytes. 【Method】 First,miRNA binding sites in the core promoter region of Notch-1 gene were predicted. Then the dual-luciferase assay was conducted to determine whether the miRNA（s） could induce Notch-1 promoter activation. Furthermore, mutations were introduced to the putative binding site of the miRNA in Notch-1 promoter to confirm the target site of miRNA. Next, RT-q PCR and Western Blot assay were used to determine whether the miRNA could modulate the endogenous mRNA and protein expression of Notch-1 gene. Then we transfected miRNA into H9 cell and determined its effect on cell apoptosis under the condition of H2O2 treatment. By knocking down Notch-1, we further determined whether the miRNA induced anti-apoptosis is dependent of the Notch-1 pathway. 【Results】 We found that miR-4279 had a putative binding site on the core promoter region of Notch-1 gene; and miR-4279 could increase the promoter activity of Notch-1. Furthermore, mutation assay suggests that this site is the actual target of miR-4279 for promoter activation. Overexpressing miR-4279 significantly increased Notch-1 mRNA and protein expressions in H9 cells. Moreover, miR-4279 could readily inhibit H9cells＇ apoptosis induced by H2O2. The knock-down assay of Notch-1 showed that the miR-4279 induced anti-apoptosis effect is dependent on the Notch-1 signaling pathway. 【Conclusion】 Mi R-4279 upregulates Notch-1 gene expression through targeting the core promoter region, which confers the anti-apoptosis ability to lymphocytes.