Department of Periodontics, School of Dental Medicine, University of Pennsylvania, 240 South 40th Street, Philadelphia, PA, 19104, USA.
Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Apoptosis. 2019 Feb;24(1-2):108-118. doi: 10.1007/s10495-018-01513-9.
MicroRNAs are small functional RNAs that modulate various biological processes in cells by interfering with gene translation. We have previously demonstrated that certain miRNAs play a crucial role in the innate immune responses of human oral epithelial cells to Porphyromonas gingivalis. While addressing the mechanisms of P. gingivalis induced apoptosis in these cells, we discovered that certain miRNAs are upregulated upon stimulation with live bacteria. These upregulated miRNAs include hsa-miR-584, hsa-miR-572, hsa-miR-210, hsa-miR-492, hsa-miR-623 and hsa-miR-663. Further analysis revealed an unexpected role for hsa-miR-663 (miR-663). To further evaluate miR-663 function, we overexpressed miR-663 in epithelial cells which resulted in cellular apoptosis. The bioinformatics analysis of the miR-663 target prediction, revealed a strong binding affinity to a 3' UTR region of Apoptosis Antagonizing Transcription Factor (AATF) mRNA. To demonstrate the binding of miR-663 to AATF mRNA, the putative miR-663 target site within the 3'-UTR region of AATF was cloned in luciferase vector and transfected to HEK293T cells. Luminescence data showed the downregulation of luciferase activity in cells that had the full length target region of the putative binding site, confirming that AATF is one of the targets for miR-663. This prompted us to further evaluate its role in a cancer cell line (MCF-7) to determine miR-663s' apoptotic function. The overexpression of miR-663 led to a significant increase in apoptosis of MCF-7 cells. Taken together, miR-663 may function as an 'apoptomiR' by inhibiting the anti-apoptotic gene AATF to induce apoptosis. These findings could have therapeutic implications for epithelial cell targeting in cancer therapy.
微小 RNA 是一类小的功能性 RNA，通过干扰基因翻译来调节细胞中的各种生物学过程。我们之前已经证明，某些 miRNA 在人类口腔上皮细胞对牙龈卟啉单胞菌的固有免疫反应中起着至关重要的作用。在研究这些细胞中牙龈卟啉单胞菌诱导凋亡的机制时，我们发现某些 miRNA 在受到活菌刺激时会上调。这些上调的 miRNA 包括 hsa-miR-584、hsa-miR-572、hsa-miR-210、hsa-miR-492、hsa-miR-623 和 hsa-miR-663。进一步的分析揭示了 hsa-miR-663（miR-663）的一个意外作用。为了进一步评估 miR-663 的功能，我们在上皮细胞中过表达 miR-663，导致细胞凋亡。miR-663 靶标预测的生物信息学分析显示，miR-663 与凋亡拮抗转录因子（AATF）mRNA 的 3'UTR 区域具有很强的结合亲和力。为了证明 miR-663 与 AATF mRNA 的结合，将 AATF mRNA 3'-UTR 区域内的假定 miR-663 靶位点克隆到荧光素酶载体中，并转染至 HEK293T 细胞。发光数据显示，在具有假定结合位点全长靶区的细胞中，荧光素酶活性下调，证实 AATF 是 miR-663 的靶标之一。这促使我们进一步在癌细胞系（MCF-7）中评估其作用，以确定 miR-663 的凋亡功能。miR-663 的过表达导致 MCF-7 细胞凋亡显著增加。总之，miR-663 可能通过抑制抗凋亡基因 AATF 发挥“凋亡 miRNA”的作用，从而诱导细胞凋亡。这些发现可能对癌症治疗中针对上皮细胞的治疗具有重要意义。
