Herbert Katharine J, Cook Anthony L, Snow Elizabeth T
School of Health Sciences, University of Tasmania, Launceston, TAS 7250, Australia.
School of Health Sciences, University of Tasmania, Launceston, TAS 7250, Australia.
J Dermatol Sci. 2014 May;74(2):142-9. doi: 10.1016/j.jdermsci.2014.01.008. Epub 2014 Jan 31.
Together with p53, the NAD-dependent lysine deacetylase SIRT1 and the microRNA miR-34a form a feedback loop which self-regulates SIRT1 expression and modulates p53-dependent responses. In addition to its well-described role in mediating transcriptional responses to genotoxic stress, p53 may also regulate microRNA processing and maturation.
This study explored the functional relationship among p53, SIRT1 and miR-34a, and the influence of p53 and SIRT1 on microRNA biogenesis and maturation in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines.
RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors were used to modulate activity and expression of SIRT1 and p53. Changes in microRNA and mRNA were analysed by qRT-PCR and protein expression was determined by immunoblotting.
Mature miR-34a decreased in p53-suppressed NHEK cells, whereas ablation of SIRT1 reduced the primary transcript (pri-miR-34a). When either SIRT1 expression or activity was inhibited in combination with p53 ablation, pri-miR-34a levels increased and mature miR-34a levels decreased. Under these same conditions, additional p53-regulated microRNAs (miRs 16-1/15, 145 and 107) also failed to mature. In HaCaT cells, primary microRNA transcripts for miR-16-1/15, miR-145 miR200c/141 and miRNA-107, but not miR-34a, were approximately 8-fold higher than in NHEK cells. However, the levels of mature microRNA sequences in HaCaT cells were only 1.5-2 fold higher (miR-16-1, miR-145), unchanged (miR-107) or decreased (miR-200c/141, miR-34a) compared to NHEK cells.
Our results suggest that p53 mutations interfere with efficient microRNA biogenesis in keratinocytes, and that SIRT1 functions in combination with p53 in this process.
烟酰胺腺嘌呤二核苷酸（NAD）依赖性赖氨酸脱乙酰酶SIRT1与p53以及微小RNA miR-34a共同形成一个反馈环，该反馈环可自我调节SIRT1的表达并调节p53依赖性反应。除了在介导对基因毒性应激的转录反应中所描述的作用外，p53还可能调节微小RNA的加工和成熟。
本研究探讨了p53、SIRT1和miR-34a之间的功能关系，以及p53和SIRT1对原代角质形成细胞（NHEK）和p53突变的角质形成细胞（HaCaT）系中微小RNA生物合成和成熟的影响。
使用RNA干扰、微小RNA靶位点阻断寡核苷酸和小分子抑制剂来调节SIRT1和p53的活性和表达。通过定量逆转录聚合酶链反应（qRT-PCR）分析微小RNA和信使核糖核酸（mRNA）的变化，并通过免疫印迹法测定蛋白质表达。
在p53受抑制的NHEK细胞中，成熟的miR-34a减少，而SIRT1的缺失则降低了初级转录本（pri-miR-34a）。当SIRT1的表达或活性与p53缺失联合受到抑制时，pri-miR-34a水平升高，而成熟的miR-34a水平降低。在相同条件下，其他p53调节的微小RNA（miRs 16-1/15、145和107）也未能成熟。在HaCaT细胞中，miR-16-1/15、miR-145、miR200c/141和miRNA-107的初级微小RNA转录本，但不包括miR-34a，比NHEK细胞中的大约高8倍。然而，与NHEK细胞相比，HaCaT细胞中成熟微小RNA序列的水平仅高1.5 - 2倍（miR-16-1、miR- miR-145）、不变（miR-107）或降低（miR-200c/141、miR-34a）。
我们的结果表明，p53突变会干扰角质形成细胞中高效的微小RNA生物合成，并且SIRT1在此过程中与p53共同发挥作用。
