Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach
Identification of a microRNA (miR-663a) induced by ER stress and its target gene PLOD3 by a combined microRNome and proteome approach
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DOI：
10.1007/s10565-016-9335-z
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发表时间：
2016-08-01
影响因子：
6.1
通讯作者：
Remondelli, Paolo
中科院分区：
文献类型：
--
作者：
Amodio, Giuseppina;Sasso, Emanuele;Remondelli, Paolo
MicroRNA (miR) 调节基因表达以支持重要的生理功能。大量证据表明，miR在许多病理事件和细胞对各种应激的反应中发挥着至关重要的作用。为了鉴定细胞内钙稳态扰动诱导的新miR，我们通过微阵列分析了毒胡萝卜素（TG）处理的细胞的miR表达谱。为了鉴定 miR-663a 调节的基因，我们通过二维差异凝胶内电泳结合差异表达蛋白质的质谱鉴定来评估 miR-663a 过表达细胞中的蛋白质组变化。微阵列和蛋白质组分析得到生化验证的支持。微阵列结果显示24个差异表达的miR；其中，miR-663a竟然是受ER应激和PERK通路控制下的未折叠蛋白反应。蛋白质组学分析表明，PLOD3 是编码胶原蛋白修饰赖氨酰羟化酶 3 (LH3) 的基因，受 miR-663a 调节。荧光素酶报告基因检测表明，miR-663a 确实通过靶向 PLOD3 mRNA 的 3'-UTR 来降低 LH3 表达。有趣的是，miR-663a对LH3表达的抑制会减少IV型胶原蛋白的细胞外积累，因此表明miR-663a参与调节生理条件下的胶原蛋白4分泌以及响应ER应激。ER应激诱导的PERK-miR-663a途径的发现可能对理解该miR在正常和/或病理条件下功能的分子机制具有重要意义。
MicroRNAs (miRs) regulate gene expression to support important physiological functions. Significant evidences suggest that miRs play a crucial role in many pathological events and in the cell response to various stresses.With the aim to identify new miRs induced by perturbation of intracellular calcium homeostasis, we analysed miR expression profiles of thapsigargin (TG)-treated cells by microarray. In order to identify miR-663a-regulated genes, we evaluated proteomic changes in miR-663a-overexpressing cells by two-dimensional differential in-gel electrophoresis coupled to mass spectrometric identification of the differentially represented proteins. Microarray and proteomic analyses were supported by biochemical validation.Results of microarray revealed 24 differentially expressed miRs; among them, miR-663a turned out to be by ER stress and under the control of the PERK pathway of the unfolded protein response. Proteomic analysis revealed that PLOD3, which is the gene encoding for collagen-modifying lysyl hydroxylase 3 (LH3), is regulated by miR-663a. Luciferase reporter assays demonstrated that miR-663a indeed reduces LH3 expression by targeting to 3'-UTR of PLOD3 mRNA. Interestingly, miR-663a inhibition of LH3 expression generates reduced extracellular accumulation of type IV collagen, thus suggesting the involvement of miR-663a in modulating collagen 4 secretion in physiological conditions and in response to ER stress.The finding of the ER stress-induced PERK-miR-663a pathway may have important implications in the understanding of the molecular mechanisms underlying the function of this miR in normal and/or pathological conditions.