miRNAs are important regulators of cellular senescence yet the extent of their involvement remains to be investigated. We sought to identify miRNAs that are involved in cytokine-induced premature senescence (CIPS) in endothelial cells. CIPS was established in young human pulmonary microvascular endothelial cells (HMVEC-Ls) following treatment with a sublethal dose (20ng/ml) of tumor necrosis factor alpha (TNF-α) for 15days. In parallel, HMVEC-Ls were grown and routinely passaged until the onset of replicative senescence (RS). Differential expression analysis following miRNA microarray profiling revealed an overlapped of eight deregulated miRNAs in both the miRNA profiles of RS and TNF-α-induced premature senescence cells. Amongst the deregulated miRNAs were members of the miR 17-92 cluster which are known regulators of angiogenesis. The role of hsa-miR-20b in TNF-α-induced premature senescence, a paralog member of the miR 17-92 cluster, was further investigated. Biotin-labeled hsa-miR-20b captured the enriched transcripts of retinoblastoma-like 1 (RBL1), indicating that RBL1 is a target of hsa-miR-20b. Knockdown of hsa-miR-20b attenuated premature senescence in the TNF-α-treated HMVEC-Ls as evidenced by increased cell proliferation, increased RBL1 mRNA expression level but decreased protein expression of p16INK4a, a cellular senescence marker. These findings provide an early insight into the role of hsa-miR-20b in endothelial senescence.

译文

mirna是细胞衰老的重要调节剂,但其参与程度仍有待研究。我们试图鉴定参与细胞因子诱导的内皮细胞过早衰老 (CIPS) 的miRNAs。用亚致死剂量 (20ng/ml) 的肿瘤坏死因子 α (TNF-α) 治疗15天后,在年轻的人肺微血管内皮细胞 (HMVEC-Ls) 中建立了CIPS。同时,HMVEC-Ls生长并常规传代,直到复制性衰老 (RS) 开始。miRNA微阵列分析后的差异表达分析显示,在RS和TNF-α 诱导的早衰细胞的miRNA谱中,八个解除调节的miRNA重叠。在解除管制的mirna中,miR 17-92簇的成员是已知的血管生成调节剂。进一步研究了hsa-miR-20b在TNF-α 诱导的过早衰老 (miR 17-92簇的旁系成员) 中的作用。生物素标记的hsa-miR-20b捕获了视网膜母细胞瘤样1 (RBL1) 的富集转录本,表明RBL1是hsa-miR-20b的靶标。hsa-miR-20b的敲除减弱了TNF-α 处理的HMVEC-Ls中的过早衰老,这可以通过增加细胞增殖,增加RBL1 mRNA表达水平但降低细胞衰老标志物p16INK4a的蛋白表达来证明。这些发现提供了hsa-miR-20b在内皮衰老中的作用的早期见解。

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