Song Jianwen, Liu Ping, Yang Zhensheng, Li Linli, Su Hui, Lu Ning, Peng Zhenhui
Department of Dermatology, the Second Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an.
Cell Physiol Biochem. 2012;29(3-4):331-40. doi: 10.1159/000338488. Epub 2012 Apr 3.
C-Jun plays a critical role in ultraviolet A (UVA) irradiation-induced photoaging. The exact mechanisms by which UVA irradiation up-regulates c-Jun expression in human dermal fibroblasts (HDFs) are still not completely understood. We undertook this study to investigate whether microRNA-155 (miR-155) directly regulates the expression of c-Jun in HDFs in vitro.
Expression of c-Jun mRNA and protein and miR-155 in UVA-irradiated HDFs were detected using quantitative real-time RT-PCR and Western blotting. Luciferase reporter assays were performed to examine whether a miR-155 binding site in the 3'-untranslated region (3'-UTR) of the c-Jun gene is responsible for miR-155-mediated c-Jun regulation in HEK293A cells, and expression of c-Jun mRNA and protein in UVA non-exposed and exposed HDFs trasfected with a miR-155 mimic or a miR-155 inhibitor was detected by quantitative real-time RT-PCR and Western blotting.
Expression of miR-155 was markedly reduced and that of c-Jun mRNA and protein was significantly up-regulated in UVA-irradiated HDFs. Luciferase reporter assays indicated that c-Jun is a direct target of miR-155 in HEK293A cells. In both UVA non-exposed and exposed HDFs, miR-155 mimic decreased c-Jun protein levels, while miR-155 inhibitor increased c-Jun protein levels, but both had no effect on c-Jun mRNA expression, which suggest that miR-155-induced c-Jun inhibition occurs at the post-transcriptional level.
Our results demonstrate that miR-155 directly controls c-Jun expression in HDFs at the post-transcriptional level and might function as a protective miRNA in HDFs.
c-Jun在紫外线A（UVA）照射诱导的光老化中起关键作用。UVA照射上调人皮肤成纤维细胞（HDFs）中c-Jun表达的确切机制仍未完全明确。我们开展本研究以调查微小RNA-155（miR-155）是否在体外直接调节HDFs中c-Jun的表达。
采用定量实时逆转录聚合酶链反应（qRT-PCR）和蛋白质印迹法检测UVA照射的HDFs中c-Jun mRNA、蛋白质及miR-155的表达。进行荧光素酶报告基因检测，以检查c-Jun基因3'-非翻译区（3'-UTR）中的miR-155结合位点是否负责miR-155介导的HEK293A细胞中c-Jun的调控，并通过qRT-PCR和蛋白质印迹法检测用miR-155模拟物或miR-155抑制剂转染的未暴露于UVA和暴露于UVA的HDFs中c-Jun mRNA和蛋白质的表达。
在UVA照射的HDFs中，miR-155的表达显著降低，而c-Jun mRNA和蛋白质的表达显著上调。荧光素酶报告基因检测表明，c-Jun是HEK293A细胞中miR-155的直接靶标。在未暴露于UVA和暴露于UVA的HDFs中，miR-155模拟物均降低了c-Jun蛋白水平，而miR-155抑制剂则增加了c-Jun蛋白水平，但两者均对c-Jun mRNA表达无影响，这表明miR-155诱导的c-Jun抑制发生在转录后水平。
我们的结果表明，miR-155在转录后水平直接控制HDFs中c-Jun的表达，并且可能在HDFs中作为一种保护性微小RNA发挥作用。
