miR-1246 releases RTKN2-dependent resistance to UVB-induced apoptosis in HaCaT cells
miR-1246 releases RTKN2-dependent resistance to UVB-induced apoptosis in HaCaT cells
复制标题
miR-1246 释放 RTKN2 依赖性抵抗 UVB 诱导的 HaCaT 细胞凋亡
DOI：
10.1007/s11010-014-2108-1
复制
发表时间：
2014-09-01
影响因子：
4.3
通讯作者：
Qian, Hua
中科院分区：
文献类型：
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作者：
Li, Wei;Wu, Ya-Fen;Qian, Hua
MicroRNA是一类在细胞增殖、分化、凋亡等生物学过程中发挥重要作用的非编码小分子RNA。紫外线诱导的细胞凋亡的细胞反应已被认为是由microRNA在转录后水平的调控，而这一过程背后的详细机制仍不清楚。本研究的目的是探讨miR-1246在UVB诱导的角质形成细胞HaCaT细胞凋亡中的作用，并确定miR-1246的功能靶点。采用实时荧光定量PCR检测HaCaT细胞在UVB胁迫下miR-1246和凋亡基因的表达。通过对miR-1246模拟物或miR-1246转染的细胞进行荧光激活细胞分选仪分析，定量miR-1246在UVB诱导的细胞凋亡中的功能。此外，RTKN 2和miR-1246之间的调控关系通过Western blot和荧光素酶报告基因测定来鉴定。miR-1246在UVB照射的HaCaT细胞凋亡中表达上调。miR-1246的过表达促进UVB诱导的细胞凋亡，而使用特异性抑制剂敲低miR-1246导致UVB诱导的细胞凋亡显著减少。我们进一步证明miR-1246通过在转录后水平与RTKN 2的3′-非翻译区结合来负调控RTKN 2的表达。此外，RTKN 2被观察到对UVB诱导的细胞凋亡具有抗性，并且RTKN 2在UVB诱导的HaCaT细胞凋亡期间拮抗miR-1246的促凋亡作用。这些发现表明，miR-1246通过下调RTKN 2表达促进UVB诱导的细胞凋亡，并且UVB上调的miR-1246通过在角质形成细胞中转录后靶向RTKN 2释放对UVB诱导的细胞凋亡的RTKN 2依赖性抗性。
MicroRNAs are a kind of small non-coding RNAs that play important roles in various biological processes such as cell proliferation, differentiation, and apoptosis. Cellular responses to UV-induced apoptosis have been suggested to be regulated by microRNAs at the posttranscriptional level, while the detailed mechanisms underlying this process remain unclear. Our aim in this study was to investigate the effects of miR-1246 in UVB-induced apoptosis and to identify the functional targets of miR-1246 in keratinocyte HaCaT cells. The expression of miR-1246 and apoptotic genes in HaCaT cells experiencing UVB stress was determined using quantitative real-time PCR. miR-1246 functions in UVB-induced apoptosis were quantified via fluorescence-activated cell sorter analysis of miR-1246 mimic or inhibitor-transfected cells. Additionally, the regulatory relationship between RTKN2 and miR-1246 was identified by Western blot and luciferase reporter assays. miR-1246 was upregulated accompanying with UVB-irradiated apoptosis in HaCaT cells. Overexpression of miR-1246 promoted UVB-induced apoptosis, while knockdown of miR-1246, using a specific inhibitor, resulted in a significant reduction in UVB-elicited apoptosis. We further demonstrate that miR-1246 negatively regulated the expression of RTKN2 through binding to the 3′-untranslated region of RTKN2 at the posttranscriptional level. Moreover, RTKN2 was observed to be resistant to UVB-induced apoptosis and RTKN2 antagonized the pro-apoptotic effects of miR-1246 during UVB-induced apoptosis in HaCaT cells. These findings suggested that miR-1246 promotes UVB-induced apoptosis by downregulating RTKN2 expression and that UVB-upregulated miR-1246 released RTKN2-dependent resistance to UVB-induced apoptosis by targeting RTKN2 post-transcriptionally in keratinocyte cells.