TRAIL诱导黑色素瘤细胞中miRNA-221的表达

TRAIL诱导黑色素瘤细胞中miRNA-221的表达
侯丽丽;程冰;张旭东;王丽;张林杰
【期刊名称】《安徽医科大学学报》
【年(卷),期】2011(46)2
【摘要】目的研究TRAIL诱导的黑色素瘤细胞中miRNA-221的表达及其在细胞凋亡抵抗中的意义.方法TRAIL(100 μg/L)分别处理黑色素瘤细胞系sk-Mel-28、IgR3、Mel-RM、MM200、Me4405,6 h后收集细胞,实时荧光定量PCR检测各细胞系中miRNA-221的mRNA水平,同时检测TRAIL(100 μg/L)诱导Mel-RM 细胞不同时间点miRNA-221的mRNA水平变化;脂质体转染反义miRNA-221下调TRAIL诱导的Mel-RM细胞中miRNA-221的表达,同时设对照组和转染无意序列组;流式细胞术PI单染法检测细胞凋亡率;Western blot检测p-ERK及p27蛋白表达.结果TRAIL(100 μg/L)诱导Mel-RM细胞中miRNA-221的mRNA水平明显高于其他细胞系(2-ΔΔCt值为18.553 4±1.514 6).随TRAIL诱导时间的延长,Mel-RM细胞中miRNA-221的表达上调.TRAIL(100 μg/L)诱导的Mel-RM细胞中,反义miRNA-22l转染组凋亡率高于对照组和转染无意序列组.miRNA-221靶基因p27在U0126(MEK1/2抑制剂)和TRAIL共处理的Mel-RM细胞中的表达较TRAIL单独处理组上调.结论抑制miRNA-221的表达可能具有提高TRAIL诱导的黑色素瘤细胞凋亡敏感性的作用,抑制ERK的激活可能下调miRNA-221的表达,从而上调miRNA-221的靶基因p27蛋白的表达.%Objective To study the expression of miRNA-221 in TRAIL-induced melanoma cells and its role in resistance of melanoma cells to TRAIL-induced apoptosis. Methods Melanoma cell lines sk-Mel-28, IgR3, Mel-RM,MM200 and Me4405 were
treated by TRAIL( 100 μg/L)for 6 h respertively, then quantitative fluorescent PCR was used to measure the miRNA-221 levels in diverse types of melanoma cells and the expression of miRNA-221 in different intervals of TRAIL-induced Mel-RM cells. Oligofectamine was used to transfect miRNA-221 antisense oligonucleotides to knock down the expression of miRNA-221 in Mel-RM cells induced by TRAIL, setting the scr-antimiR treated group and control group. Apoptosis was measured by flow cytometry using propidium iodide staining.Protein levels of p-ERK and p27 were detected by Western blot. Results The expression of miRNA-221 induced by TRAIL( 100 μg/L) was higher in Mel-RM cells with the average value of 2 -ΔΔct being 18. 5534 ± 1. 5146 than that in other cell lines. And it was up-regulated induced by TRAIL in Mel-RM cells. The apoptosis of Mel-RM cells was higher in antimiR-221 treated group than that in scr-antimiR treated group and control group. The target gene of miRNA-221,p27 was higher in combination of MEK1/2 inhibitor U0126 and TRAIL treated group of Mel-RM cells than that in TRAIL alone treated group.Conclusion Reduction of miRNA-221 expression may improve the sensitivity of melanoma cells to TRAIL-induced apoptosis. Down-regulation of miRNA-221 expression,which may be accomplished by inhibiting ERK, can increase its target gene p27.
【总页数】5页(P120-124)
【作者】侯丽丽;程冰;张旭东;王丽;张林杰
【作者单位】安徽医科大学免疫学教研室,合肥,230032;合肥微尺度物质科学国家实验室,中国科学技术大学生命科学学院,合肥,230027;安徽医科大学免疫学教研室,合肥,230032;安徽医科大学免疫学教研室,合肥,230032;安徽医科大学免疫学教研室,合肥,230032
【正文语种】中文
【中图分类】R342.7;R329.25;R394;R739.5
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