中华皮肤科杂志 ›› 2010, Vol. 43 ›› Issue (5): 358-361.

• 论著 • 上一篇    下一篇

let-7a微小RNA在黑素瘤中的表达及其对A375细胞增殖的影响

王震英1,王焱2,薛燕宁3,徐秀莲2,曾学思2,孙建方2   

  1. 1. 山东省立医院皮肤科
    2. 南京 中国医学科学院北京协和医学院皮肤病研究所
    3. 江苏省中医院皮肤科
  • 收稿日期:2009-10-27 修回日期:2009-12-15 出版日期:2010-05-15 发布日期:2012-04-12
  • 通讯作者: 王震英 E-mail:zhenyingwang2002@yahoo.com.cn

Expression of microRNA let-7a in melanoma and its effect on the proliferation of A375 human melanoma cell line

2,Xiu-Lian Xu2,   

  • Received:2009-10-27 Revised:2009-12-15 Online:2010-05-15 Published:2012-04-12

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摘要：

目的 探讨甲醛固定石蜡包埋的黑素瘤组织中let-7a微小RNA（microRNA）的表达情况及其对黑素瘤A375细胞增殖的影响。方法 选取黑素瘤蜡块13份，先天性色素痣蜡块10份，抽提microRNA，采用茎环引物逆转录为cDNA，然后采用Taqman MGB探针，相应的引物，进行实时荧光定量PCR。继而采用cy3标记的siRNA阴性对照物转染A375细胞，荧光显微镜下观察转染效率，最后将let-7a的模拟物转染入A375细胞，MTT实验检测转染后细胞增殖的变化。结果 从石蜡包埋组织中成功抽提出microRNA，而且定量PCR结果显示let-7a在黑素瘤中的表达明显低于色素痣组织（t = 2.364，Ｐ < 0.05）。荧光显微镜下观察siRNA转染效率约80% ~ 90%，hsa-let-7a转染A375细胞后，使其增殖受抑，转染后24 h抑制率达32.7%。结论 let-7a在黑素瘤中表达下降，过表达let-7a可使A375细胞增殖受到抑制，提示其在黑素瘤中可能起抑癌基因样作用。

关键词: 细胞增殖

Abstract:

Objective To investigate the expression of microRNA let-7a in formalin-fixed paraffin-embedded （FFPE） melanoma tissue and its effect on the proliferation of A375 cell lines. Methods Tissue samples were obtained from 13 patients with malignant melanoma and 10 with congenital pigmented nevus, then fixed with formalin and embeded with paraffin. microRNAs were isolated from these samples and reversely transcripted to cDNA with stem-loop primer. Then, real time quantitative PCR was performed with Taqman MGB probe and matched primers to measure the expression of microRNA let-7a. cy3-Labeled negative control siRNA was transfected into A375 cells followed by the examination of transfection efficiency under fluorescense microscopy. Subsequently, hsa-let-7a mimics were transfected into A375 cells to observe their effect on the proliferation of these cells. Results microRNAs were successfully isolated from FFPE tissues, and quantitative PCR showed a significant reduction in the expression of microRNA let-7a in melanoma tissue compared with nevus tissue（t = 2.364, P < 0.05）. The transfection efficiency was found to be about 80% ~ 90%. After transfection with hsa-let-7a mimics, the proliferation of A375 cells was inhibited and the inhibition rate amounted to 32.7% at 24 hours. Conclusions The expression of microRNA-let-7a is attenuated in melanoma, and overexpression of let-7a can inhibit the proliferation of A375 cells, which implies that let-7a functions as a tumor suppressor gene in melanoma.

Key words: cell proliferation