在乳腺癌细胞中，miR-20a和miR-20b通过靶向RB1CC1/FIP200对自噬进行负调控。
MiR-20a and miR-20b negatively regulate autophagy by targeting RB1CC1/FIP200 in breast cancer cells.
作者信息
Li Shufeng, Qiang Qian, Shan Haitao, Shi Minke, Gan Guangming, Ma Fang, Chen Baojun
机构信息
Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Department of Biochemistry and Molecular Biology, Medical School of Southeast University, Nanjing 210009, China.
Key Laboratory of Developmental Genes and Human Disease in Ministry of Education, Department of Biochemistry and Molecular Biology, Medical School of Southeast University, Nanjing 210009, China.
出版信息
Life Sci. 2016 Feb 15;147:143-52. doi: 10.1016/j.lfs.2016.01.044. Epub 2016 Jan 29.
AIMS
RB1CC1/FIP200 was essential for autophagosome formation. Therefore, RB1CC1/FIP200 cellular levels are critical for the activation of the autophagy pathways. Following the screen of miRNAs affecting RB1CC1/FIP200 level and rapamycin-induced autophagy, we discovered miR-20a and miR-20b could regulate autophagy by targeting RB1CC1/FIP200.
MAIN METHODS
Inhibitory effect of miR-20a and 20b on basal and rapamycin-stimulated autophagy was demonstrated using various autophagic tests including GFP-LC3 puncta analysis, LC3II/LC3I gel shift and TEM observation.
KEY FINDINGS
We discovered RB1CC1/FIP200 as cellular targets of miR-20a and miR-20b. Upon miR-20a and miR-20b overexpression, both mRNA and protein levels of RB1CC1/FIP200 decreased. miR-20a and miR-20b target sequences present in the 3' UTR of RB1CC1/FIP200 mRNAs and introduction of mutations abolished the miR-20a and miR-20b responsiveness. In MCF7 and MDA-MB-231 breast cancer cells, miR-20a and miR-20b over-expression attenuated basal and rapamycin-induced autophagy; while suppression of miR-20a or miR-20b by specific antagomir showed normal rapamycin-induced autophagic activity.
SIGNIFICANCE
To our knowledge, this is the first study showing the significance of miR-20a and miR-20b regulating autophagy by targeting RB1CC1/FIP200.
目的
RB1CC1/FIP200对自噬体形成至关重要。因此，RB1CC1/FIP200的细胞水平对自噬途径的激活至关重要。在筛选影响RB1CC1/FIP200水平的微小RNA（miRNA）以及雷帕霉素诱导的自噬后，我们发现miR-20a和miR-20b可通过靶向RB1CC1/FIP200来调节自噬。
主要方法
使用包括绿色荧光蛋白-微管相关蛋白1轻链3（GFP-LC3）斑点分析、LC3II/LC3I凝胶迁移和透射电子显微镜（TEM）观察在内的各种自噬检测方法，证明了miR-20a和20b对基础和雷帕霉素刺激的自噬的抑制作用。
关键发现
我们发现RB1CC1/FIP200是miR-20a和miR-20b的细胞靶点。在miR-20a和miR-20b过表达时，RB1CC1/FIP200的mRNA和蛋白质水平均下降。RB1CC1/FIP200 mRNA的3'非翻译区（UTR）中存在miR-20a和miR-20b的靶序列，引入突变可消除miR-20a和miR-20b的反应性。在MCF7和MDA-MB-231乳腺癌细胞中，miR-20a和miR-20b的过表达减弱了基础和雷帕霉素诱导的自噬；而通过特异性拮抗剂抑制miR-20a或miR-20b则显示出正常的雷帕霉素诱导的自噬活性。
意义
据我们所知，这是第一项表明miR-20a和miR-20b通过靶向RB1CC1/FIP200调节自噬的重要性的研究。
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