Ren Hong, Zhang Yueqiao, Zhu Hongzhou
Department of Radiology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.
Department of Interventional, Zhejiang Cancer Hospital, Hangzhou, China.
Thorac Cancer. 2018 Mar;9(3):408-414. doi: 10.1111/1759-7714.12597. Epub 2018 Jan 29.
S-phase kinase-associated protein 2 (Skp2) takes great part in the development of multiple tumors. However, the post-transcriptional modulation mechanism of Skp2 remains unclear. Here, we present a new regulatory microRNA of Skp2, miR-339, which directly targets Skp2 to inhibit cell proliferation in lung cancer.
The expression of miR-339 or Skp2 in lung cancer samples was tested by real time-PCR. The correlation between miR-339 and Skp2 in lung cancer samples was analyzed by Pearson's correlation coefficient. The effect of miR-339 or anti-miR-339 on Skp2 was evaluated by immunoblotting. The luciferase reporter gene assay was used to test the targeting of miR-339 on Skp2. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide and colony formation analysis were applied to examine the function of miR-339 targeting Skp2 in lung cancer cells.
The negative correlation of miR-339 with Skp2 was found in clinical human lung cancer tissues. Furthermore, Skp2 expression was obviously abated by miR-339 in lung cancer A549 cells. Mechanistically, we used bioinformatics to predict that miR-339 could target the 3'-untranslated region of Skp2 mRNA. Luciferase reporter gene assay demonstrated that miR-339 could decrease the luciferase activities of the 3'-untranslated region vector of Skp2. In terms of function, ectopic miR-339 expression significantly suppressed cell proliferation in lung cancer. Overexpressed Skp2 accelerated miR-339-bated proliferation of lung cancer cells. MiR-339 inhibitor promoted cell proliferation in lung cancer, but Skp2 RNA interference reversed miR-339 inhibitor-driven cell proliferation.
MiR-339 targets the 3'-untranslated region of Skp2 mRNA to depress the proliferation of lung cancer cells.
S期激酶相关蛋白2（Skp2）在多种肿瘤的发生发展中起重要作用。然而，Skp2的转录后调控机制仍不清楚。在此，我们提出一种新的Skp2调控微小RNA，即miR-339，它直接靶向Skp2以抑制肺癌细胞增殖。
采用实时定量聚合酶链反应检测肺癌样本中miR-339或Skp2的表达。通过Pearson相关系数分析肺癌样本中miR-339与Skp2的相关性。采用免疫印迹法评估miR-339或抗miR-339对Skp2的影响。采用荧光素酶报告基因检测法检测miR-339对Skp2的靶向作用。应用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐和集落形成分析检测miR-339靶向Skp2在肺癌细胞中的功能。
在临床人肺癌组织中发现miR-339与Skp2呈负相关。此外，miR-339在肺癌A549细胞中明显降低Skp2的表达。机制上，我们利用生物信息学预测miR-339可靶向Skp2 mRNA的3'-非翻译区。荧光素酶报告基因检测表明，miR-339可降低Skp2 3'-非翻译区载体的荧光素酶活性。在功能方面，异位表达miR-339可显著抑制肺癌细胞增殖。过表达Skp2可加速miR-339抑制的肺癌细胞增殖。miR-339抑制剂可促进肺癌细胞增殖，但Skp2 RNA干扰可逆转miR-339抑制剂驱动的细胞增殖。
miR-339靶向Skp2 mRNA的3'-非翻译区以抑制肺癌细胞增殖。
