The p53/miR-193a/EGFR feedback loop function as a driving force for non-small cell lung carcinoma tumorigenesis
The p53/miR-193a/EGFR feedback loop function as a driving force for non-small cell lung carcinoma tumorigenesis
复制标题
DOI：
10.1177/1758835919850665
复制
发表时间：
2019-05-01
影响因子：
4.9
通讯作者：
Pan, Yue-Yin
中科院分区：
文献类型：
--
作者：
Wang, Wei;Shen, Xia-Bo;Pan, Yue-Yin
背景：非小细胞肺癌(NSCLC)治愈率低、致死率高，是严重威胁人类健康的世界性疾病。越来越多的证据表明，表皮生长因子受体(EGFR)在肿瘤的发生和发展中起着重要的作用，被认为是一种重要的癌症驱动蛋白。然而，在非小细胞肺癌发生过程中，EGFR的表达是如何被调节的还没有完全阐明。方法：分别用Western blotting和qRT-PCR方法检测NSCLC临床标本中EGFR蛋白的表达。结合microRNA(MiRNA)靶标预测软件和下拉试验，我们预测了靶向EGFR的microRNAs(MiRNAs)。接下来，使用三个非小细胞肺癌细胞系，A549(P53 WT)，H322(p53突变体)和H1299(p53缺失)，证明了miR-193a直接靶向EGFR。此外，我们还利用细胞计数试剂盒(EDU)、Transwell和细胞凋亡实验研究了miR-193a体外抑制EGFR的生物学效应。然后，利用芯片和荧光素酶分析，我们证明了miR-193a在转录水平上被p53直接激活，并且p53诱导的miR-193a与EGFR形成了一个双负反馈环。结果：我们发现在非小细胞肺癌中EGFR基因和蛋白表达上调。我们预测EGFR是miR-193a的靶标，并证实miR-193a直接与EGFR mRNA的3‘-UTR结合。此外，miR-193a通过直接下调EGFR，抑制NSCLC的增殖和侵袭，促进NSCLC的凋亡。然后，我们证明了P53直接激活miR-193a的转录，而EGFR作为转录抑制因子负调控miR-193a的表达，形成一个反馈环。该环促进了NSCLC细胞的增殖和迁移，并加速了异种移植小鼠的肿瘤生长。结论：本研究强调了非小细胞肺癌患者的双负反馈回路。反馈环是至关重要的，因为过度表达EGFR强烈地加速了肿瘤的生长，而miR-193a修复抑制了体内的肿瘤生长。我们的发现与一种新的观点是一致的，即miRNAs和蛋白质调节器在关键的生物过程中形成调控网络，它们的调节失调会导致细胞功能障碍。总之，这项研究对非小细胞肺癌进展的分子机制提供了重要的见解，并可能有助于开发治疗非小细胞肺癌的新疗法。
Background: Non-small cell lung carcinoma (NSCLC) is a major worldwide health threat due to its low cure rate and high lethality. Emerging evidence suggests that epidermal growth factor receptor (EGFR) plays vital roles in cancer initiation and progression, and is considered an important cancer-driving protein. However, how EGFR expression is regulated during NSCLC development remains to be fully elucidated. Methods: In NSCLC clinical samples, EGFR protein levels were measured by western blotting and qRT-PCR, respectively. Combining microRNA (miRNA) target prediction software and the pulldown assay, we predicted microRNAs (miRNAs) that targeted EGFR. Next, three NSCLC cell lines, A549 (p53 WT), H322 (p53 mutant), and H1299 (p53 null), were used to demonstrate the direct targeting of EGFR by miR-193a. In addition, we investigated the biological effects of EGFR inhibition by miR-193a in vitro using Cell Counting Kit-8, 5-Ethynyl-2 '-deoxyuridine (EdU), transwell, and apoptosis assays. Then, using ChIP and luciferase assays, we demonstrated that miR-193a was directly activated by p53 at the transcriptional level and that p53-induced-miR-193a and EGFR form a double-negative feedback loop. Results: We found that EGFR mRNA and protein were upregulated in NSCLC. We predicted that EGFR was a target of miR-193a and validated that miR-193a bound directly to the 3 '-UTR of the EGFR mRNA. Moreover, miR-193a inhibited NSCLC proliferation and invasion, and promotes NSCLC apoptosis by directly downregulating EGFR. Then, we demonstrated that p53 directly activated miR-193a transcription, whereas EGFR functioned as a transcriptional repressor to negatively control miR-193a expression, forming a feedback loop. The loop promoted NSCLC cell proliferation and migration and accelerated tumor growth in xenograft mice. Conclusions: This study highlights a double-negative feedback loop in NSCLC. The feedback loop is crucial because overexpressing EGFR strongly accelerated tumor growth, while miR-193a restoration blocked tumor growth in vivo. Our findings are in line with the emerging opinion that miRNAs and protein regulators form regulatory networks in critical biological processes and that their dysregulation can lead to cellular dysfunction. In conclusion, this study provides important insights into the molecular mechanisms of NSCLC progression and may help inform the development of new therapeutics for managing NSCLC.