Li Juan, Li Ping, Chen Tengfei, Gao Ge, Chen Xiaonan, Du Yuwen, Zhang Ren, Yang Rui, Zhao Wei, Dun Shaozhi, Gao Feng, Zhang Guojun
Department of Respiratory Medicine, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, Henan, China.
Tumour Biol. 2015 Feb;36(2):685-92. doi: 10.1007/s13277-014-2698-y. Epub 2014 Oct 7.
MicroRNAs are implicated in the regulation of various cellular processes, including proliferation, differentiation, cell death, and cell mobility, and can function either as oncogenes or tumor suppressors in tumor progression. The effects of the expression of miR-96 in non-small cell lung cancer (NSCLC) remain unclear. In our study, qRT-PCR (quantitative reverse transcription PCR) was performed to identify the miR-96 expression level in 68 paired NSCLC and adjacent normal lung tissues. Trans-well, cell counting kit-8, and apoptosis assays were used to evaluate the effects of miR-96 expression on cell invasion, proliferation, and apoptosis. Dual-luciferase reporter assay and Western blotting were used to verify whether FOXO3 was a potential major target gene of miR-96. Finally, the effect of FOXO3 on miR-96-induced cell survival was determined by transfection of the genes expressing FOXO3 lacking 3'UTR and miR-96. The expression level of miR-96 in NSCLC tissues was higher than that in adjacent normal lung tissues, and this increased expression was significantly associated with lymph node metastasis. In contrast to the cells in the blank and negative control groups, the number of cells migrating through the matrigel was significantly lower and the incidence of apoptosis was significantly higher in cells transfected with a miR-96 inhibitor. Western blotting and dual-luciferase reporter assays demonstrated that miR-96 can bind to the putative seed region in FOXO3 mRNA 3'UTR, and can significantly lower the expression of FOXO3. The introduction of FOXO3 cDNA without 3'UTR restored miR-96 induced cell apoptosis and invasion. MiR-96 is up-regulated in NSCLC tissues. Downregulation of miR-96 inhibits invasion and promotes apoptosis in NSCLC cells A549 and SPC-A-1 by targeting FOXO3. Therefore, our study improves our understanding of the mechanisms underlying NSCLC pathogenesis and may promote the development of novel targeted therapies.
微小RNA参与调控多种细胞过程，包括增殖、分化、细胞死亡和细胞迁移，并且在肿瘤进展过程中既可以作为癌基因发挥作用，也可以作为肿瘤抑制因子发挥作用。miR-96在非小细胞肺癌（NSCLC）中的表达影响尚不清楚。在我们的研究中，采用qRT-PCR（定量逆转录PCR）检测68对NSCLC组织及其相邻正常肺组织中miR-96的表达水平。运用Trans-well实验、细胞计数试剂盒-8实验和凋亡实验评估miR-96表达对细胞侵袭、增殖和凋亡的影响。采用双荧光素酶报告基因实验和蛋白质免疫印迹法验证FOXO3是否为miR-96潜在的主要靶基因。最后，通过转染缺失3'UTR的FOXO3表达基因和miR-96，确定FOXO3对miR-96诱导的细胞存活的影响。NSCLC组织中miR-96的表达水平高于相邻正常肺组织，且这种表达增加与淋巴结转移显著相关。与空白对照组和阴性对照组的细胞相比，转染miR-96抑制剂的细胞穿过基质胶迁移的细胞数量显著减少，凋亡发生率显著升高。蛋白质免疫印迹法和双荧光素酶报告基因实验表明，miR-96可与FOXO3 mRNA 的3'UTR中的假定种子区域结合，并可显著降低FOXO3的表达。导入缺失3'UTR的FOXO3 cDNA可恢复miR-96诱导的细胞凋亡和侵袭。miR-96在NSCLC组织中上调。下调miR-96通过靶向FOXO3抑制NSCLC细胞A549和SPC-A-1的侵袭并促进其凋亡。因此，我们的研究增进了我们对NSCLC发病机制的理解，并可能促进新型靶向治疗的发展。
