lncRNA⁃DINO靶向miR⁃141调控Keap1⁃Nrf2通路促进非小细胞肺癌化疗耐药的机制研究

lncRNA-DINO targets miR-141 to regulate the Keap1-Nrf2 pathway to promote non-small cells study on the mechanism of chemoresistance of lung cancer cells

目的研究lncRNA⁃DINO在非小细胞肺癌(NSCLC)化疗耐药中的作用机制。方法对比耐药的和敏感的NSCLC临床标本,用RT⁃PCR及Western实验检测DINO、miR⁃141、Keap1、Nrf2的表达,探索DINO、miR⁃141的表达与化疗耐药的关系,其次,将A549细胞株和A549/DDP细胞株进行细胞培养,使用Trizol法提取总RNA,用LncRNA芯片对比检测两组细胞株间LncRNA的表达差异。通过在线软件(RNAhybrid)预测和计算,评估DINO与miR⁃141结合可能性的高低。最后将A549细胞株和A549/DDP细胞株进行细胞培养,提取总RNA,RT⁃PCR验证两组细胞株间的DINO、miR⁃141、Keap1、Nrf2、HO⁃1、NQO1。结果耐药的A549/DDP细胞株中DINO表达较敏感的A549细胞显著降低(P<0.05),且DINO是通过与miR⁃141结合而发挥作用的。RT⁃PCR对比检测发现,与A549细胞相比,过表达miR⁃141的A549⁃miR⁃141细胞中Keap1基因表达明显下降(P<0.05),而PTEN和MSH2表达无明显变化。与A549细胞相比,A549⁃miR⁃141细胞中Keap1基因的表达下调,而Nrf2基因和其下游HO⁃1、NQO1基因表达显著上调(P<0.05)。结论非小细胞肺癌中lncRNA⁃DINO可靶向结合miR⁃141,进而调控Keap1⁃Nrf2通路而导致细胞的化疗耐药。

Objective To study the mechanism of lncRNA-DINO in chemoresistance of non-small cell lung cancer(NSCLC).Methods compare drug-resistant and sensitive NSCLC clinical specimens,detect the expression of DINO,miR-141,Keap1 and Nrf2 by RT-PCR and Western experiment,and explore the relationship between the expression of DINO and miR-141 and chemotherapy resistance.Secondly,A549 cell line and A549/DDP cell line were cultured,and total RNA was extracted by Trizol(Invitrogen company),The expression difference of lncrna between the two groups of cell lines was detected by lncrna chip.Through the prediction and calculation of online software(rnahybrid),the possi-bility of DINO binding with miR-141 was evaluated.Finally,A549 cell line and A549/DDP cell line were cultured.Total RNA was extracted by Trizol(Invitrogen company).The DINO,miR-141,Keap1,Nrf2,HO-1 and NQO1 between the two groups were verified by RT-PCR.Results the expression of DINO in drug⁃resistant A549/DDP cell lines was significantly lower than that in sensitive A549 cells(P<0.05),and DINO played a role by binding to miR⁃141.RT⁃PCR showed that compared with A549 cells,the expression of Keap1 gene in a549⁃mir⁃141 cells overexpressing miR⁃141 decreased significantly(P<0.05),while the expression of PTEN and MSH2 did not change significantly.Compared with A549 cells,the expression of Keap1 gene was down⁃regulated in a549⁃mir⁃141 cells,while the expression of Nrf2 gene and its downstream HO⁃1 and NQO1 genes were significantly up⁃regulated(P<0.05).Conclusion In non⁃small cell lung cancer,lncRNA⁃DINO can target and bind miR⁃141,and then reg⁃ulate keap1⁃nrf2 pathway,resulting in chemoresistance.