长链非编码RNABRE-AS1在非小细胞肺癌中的表达及其调控功能

Expression of long non-coding RNA BRE-AS1 and its function in non-small cell lung cancer

  • 摘要:
    背景 非小细胞肺癌(NSCLC)是严重危害人类健康的恶性肿瘤, 长链非编码RNA (lncRNA)可能在其发生发展中发挥重要作用。
    目的 探讨lncRNA BRE-AS1在NSCLC中的表达、生物学功能和调控作用, 为进一步了解NSCLC发病机制提供理论依据。
    方法 利用实时定量聚合酶链反应(RT-qPCR)技术对91例来自南京胸科医院的原发性NSCLC患者的癌组织和癌旁组织以及NSCLC细胞中BRE-AS1表达水平进行检测, 应用TCGA RNASeqV2系统分析TCGA数据库中BRE-AS1在1 016例NSCLC组织和110例正常组织中的表达水平。应用受试者工作特征(ROC)曲线评价BRE-AS1对NSCLC的诊断价值, 应用t检验分析不同临床特征NSCLC患者的BRE-AS1表达水平。采用慢病毒转染在NSCLC细胞A549中构建BRE-AS1过表达稳转细胞株, 分别以CCK8法和流式细胞术检测BRE-AS1对细胞增殖、周期及凋亡的影响。通过功能富集分析BRE-AS1的下游信号通路并用Western blotting法检测信号通路中关键蛋白表达水平。
    结果 RT-qPCR和TCGA数据库分析结果显示BRE-AS1在NSCLC组织RT-qPCR: 差异表达倍数(FC)=-13.15, TCGA: FC=-4.85, P < 0.05和NSCLC细胞A549(FC=-4.87, P < 0.05)中表达下调。ROC分析结果显示BRE-AS1对NSCLC具有较强的诊断能力(NSCLC组织: 曲线下面积为0.916, 95%CI: 0.872~0.960;TCGA数据库: 曲线下面积为0.853, 95%CI: 0.809~0.897)。CCK8结果显示, 在A549细胞中过表达BRE-AS1后, 细胞增殖能力在24、36、48 h降低(P < 0.01);同时流式细胞术分析显示, 过表达BRE-AS1阻滞A549细胞周期于G2/M期, 并诱导细胞凋亡率上升(P < 0.05)。功能富集分析发现, 磷酸肌醇3-激酶(PI3K)-蛋白激酶B (AKT)-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路与NSCLC发生发展相关(P < 0.05), 进一步Western blotting法分析发现, 过表达BRE-AS1后, A549细胞中AKT3、p-AKT3、mTOR、p-mTOR表达以及p-AKT3/AKT3、p-mTOR/mTOR均下降, PTEN表达上升, 提示过表达BRE-AS1可抑制A549细胞中PI3K-AKT-mTOR信号通路。
    结论 BRE-AS1在NSCLC中表达下调, 具有较高诊断价值。BRE-AS1可能通过抑制PI3KAKT-mTOR信号传导途径降低A549细胞生长活力, 阻滞细胞周期, 诱导细胞凋亡, 进而影响NSCLC发生发展。

     

    Abstract:
    Background Non-small cell lung cancer (NSCLC) is a malignant tumor which seriously affects human health. Long non-coding RNA (lncRNA) may play an important role in its occurrence and development.
    Objective This study is designed to explore the expression, biological function, and regulation mechanism of lncRNA BRE-AS1 in NSCLC and provide a theoretical basis for further understanding of the pathogenesis of NSCLC.
    Methods Real-time quantitative polymerase chain reaction (RT-qPCR) technology was used to detect the expression level of BRE-AS1 in NSCLC cell lines and in tumor and adjacent non-tumor tissues from 91 NSCLC patients of Nanjing Chest Hospital. The TCGA RNASeqV2 system was used to analyze the expression level of BRE-AS1 in 1 016 NSCLC tissues and 110 normal lung tissues from the TCGA database. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of BRE-AS1 for NSCLC, and t test was used to compare the expression levels of BRE-AS1 in NSCLC patients with different clinical characteristics. Lentiviral transfection was used to construct a stable BRE-AS1 overexpression cell strain in NSCLC cells A549, and the effects of BRE-AS1 on cell proliferation, cycle, and apoptosis were detected by CCK8 and flow cytometry assay. The potential signaling pathways regulated by BRE-AS1 were evaluated by functional enrichment analysis and the expression levels of key proteins in the signaling pathways were detected by Western blotting.
    Results The results of RT-qPCR and TCGA database analysis showed that the expression of BRE-AS1 was significantly down-regulated in NSCLC tissuesRT-qPCR: fold change (FC)=-13.15, TCGA: FC=-4.85, P < 0.05 and NSCLC cells A549 (FC=-4.87, P < 0.05). The results of ROC analysis showed that the expression levels of BRE-AS1 distinguished between tumor and non-tumor with high diagnostic power (tissues of NSCLC patients: area under curve=0.916, 95% CI: 0.872-0.960; TCGA database: area under curve=0.853, 95% CI: 0.809-0.897). The results of CCK8 showed that the cell proliferation ability was significantly reduced at 24, 36, and 48 h after overexpression of BRE-AS1 in A549 cells (P < 0.01). The results of flow cytometry analysis showed that overexpression of BRE-AS1 arrested A549 cell cycle at G2/M phase and induced an increase of cell apoptosis rate (P < 0.05). The results of functional enrichment analysis found that phosphatidylinositide 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) signaling pathway was significantly related to the occurrence and development of NSCLC (P < 0.05). The Western blotting results showed that overexpression of BRE-AS1 in A549 cells decreased the expressions of AKT3, p-AKT3, mTOR, p-mTOR, p-AKT3/AKT3, and p-mTOR/mTOR, and increased the expression of PTEN, suggesting that overexpression of BRE-AS1 inhibit the PI3K-AKT-mTOR signaling pathway in A549 cells.
    Conclusion BRE-AS1 is significantly down-regulated in NSCLC and has a high diagnostic value. BRE-AS1 may reduce A549 cell viability, block cell cycle, and induce cell apoptosis by inhibiting the PI3K-AKT-mTOR signaling pathway to affect NSCLC occurrence and development.

     

/

返回文章
返回