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.