Department of Pediatrics, Zhongshan Guzhen People's Hospital, Zhongshan, China.
Department of Emergency Medicine, Zhongshan Guzhen People's Hospital, Zhongshan, China.
Panminerva Med. 2022 Sep;64(3):374-383. doi: 10.23736/S0031-0808.20.03976-2. Epub 2020 Jun 4.
The aim of this study is to explore the effect of micro ribonucleic acid (miR)-181a on the radiosensitivity of non-small cell lung cancer (NSCLC) and its potential mechanism of action.
The differentially expressed miRNAs were screened in lung cancer tissues of radiotherapy-resistant and non-radiotherapy-resistant NSCLC patients, and verified via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Next, the effects of different miRNA expressions on patients' survival time were discussed, and target genes of miR-181a were predicted. The effect of miR-181a expression on radiosensitivity was determined using cell counting kit-8 (CCK-8) assay and flow cytometry. The direct target of miR-181a was verified via luciferase reporter assay. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) was overexpressed using lentiviruses, and then whether miR-181a reduces radiosensitivity via targeting PTEN was detected via CCK-8 assay and flow cytometry. Finally, Western blotting was performed to detect the protein expression of PTEN.
The screening results of microarray expression profile assay revealed that 15 miRNAs had significant differences in lung cancer tissues of radiotherapy-resistant NSCLC patients compared with those in non-radiotherapy-resistant NSCLC patients. The results of RT-qPCR showed that hsa-miR-181a, hsa-miR-199b, hsa-miR-489 and hsa-miR-589 were significantly up-regulated in the lung cancer tissues of radiotherapy-resistant NSCLC patients compared with those in non-radiotherapy-resistant NSCLC patients. In addition, it was found that the survival time of NSCLC patients was obviously prolonged in hsa-miR-181a low-expression group and hsa-miR-589 high-expression group, but hsa-miR-489 and hsa-miR-199b had no significant influence on the survival time of NSCLC patients. According to KEGG enrichment analysis, the target genes of miR-181a were evidently enriched in the phosphatidylinositol 3-hydroxy kinase (PI3K)/protein kinase B (AKT) signaling pathway, NSCLC signaling pathway and other cancer signaling pathways. Under the radiation dose of 2, 4, 6 and 8 Gy, the survival rate of A549 cells rose in miR-181a mimic group, but declined in miR-181a inhibitor group. Moreover, compared with that in model group, the radiotherapy-induced apoptosis was markedly inhibited in miR-181a mimic group, but markedly promoted in miR-181a inhibitor group. It was also observed that the response of cells to radiotherapy-induced apoptosis was remarkably weakened in miR-181a mimic + PTEN overexpression group compared with that in miR-181a mimic group. Finally, miR-181a mimic group had a significantly lower protein expression of PTEN and significantly higher protein expressions of CXC chemokine receptor 4 (CXCR4), phosphorylated signal transducer and activator of transcription 3 (p-STAT3), p-AKT1 and p-mammalian target of rapamycin (mTOR) than model group, while miR-181a inhibitor group had the opposite protein expressions. The protein expressions of CXCR4, p-STAT3, p-AKT1 and p-mTOR were obviously lower in miR-181a mimic + PTEN overexpression group than those in miR-181a mimic group.
MiR-181a reduces the radiosensitivity of NSCLC via inhibiting PTEN expression.
本研究旨在探讨微小 RNA（miR）-181a 对非小细胞肺癌（NSCLC）放射敏感性的影响及其潜在作用机制。
通过逆转录定量聚合酶链反应（RT-qPCR）筛选放疗抵抗和非放疗抵抗 NSCLC 患者肺癌组织中差异表达的 miRNAs，并进行验证。接下来，讨论了不同 miRNA 表达对患者生存时间的影响，并预测了 miR-181a 的靶基因。使用细胞计数试剂盒-8（CCK-8）检测和流式细胞术确定 miR-181a 表达对放射敏感性的影响。通过荧光素酶报告实验验证 miR-181a 的直接靶标。使用慢病毒过表达磷酸酶和张力蛋白同源物缺失的第十号染色体（PTEN），然后通过 CCK-8 检测和流式细胞术检测 miR-181a 是否通过靶向 PTEN 降低放射敏感性。最后，通过 Western blot 检测 PTEN 的蛋白表达。
微阵列表达谱检测的筛选结果显示，放疗抵抗 NSCLC 患者的肺癌组织中 15 种 miRNA 存在显著差异。RT-qPCR 结果显示，与非放疗抵抗 NSCLC 患者相比，放疗抵抗 NSCLC 患者的 hsa-miR-181a、hsa-miR-199b、hsa-miR-489 和 hsa-miR-589 明显上调。此外，研究发现 NSCLC 患者的 hsa-miR-181a 低表达组和 hsa-miR-589 高表达组的生存时间明显延长，但 hsa-miR-489 和 hsa-miR-199b 对 NSCLC 患者的生存时间没有显著影响。根据 KEGG 富集分析，miR-181a 的靶基因在磷脂酰肌醇 3-激酶（PI3K）/蛋白激酶 B（AKT）信号通路、NSCLC 信号通路和其他癌症信号通路中明显富集。在 2、4、6 和 8 Gy 的辐射剂量下，miR-181a 模拟物组 A549 细胞的存活率上升，但 miR-181a 抑制剂组的存活率下降。此外，与模型组相比，miR-181a 模拟物组中放射诱导的细胞凋亡明显受到抑制，但 miR-181a 抑制剂组中放射诱导的细胞凋亡明显受到促进。还观察到，与 miR-181a 模拟物组相比，miR-181a 模拟物+PTEN 过表达组的细胞对放射诱导的凋亡的反应明显减弱。最后，miR-181a 模拟物组的 PTEN 蛋白表达明显降低，而 CXC 趋化因子受体 4（CXCR4）、磷酸化信号转导和转录激活因子 3（p-STAT3）、p-AKT1 和哺乳动物雷帕霉素靶蛋白（mTOR）的蛋白表达明显升高，而模型组的蛋白表达则相反。与 miR-181a 模拟物组相比，miR-181a 模拟物+PTEN 过表达组的 CXCR4、p-STAT3、p-AKT1 和 p-mTOR 蛋白表达明显降低。
miR-181a 通过抑制 PTEN 表达降低 NSCLC 的放射敏感性。
