Amri Jamal, Molaee Neda, Karami Hadi
Molecular and Medicine Research Center, Faculty of Medicine, Arak University of Medical Sciences, Arak, Iran.
Traditional and Complementary Medicine Research Center, Faculty of Medicine, Arak University of Medical Sciences, Arak, Iran.
Asian Pac J Cancer Prev. 2019 Nov 1;20(11):3361-3367. doi: 10.31557/APJCP.2019.20.11.3361.
Despite the dramatic efficacy of erlotinib, an EGFR tyrosine kinase inhibitor (TKI), most of non-small cell lung cancer (NSCLC) patients ultimately acquire resistance to this agent. Different studies indicated that miRNA-125a-5p is down-regulated in human lung cancer cells and may function as a tumor suppressor by targeting EGFR. However, the biological function of miRNA-125a-5p in NSCLC resistance to EGFR-TKIs is not fully understood. In this study the effect of miRNA-125a-5p on cell proliferation, apoptosis and sensitivity of the A549 lung cancer cells to erlotinib was investigated.
After miRNA-125a-5p transfection, the expression levels of EGFR mRNA were measured by QRT-PCR. Trypan blue assays were performed to evaluate the proliferation of the A549 lung cancer cells. The cytotoxic effects of miRNA-125a-5p and erlotinib, alone and in combination, were determined using MTT assay. Combination index study was performed using the method of Chou-Talalay. Apoptosis was assessed using an ELISA cell death assay kit.
MiRNA-125a-5p clearly reduced the expression of EGFR mRNA in a time dependent manner, causing marked cell proliferation inhibition and spontaneous apoptosis (p<0.05, relative to control). Pretreatment with miRNA-125a-5p synergistically increased the cytotoxic effect of erlotinib and decreased its IC50. Furthermore, miRNA-125a-5p significantly enhanced the apoptotic effect of erlotinib. Negative control miRNA had no significant effect on biological parameter of the tumor cells.
Our data suggest that suppression of EGFR by miRNA-125a-5p can effectively trigger apoptosis and overcome EGFR-TKs resistance of lung cancer cells. Therefore, miRNA-125a-5p may be a potential therapeutic adjuvant in patients with lung cancer.
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尽管表皮生长因子受体（EGFR）酪氨酸激酶抑制剂（TKI）厄洛替尼疗效显著，但大多数非小细胞肺癌（NSCLC）患者最终会对该药物产生耐药性。不同研究表明，miRNA - 125a - 5p在人肺癌细胞中表达下调，可能通过靶向EGFR发挥肿瘤抑制作用。然而，miRNA - 125a - 5p在NSCLC对EGFR - TKIs耐药中的生物学功能尚未完全明确。本研究探讨了miRNA - 125a - 5p对A549肺癌细胞增殖、凋亡及对厄洛替尼敏感性的影响。
转染miRNA - 125a - 5p后，采用实时定量聚合酶链反应（QRT - PCR）检测EGFR mRNA表达水平。进行台盼蓝试验评估A549肺癌细胞的增殖情况。采用噻唑蓝（MTT）法测定miRNA - 125a - 5p和厄洛替尼单独及联合使用时的细胞毒性作用。采用Chou - Talalay方法进行联合指数研究。使用ELISA细胞死亡检测试剂盒评估凋亡情况。
miRNA - 125a - 5p能明显以时间依赖性方式降低EGFR mRNA表达，导致显著的细胞增殖抑制和自发凋亡（相对于对照组，p < 0.05）。miRNA - 125a - 5p预处理可协同增强厄洛替尼的细胞毒性作用并降低其半数抑制浓度（IC50）。此外，miRNA - 125a - 5p显著增强了厄洛替尼的凋亡作用。阴性对照miRNA对肿瘤细胞的生物学参数无显著影响。
我们的数据表明，miRNA - 125a - 5p抑制EGFR可有效诱导凋亡并克服肺癌细胞对EGFR - TK的耐药性。因此，miRNA - 125a - 5p可能是肺癌患者潜在的治疗辅助药物。
