Shandong University, Jinan, Shandong, P.R. China.
Faculty of Pharmacy, Bengbu Medical College, Bengbu, Anhui, P.R. China.
Int J Oncol. 2015 Oct;47(4):1379-92. doi: 10.3892/ijo.2015.3144. Epub 2015 Aug 31.
Chemoresistance is an inevitable occurrence in lung adenocarcinoma, which has been associated with decreased expression of the phosphatase and tensin homolog deleted on chromosome ten (PTEN). Therefore, it is important to identify novel molecular mechanisms to suppress chemoresistance in lung adenocarcinoma cells. Paclitaxel- and cisplatin-resistant A549 lung carcinoma cell derivatives were developed by long-term serial culture. The metastatic properties of the cells were assessed using wound-healing assays, migration assays, invasion assays, morphological examination, and western blot analysis/RT-PCR of genes associated with the epithelial-mesenchymal transition (EMT). To identify novel regulators of EMT in A549 cells, differentially expressed miRNAs in drug-resistant cells were identified by microarray analysis. The role of miR-181a was established by transfection with specific mimic and inhibitor followed by functional assays. Luciferase assays were performed to assess the ability of miR-181a to target the PTEN promoter, and regulation of PTEN expression by miR-181a was assessed by western blot analysis and RT-PCR. Paclitaxel- and cisplatin-resistant A549 cells acquired metastatic properties and EMT phenotype and had reduced PTEN expression as compared to sensitive cells. miR‑181a was identified as a differentially expressed miRNA in drug-resistant A549 cells, and miR-181a mimic and inhibitor were shown to affect migration, invasion, morphology and expression of EMT-associated genes. PTEN was identified as a direct target of miR-181a. Our findings demonstrate that miR-181a expression in lung adenocarcinoma is associated with EMT progression, potentially through targeting of PTEN. Regulation of miR-181a may provide a novel strategy for overcoming resistance to paclitaxel and cisplatin in lung adenocarcinoma.
化学耐药性是肺腺癌中不可避免的现象，它与磷酸酶和张力蛋白同源物缺失的染色体 ten（PTEN）表达降低有关。因此，重要的是要确定新的分子机制来抑制肺腺癌细胞的化学耐药性。通过长期连续培养开发了紫杉醇和顺铂耐药的 A549 肺癌细胞衍生物。使用划痕愈合测定、迁移测定、侵袭测定、形态学检查和与上皮-间充质转化（EMT）相关的基因的 Western blot 分析/RT-PCR 评估细胞的转移特性。为了鉴定 A549 细胞中 EMT 的新调节剂，通过微阵列分析鉴定药物耐药细胞中差异表达的 miRNAs。通过转染特异性模拟物和抑制剂并进行功能测定来确定 miR-181a 的作用。进行荧光素酶测定以评估 miR-181a 靶向 PTEN 启动子的能力，并通过 Western blot 分析和 RT-PCR 评估 miR-181a 对 PTEN 表达的调节。与敏感细胞相比，紫杉醇和顺铂耐药的 A549 细胞获得了转移特性和 EMT 表型，并且 PTEN 表达降低。miR-181a 被鉴定为药物耐药 A549 细胞中差异表达的 miRNA，miR-181a 模拟物和抑制剂被证明会影响迁移、侵袭、形态和 EMT 相关基因的表达。PTEN 被鉴定为 miR-181a 的直接靶标。我们的研究结果表明，肺腺癌中 miR-181a 的表达与 EMT 进展有关，可能通过靶向 PTEN。miR-181a 的调节可能为克服肺腺癌对紫杉醇和顺铂的耐药性提供一种新策略。
