RESUMEN
MicroRNA (miRNA or miR)10b is an oncogenic miRNA associated with metastasis that is present in various types of tumor, including lung cancer. However, whether miR10b is involved in different malignant characteristics, such as drug resistance or stemness, remains unclear. Therefore, the present study investigated whether miR10b is an upstream regulator of p53. Ectopic expression of miR10bagomir decreased the expression of p53 and its downstream effectors, such as Bax and p53 upregulated modulator of apoptosis. Two noncanonical sites, including 1,5801,587 and 2,0292,035, located in p53 3'untranslated region (UTR) were affected by the presence of miR10b. In functional assays, upregulation of the p53 signaling pathway following cisplatin treatment was associated with decreased levels of miR10b and upregulation of the luciferase activity of wildtype, but not 1,584, 2,032dualmutant, p53 3'UTR. The ectopic expression of miR10bagomir attenuated the stability of p53 3'UTR and the expression of p53 and its downstream effectors induced by cisplatin. By contrast, the knockdown of miR10b induced the stability of p53 3'UTR and increased levels of p53 and the sensitivity of A549 cells to cisplatin treatment. Similar results were also observed for Beas 2B cells. In the clinical investigation, p53 exhibited two distinct associations (cocurrent and countercurrent) with miR10b in patients with lung cancer. Patients with lung cancer with low p53 and high miR10b levels exhibited the poorest prognosis, while those with high p53 and low miR10b exhibited the most favorable prognosis. These findings indicate a novel pathway in which cisplatin induces the levels of p53 by increasing mRNA stability via miR10b, indicating a novel oncogenic role of miR10b in promoting the malignant characteristics of nonsmall cell lung carcinoma.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/genética , Resistencia a Antineoplásicos , Neoplasias Pulmonares/genética , MicroARNs/genética , Proteína p53 Supresora de Tumor/genética , Regiones no Traducidas 3' , Células A549 , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Cisplatino/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
MicroRNAs (miRNAs/miRs) are known to play a key role in tumorigenesis and usually serve as therapeutic targets in cancer treatment. In the present study, the inhibitory effects and the targeting miRNAs of withaferin A (WA) were investigated in human lung cancer cells. Different lung cancer cell lines were administrated with different concentrations of WA for different time interval followed by western blot or reverse transcription-quantitative PCR analyses to determine the underlying signaling pathway. The results demonstrated that WA decreased the viability of lung cancer cells in a caspase-dependent manner. Further investigations indicated that treatment with WA induced the expression of proapoptotic molecules, p53 and Bax, and decreased Bcl-2 expression in A549 cells. Notably, the results demonstrated that WA also decreased the motility of lung cancer cells in a dose-dependent manner, at a relatively lower concentration. Western blot analysis revealed increased E-cadherin and decreased vimentin expression levels in lung cancer cells treated with WA. In addition, two oncomiRs, including miR-10b and miR-27a, which regulate the expression of E-cadherin and Bax, respectively, were downregulated in the presence of WA. The ectopic expression of miR-10b mimics was able to recover the WA-decreased motility of lung cancer cells, which was accompanied by a reduction in E-cadherin expression. Conversely, the ectopic expression of miR-27a mimics decreased the expression of Bax and recovered the viability of lung cancer cells attenuated by WA. In addition, the ectopic expression of p53-wild type decreased the expression levels of both miR-10b and miR-27a, whereas p53 knockdown induced their expression. Transient knockdown of p53 decreased the inhibitory effects of WA in the motility and viability of lung cancer cells, suggesting an association between WA-p53-miR-10b/27a and motility/viability. Further investigations demonstrated that p53 knockdown in lung cancer stable cell lines exhibited higher levels of both miR-10b and miR-27a, and higher motility and viability following treatment with WA. However, suppression of miR-10b and miR-27a effectively decreased motility and viability, respectively, following treatment with WA. Taken together, the results of the present study suggest that WA inhibits the functionality of lung cancer cells by decreasing the expression levels of both miR-10b and miR-27a in a p53-dependent manner.