Inhibition of microRNA-196a might reverse cisplatin resistance of A549/DDP non-small-cell lung cancer cell line.

We aimed to explore the possible mechanism of microRNA-196a (miR-196a) inhibition and reversion of drug resistance to cisplatin (DDP) of the A549/DDP non-small-cell lung cancer (NSCLC) cell line. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expression differences of miR-196a in the drug-resistant A549/DDP NLCLC cell line and the parental A549 cell line, and expressions of miR-196a in the A549/DDP NLCLC cell line transfected with miR-196a inhibitor (anti-miR-196a group) and the miR-196a negative control (miR-NC) group and blank group (without transfection). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was applied in examining the cell viability of A549/DDP cell line before and after transfection. Clonogenic assay was used to detect cell proliferation ability. Flow cytometry was applied in detecting apoptosis rate of assayed tumor cell and rhodamine-123 changes in cells. Western blot was applied in detecting proteins of drug-resistant related gene in A549/DDP cell line. Significantly higher expression of miR-196a was detected in the drug-resistant A549/DDP cell line than that in the parental A549 cell line (P < 0.05). However, miR-196a expression in the anti-miR-196a group decreased obviously compared to that in the blank group and the miR-NC group (both P < 0.05); The value of IC50 in the anti-miR-196a group showed remarkably lower than that in the blank group and the miR-NC group (both P < 0.05); Rh-123 absorbing ability in the anti-miR-196a group increased 2.51 times and 2.49 times respectively compared to that in the blank group and the miR-NC group (both P < 0.05). No statistical differences in the apoptosis rate of A549/DDP cell line in the early stage were found among the three groups (all P > 0.05), but the late-stage apoptosis rate in the anti-miR-196a group was significantly higher than that in the blank group and the miR-NC group (both P < 0.05); The expressions of human multidrug resistance 1 (MDR1), multidrug resistance protein 1 (MRP1), excision repair cross-complementation 1 (ERCC1), survivin, and B cell lymphoma 2 (Bcl-2) decreased significantly while RhoE increased significantly in the anti-miR-196a group than the blank group and the miR-NC group (all P < 0.05). Inhibition of miR-196a could reverse cisplatin resistance of A549/DDP cell lines, which might relate with inhibition of drug efflux, down-regulation of drug-resistant protein expression, cell apoptosis, and cell proliferation suppression.

TUSC3 induces autophagy in human non-small cell lung cancer cells through Wnt/ß-catenin signaling.

We investigated the effects of tumor suppressor candidate 3 (TUSC3) on autophagy in human non-small cell lung cancer (NSCLC) cells. A total of 118 NSCLC patients (88 males and 30 females) who underwent surgery at our institute were enrolled in the study. Immunohistochemical analysis revealed that TUSC3 protein expression was lower in NSCLC specimens than adjacent normal tissue. Correspondingly, there was greater methylation of TUSC3 in NSCLC than adjacent normal tissue. After transient transfection of A549 NSCLC cells with constructs designed to up-regulate or down-regulate TUSC3 expression, we analyzed the effects of inhibiting the Wnt pathway (XAV939) and autophagy (chloroquine, CQ) on the behavior of NSCLC cells. We also performed TOP/FOP-Flash reporter assays, MTT assays, Annexin V-FITC/propidium iodide staining, and acridine orange staining to evaluate Wnt/ß-catenin signaling, cell proliferation, apoptosis, and autophagy, respectively. Expression of Wnt/ß-catenin pathway components and autophagy-related proteins was analyzed using qRT-PCR and Western blotting. We found that TUSC3 inhibited cell proliferation and promoted both apoptosis and autophagy in A549 cells. In addition, TUSC3 increased expression of autophagy-related proteins. It also increased expression of Wnt/ß-catenin signaling pathway components and promoted nuclear transfer of ß-catenin, resulting in activation of Wnt/ß-catenin signaling. TUSC3 thus induces autophagy in human NSCLC cells through activation of the Wnt/ß-catenin signaling pathway.