Lung cancer remains the leading cause of cancer-related mortality for both men and women. Tumor recurrence and metastasis is the major cause of lung cancer treatment failure and death. The microRNA‑200 (miR-200) family is a powerful regulator of the epithelial-mesenchymal transition (EMT) process, which is essential in tumor metastasis. Nevertheless, miR-200 family target genes that promote metastasis in non-small cell lung cancer (NSCLC) remain largely unknown. Here, we sought to investigate whether the microRNA-200 family regulates our previously identified NSCLC prognostic marker genes associated with metastasis, as potential molecular targets. Novel miRNA targets were predicted using bioinformatics tools based on correlation analyses of miRNA and mRNA expression in 57 squamous cell lung cancer tumor samples. The predicted target genes were validated with quantitative RT-PCR assays and western blot analysis following re-expression of miR-200a, -200b and -200c in the metastatic NSCLC H1299 cell line. The results show that restoring miR-200a or miR-200c in H1299 cells induces downregulation of DLC1, ATRX and HFE. Reinforced miR-200b expression results in downregulation of DLC1, HNRNPA3 and HFE. Additionally, miR-200 family downregulates HNRNPR3, HFE and ATRX in BEAS-2B immortalized lung epithelial cells in quantitative RT-PCR and western blot assays. The miR-200 family and these potential targets are functionally involved in canonical pathways of immune response, molecular mechanisms of cancer, metastasis signaling, cell-cell communication, proliferation and DNA repair in Ingenuity pathway analysis (IPA). These results indicate that re-expression of miR-200 downregulates our previously identified NSCLC prognostic biomarkers in metastatic NSCLC cells. These results provide new insights into miR-200 regulation in lung cancer metastasis and consequent clinical outcome, and may provide a potential basis for innovative therapeutic approaches for the treatment of this deadly disease.

Smoking is responsible for 90% of lung cancer cases. There is currently no clinically available gene test for early detection of lung cancer in smokers, or an effective patient selection strategy for adjuvant chemotherapy in lung cancer treatment. In this study, concurrent coexpression with multiple signaling pathways was modeled among a set of genes associated with smoking and lung cancer survival. This approach identified and validated a 7-gene signature for lung cancer diagnosis and prognosis in smokers using patient transcriptional profiles (n=847). The smoking-associated gene coexpression networks in lung adenocarcinoma tumors (n=442) were highly significant in terms of biological relevance (network precision = 0.91, FDR<0.01) when evaluated with numerous databases containing multi-level molecular associations. The gene coexpression network in smoking lung adenocarcinoma patients was confirmed in qRT-PCR assays of the identified biomarkers and involved signaling pathway genes in human lung adenocarcinoma cells (H23) treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Furthermore, the western blotting results of p53, phospho‑p53, Rb and EGFR in NNK-treated H23 and transformed normal human lung epithelial cells (BEAS-2B) support their functional involvement in smoking-induced lung cancer carcinogenesis and progression.