Fig 1: Predicted miR‐1179 targets in NSCLC cells and in NSCLC patients. (A) Expression of the predicted miR‐1179 targets CCNE1, NUAK2 and SPOCK1 was measured in miR‐1179 mimic‐transfected NCI‐H1993 and NCI‐H2073 cells. Fold changes relative to control cells are shown. *p < 0.05. (B) Stably transfected A549pCMV‐miR‐1179 cells were treated with an miR‐1179 inhibitor, and RT‐PCR showed upregulated expression of CCNE1, NUAK2 and SPOCK1. *p < 0.05. (C) Western blot analysis of cyclin E1 in NCI‐H1993 and NCI‐H2073 cells transfected with control RNA (–) or miR‐1179 (+). (D) Luciferase activity in cells that were cotransfected with a luciferase reporter construct containing either the wild‐type (WT) or the mutated (MUT) 3′‐UTR of CCNE1 and with miR‐1179 mimics. RLU, relative luminescence units.
Fig 2: MS‐HRM analyses of the (A) miR‐10b, (B) miR‐129‐2, (C) miR‐137, (D) miR‐572, (E) miR‐1179 and (F) miR‐3150b genes in TU samples and NL samples from 104 NSCLC patients. Statistically significant differences in miRNA methylation between TU samples and NL samples were found, and are summarized by violin plots. In addition, ROC curves demonstrating the discrimination between TU samples and NL samples of 104 NSCLC patients on the basis of miRNA methylation are shown. ***p < 0.0001.
Fig 3: The top 30 predicted targets of miR‐1179 and their relationships with certain molecular pathways. Targets are ranked, on the basis of their prediction score, from red (highest score) to light blue (lowest score).
Fig 4: Effect of epigenetically active drugs on miR‐1179 gene expression and of miR‐1179 transfection on cell viability/proliferation of NSCLC cells. (A) A549 cells were treated with either Aza‐dC, TSA, or a combination of Aza‐dC and TSA. miR‐1179 gene expression was found to be upregulated in drug‐treated cells, as determined by RT‐PCR. The fold change in expression of drug‐treated cells as compared with untreated cells is shown. *p < 0.05; ***p < 0.001; ns, not significant. (B) Reduced viability of miR‐1179 mimic‐transfected NCI‐H2073 cells as compared with control cells was observed. Experiments were performed in triplicate. Error bars indicate standard deviations. RFU, relative fluorescence units; *p < 0.05; **p < 0.01. (C) Reduced proliferation of miR‐1179 mimic‐transfected NCI‐H2073 cells as compared with controls was found with the xCELLigence RTCA system. Cells were plated in triplicate. Error bars indicate standard deviations. (D) Increased proliferation of stably miR‐1179‐transfected A549 cells treated with miR‐1179 inhibitors as compared with controls was found with the xCELLigence RTCA system. Cells were plated in triplicate. Error bars indicate standard deviations. Cell index, quantitative measure of cell number.
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