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. 2019 Aug 23;16(12):1721–1732. doi: 10.1080/15476286.2019.1657351

Figure 7.

Figure 7.

Restoration of miR-146a expression in A549 cells results in decreased production and release of CCL2. (a) The NSCLC KM Plotter Tool (www.kmplot.com) was used to generate survival curves based on a patient’s overall survival in months and their CCL2 expression level (low or high) relative to the median value. No significant correlation between CCL2 expression and overall survival in 524 lung squamous cell carcinoma patients was observed (p = 0.12), but there was a highly significant correlation between high CCL2 expression and overall survival in 720 lung adenocarcinoma patients (p = 2.3 x 10−4). (b) A549, H1975, H1299, and Beas2B cells were allowed to grow undisturbed for 48 hours. Cell-free, serum-free supernatants were collected, then an enzyme-linked immunosorbent assay (ELISA) was used to measure baseline CCL2 concentrations. A549 cells produced much higher amounts of CCL2 compared to H1975, H1299, and Beas2B cells. H1975 cells produced less CCL2 than A549 cells, but still significantly more than H1299 and Beas2B cells. Assay Buffer A was used as a negative control. (*) p < 0.015, n= 4; (**) p < 0.0001, n= 4. (c) A549 cells were transiently transfected with miRNA mimics at a 50 nM concentration. Cells were lysed and RNA was isolated 48 hours post-transfection. CCL2 expression was normalized to GAPDH mRNA. CCL2 mRNA levels were significantly different in cells transfected with miR-146a from levels in cells transfected with 50 nM of non-targeting miRNA. Co-transfection of the miRNA mimics with a HuR expression vector, pcDNA3.1(+)-HuR-FLAG, could rescue CCL2 mRNA levels, even in the presence of miR-146a. (*) p < 0.05, n = 3; (**) p < 0.01, n = 3. (d) Cell-free, serum-free supernatants were collected 48 hours post-transfection. ELISA analysis indicated that A549 cells transfected with synthetic miR-146a produced significantly less CCL2 compared to mock-treated cells and cells transfected with a non-targeting miRNA. Each sample’s CCL2 concentration was normalized to its total protein concentration. (*) p < 0.025, n= 3.