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. 2020 Jan 22;10(6):2553–2570. doi: 10.7150/thno.41120

Figure 2.

Figure 2

HIF-1a-mediated miR-1275 maintains stemness in vitro. (A) Sphere formation ability of stably transfected A549, H1299, and PC9 cells was examined (left panel) and quantified (right panel). Scale bar, 100 μm. (B) Overexpression of miR-1275 results in higher SP cell proportions, whereas the silencing of miR-1275 results in lower SP cell proportions. (C) RT-qPCR analysis for stemness-associated markers, including SOX2, OCT4, ABCG2, CD133, and ALDH1. (D) Subcellular localization of CD133 and ALDH1 was examined by immunofluorescence staining in the indicated cell lines. Magnification, ×200. Scale bar, 50 μm. (E) Predicted binding sites of HIF-1 on the miR-1275 promoter region (site 1, site 2, site 3). (F) Confirmation of HIF-1ɑ binding to miR-1275 promoter region by ChIP analysis in the indicated cells. (G) Pearson's correlation scatter plot of HIF-1α and miR-1275 expression in 20 LUAD clinical specimens. (H) RT-qPCR analysis showing HIF-1α knockdown by siRNA downregulates the levels of miR-1275. Each experiment was performed three times. Data are shown as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001.