HIF‐1A regulates miR‐210‐3p mediated CCL2 inhibition and monocyte infiltration in hypoxic A549 lung adenocarcinoma cells. (A) Immunoblots (left panel) with densitometric plots (right panel) showing the abundance of CCL2 and HIF‐1A proteins in A549 cells transfected with scramble or HIF‐1A RNAi plasmid. Data represent the mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, **P < 0.01 vs Scramble. (B,C) RT‐qPCR analysis of CCL2 gene expression (B), and ELISA showing CCL2 secretion into the culture medium (C) of A549 cells transfected with either scramble or HIF‐1A RNAi plasmid and incubated in 21% oxygen or 1% oxygen conditions for 24 h. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01. (D) Immunoblots (left panel) with densitometric plots (right panel) showing the abundance of CCL2 and HIF‐1A proteins in A549 cells transfected with scramble plasmid and HIF‐1A‐ΟΕ (overexpression) plasmid. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, ****P < 0.0001 vs Scramble. (E,F) RT‐qPCR analysis of relative CCL2 gene expression (E), and ELISA showing CCL2 secretion into the culture medium (F) of A549 cells transfected with either scramble plasmid or HIF‐1A‐OE (overexpression) plasmid and incubated in 21% oxygen or 1% oxygen conditions for 24 h. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ***P < 0.001, *P < 0.05, ns, nonsignificant. (G,H) Representative immunofluorescence images (G) and their quantifications (H) showing CCL2 (green) and HIF‐1A (red) protein levels in A549 cells transfected with scramble or HIF‐1A RNAi or HIF‐1A‐OE plasmid and incubated under normoxic and hypoxic conditions for 24 h, respectively. Scale bars: 15 μm. Data represent the mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ***P < 0.001, **P < 0.01. (I) RT‐qPCR analysis showing mir‐210‐3p gene expression in A549 cells transfected with scramble or HIF‐1A RNAi or HIF‐1A‐OE plasmid and incubated under normoxic and hypoxic conditions for 24 h, respectively. Data represent mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ****P < 0.0001, ***P < 0.001, *P < 0.05. (J) RT‐qPCR analysis showing relative CCL2 mRNA level in A549 cells transfected with scramble or HIF‐1A RNAi plasmid or HIF‐1A‐OE plasmid in the presence of miR‐210‐3p mimic (M) or miR‐210‐3p inhibitor (I) as indicated. Data represent mean ± SD of three independent experiments. Statistical significance was analyzed by one‐way ANOVA, **P < 0.01. (K,L) Representative images of THP‐1 monocytes migration (L) and their quantifications (K) from upper to lower surface of the transwell insert placed on the well containing A549 cells transfected with scramble or HIF‐1A RNAi or HIF‐1A‐OE plasmid in the presence of miR‐210‐3p mimic (M) or miR‐210‐3p inhibitor (I) as indicated. Scale bars: 50 μm. Data represent mean ± SD of three independent experiments. Statistical significance was analyzed by one‐way ANOVA, ****P < 0.0001.
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