Hypoxia‐induced miR‐210‐3p directly inhibits CCL2 expression attenuating monocyte infiltration in lung adenocarcinoma cell lines. (A) RT‐qPCR analysis indicating relative abundance of CCL2 mRNA levels in A549 and NCI‐H460 cell lines incubated with indicated levels of oxygen for 24 h. GAPDH was used as an internal loading control for normalization. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, **P < 0.01 vs 21% oxygen. (B) Immunoblots showing the abundance of CCL2 and HIF‐1A proteins in A549 and NCI‐H460 cells treated with indicated concentrations of CoCl2. β‐Actin was served as a loading control. (C,D) Representative immunofluorescence images (20×, scale bars: 50 μm and 63×, scale bars: 10 μm) (C) and their quantifications (D) showing HIF‐1A (red) and CCL2 (green) levels in A549 cells incubated under normoxic and hypoxic conditions for 24 h, respectively. Data represent the mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, ***P < 0.001 vs normoxia. (E) RT‐qPCR analysis showing mir‐210‐3p gene expression in A549 and NCI‐H460 cells incubated in normoxic and hypoxic conditions for 24 h. U6 snRNA was used as an internal reference control for miRNA normalization. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, ***P < 0.001 vs normoxia. (F,G) miR target reporter luciferase assay showing relative luciferase activity in control vector, wildtype, and mutated CCL2‐3′UTR transfected A549 cells treated with control mimic or miR‐210‐3p mimic (F) or in wildtype CCL2‐3′UTR transfected A549 cells treated with miR‐210‐3p inhibitor or 60 nt long miR‐210‐3p target protector under normoxic and hypoxic conditions for 24 h (G). Data represent the mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ***P < 0.001, **P < 0.01. (H) RT‐qPCR analysis of mir‐210‐3p gene expression in A549 cells transfected with control mimic or miR‐210‐3p mimic. U6 snRNA was used as an internal reference control. Data represent the mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, ****P < 0.0001 vs control mimic. (I,J) RT‐qPCR analysis of CCL2 gene expression (I) and ELISA showing CCL2 and HIF‐1A protein abundance (J) in control mimic or miR‐210‐3p mimic transfected A549 cells incubated with normoxic or hypoxic conditions for 24 h. GAPDH was used as a loading control for RT‐qPCR. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ***P < 0.001, **P < 0.01. (K) RT‐qPCR analysis of mir‐210‐3p gene expression in A549 cells transfected with control inhibitor or miR‐210‐3p inhibitor. U6 snRNA was used as an internal reference control. Data represent the mean ± SD of three independent experiments. Statistical significance was analyzed by Student's t‐test, ****P < 0.0001 vs control inhibitor. (L,M) RT‐qPCR analysis of CCL2 gene expression (L) and ELISA showing CCL2 protein abundance (M) in control inhibitor or miR‐210‐3p inhibitor transfected A549 cells treated with normoxic or hypoxic conditions for 24 h. GAPDH was used as a loading control for RT‐qPCR. Data represented as mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ***P < 0.001, **P < 0.01. (N,O) Representative images (N) and their quantifications (O) of THP‐1 monocytes migration from the upper surface to lower surface of transwell insert placed on the well containing A549 cells transfected without or with miR‐210‐3p mimic or miR‐210‐3p inhibitor and exposed to normoxic or hypoxic conditions for 16 h. Scale bars: 50 μm. Data represent the mean ± SD of three independent experiments. Statistical significance was analyzed by two‐way ANOVA, ****P < 0.0001, ***P < 0.001, **P < 0.01.
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