LIN28B Aggravates the Inflammation in Sepsis‐Induced Lung Injury by Stabilizing GATA6 mRNA and Upregulating TRAF6

Figures

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  Figure 1. Knockdown of LIN28B inhibited LPS‐induced inflammation and injury in HPMEC. HPMECs were treated with LPS. (A) CCK‐8 was employed to detect the effects of LPS on HPMEC viability. (B, C) Flow cytometry and TUNEL staining (Scale bar, 100 μm) were applied to assess apoptosis. (D) Levels of inflammatory factors (TNF‐α, IL‐1β, IL‐6) were determined by ELISA assay. (E) Expressions of LIN28B, Bax, Bcl‐2 and cleaved caspase‐3 were detected utilizing western blot. Next, HPMECs were transfected with sh‐NC or sh‐LIN28B and then treated with LPS. (F) Cell viability was detected by CCK‐8. (G, H) Flow cytometry and TUNEL staining were used to detect apoptosis (Scale bar, 100 μm). (I) Inflammatory factor levels were confirmed by ELISA assay. (J) LIN28B, Bax, Bcl‐2, and cleaved caspase‐3 levels were identified by western blot. Values were expressed as mean ± SD of three separate determinations. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 2. LIN28B bound to GATA6 and enhanced its mRNA stability. (A, B) The relationship between LIN28B and GATA6 was verified by RIP and RNA pull‐down assays. Next, HPMECs were transfected with sh‐LIN28B, oe‐LIN28B or their negative controls. (C) RT‐qPCR and western blot were applied to detect GATA6 levels. (D) GATA6 mRNA level was analyzed by RT‐qPCR after treatment with 5 μg/mL actinomycin D for 0, 1.5, 3, and 6 h. Each bar represents mean ± SD calculated from 3 independent experiments. *p < 0.05, ***p < 0.001.

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  Figure 3. LIN28B stabilized GATA6 mRNA to promote LPS‐induced HPMEC inflammation and injury. HPMECs were transfected with oe‐LIN28B and sh‐GATA6, and then treated with LPS. (A) Western blot was applied to detect LIN28B and GATA6 protein levels. (B) Cell viability was tested by CCK‐8. (C, D) Cell apoptosis was detected using flow cytometry and TUNEL staining (Scale bar, 100 μm). (E) ELISA assay was employed to quantify inflammatory factor levels. (F) Expressions of LIN28B, GATA6, Bax, Bcl‐2 and cleaved caspase‐3 were measured by western blot. Values were expressed as mean ± SD of three separate determinations. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 4. GATA6 transcriptionally activated TRAF6 to promote LPS‐induced HPMEC inflammation and injury. (A) The binding site of GATA6 on the TRAF6 promoter region was predicted by the JASPAR database. (B, C) ChIP and dual luciferase reporter assays were used to confirm the binding association between GATA6 and the TRAF6 promoter. HPMECs were transfected with oe‐GATA6 and sh‐TRAF6, and then treated with LPS. (D) Western blot was applied to detect GATA6 and TRAF6 protein levels. (E) Cell viability was checked utilizing CCK‐8. (F, G) For the detection of apoptosis, flow cytometry analysis and TUNEL staining techniques were used (Scale bar, 100 μm). (H) Levels of inflammatory factors were evaluated utilizing the ELISA assay. (I) To investigate GATA6, TRAF6, Bax, Bcl‐2, and cleaved caspase‐3 levels, the western blot was employed. Values were expressed as mean ± SD of three separate determinations. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Figure 5. LIN28B upregulated the GATA6/TRAF6 axis to enhance LPS‐triggered inflammation and injury in HPMEC. HPMECs were transfected with oe‐LIN28B and sh‐TRAF6, and then treated with LPS. (A) Cell viability was checked utilizing CCK‐8. (B, C) Flow cytometry and TUNEL staining were employed to detect apoptosis (Scale bar, 100 μm). (D) The concentrations of inflammatory mediators were quantified using the ELISA assay. (E) Western blot analysis was utilized to detect LIN28B, GATA6, TRAF6, Bax, Bcl‐2 and cleaved caspase‐3 expressions. Values were expressed as mean ± SD of three separate determinations. *p < 0.05, **p < 0.01, ***p < 0.001.