LncRNA NEAT1 Promote Inflammatory Responses in Coronary Slow Flow Through Regulating miR-148b-3p/ICAM-1 Axis

Figures

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  Figure 1. Relationship between plasma inflammation-associated biomarkers and coronary slow flow (CSF). (A) Plasma sICAM-1 levels were significantly higher in patients with CSF than in controls. (B) Plasma IL-6 levels were significantly higher in patients with CSF. (C) Plasma TNF-α levels were significantly higher in patients with CSF. (D) Correlation between the number of coronary arteries involved in CSF and plasma sICAM-1 level. (E) Receiver-operating characteristic (ROC) curve analysis of plasma sICAM-1 for diagnosing CSF; Data were analysed using Student’s t-test (A–C); one-way ANOVA for pairwise comparisons or comparisons between multiple groups with a single variable (D). Data represent the mean ± SD, **P < 0.01 vs Control group.

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  Figure 2. Expression of plasma miR-148b-3p and NEAT1 in CSF and controls and their relationship with CSF. (A) Plasma miR-148b-3p levels were significantly lower in patients with CSF. Data represent the mean ± SD. (B) Plasma miR-148b-3p levels were negatively correlated with the mean corrected thrombolysis in myocardial infarction frame count (cTFC). (C) Plasma miR-148b-3p levels were negatively correlated with sICAM-1. (D) Plasma NEAT1 levels were significantly higher in patients with CSF. Data represent the mean ± SD. (E) Plasma NEAT1 levels were positively correlated with the mean cTFC. (F) Plasma NEAT1 levels were positively correlated with sICAM-1. (G and H) ROC curve analysis of plasma miR-148b-3p and NEAT1 for diagnosing CSF. Data were analysed using Student’s t-test (A and D); Significance was determined using linear regression analysis (B, C, E and F).

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  Figure 3. Effect of oxygen glucose deprivation (OGD) on HUVEC proliferation and apoptosis and relative expression levels of NEAT1, miR-148b-3p, and ICAM-1. (A) qRT-PCR and WB analyses were performed to detect the relative expression levels of NEAT1, miR-148b-3p and ICAM-1. Data represent the mean ± SD, **P < 0.01 vs Control group. (B) CCK-8 assays were performed to measure the effect of OGD on HUVEC proliferation (n = 3 per group). Data represent mean ± SD, **P < 0.01 vs Control group. (C) EdU assays were performed to measure the effect of OGD on HUVEC proliferation (n = 3 per group). Data represent mean ± SD, **P < 0.01 vs Control group. Scale bars represent 40 μm. (D) Flow cytometric analysis of annexinV-633 staining was used to detect the effect of OGD on HUVEC apoptosis (n = 3 per group). Data represent the mean ± SD, **P < 0.01 vs Control group. All data were analysed using Student’s t-test.

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  Figure 4. The effects of ICAM-1 on the proliferation and apoptosis of OGD-treated HUVECs. (A) Expression of ICAM-1 protein (n = 3 per group) after ICAM-1 over-expression or inhibition in OGD-treated HUVECs. Data represent the mean ± SD, **P < 0.01 vs pre-ICAM-1-NC group, #P < 0.05 vs sh-ICAM-1-NC group. (B) CCK-8 assays were performed to measure the effect of ICAM-1 on the proliferation of OGD-treated HUVECs (n = 3 per group). Data represent the mean ± SD, **P < 0.01 vs pre-ICAM-1-NC group, ##P < 0.01 vs sh-ICAM-1-NC group. (C) EdU assays were performed to measure the effect of ICAM-1 on the proliferation of OGD-treated HUVECs (n =3 per group). Data represent the mean ± SD, **P < 0.01 vs pre-ICAM-1-NC group, ##P < 0.01 vs sh-ICAM-1-NC group. Scale bars represent 40 μm. (D) Flow cytometric analysis of annexinV-633 staining was used to measure the percentages of apoptotic OGD-treated HUVECs (n = 3 per group) after ICAM-1 over-expression or inhibition. Data represent the mean ± SD, **P < 0.01 vs pre-ICAM-1-NC group, ##P < 0.01 vs sh-ICAM-1-NC group. One-way ANOVA for pairwise comparisons or comparisons between multiple groups with a single variable.

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  Figure 5. Continued.

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  Figure 5. The effects of miR-148b-3p on OGD-treated HUVECs and miR-148b-3p over-expression reversed the ICAM-1-mediated on the proliferation and apoptosis of OGD-treated HUVECs. (A) qRT-PCR and WB analyses were performed to measure the expression of ICAM-1 mRNA and protein, respectively (n = 3 per group) after miR-148b-3p over-expression or inhibition in OGD-treated HUVECs. Data represent the mean ± SD, *P < 0.05 vs pre-miR-148b-3p-NC group; **P < 0.01 vs pre-miR-148b-3p-NC group; ##P < 0.01 vs anti-miR-148b-3p-NC group. (B) CCK-8 assays were performed to measure the effect of miR-148b-3p on the proliferation of OGD-treated HUVECs (n = 3 per group). Data represent the mean ± SD, **P < 0.01 vs pre-miR-148b-3p-NC group; #P < 0.05 vs anti-miR-148b-3p-NC group. (C) EdU assays were performed to measure the effect of miR-148b-3p on the proliferation of OGD-treated HUVECs (n = 3 per group). Data represent the mean ± SD, **P < 0.01 vs pre-miR-148b-3p-NC group; ##P < 0.01 vs anti-miR-148b-3p-NC group. Scale bars represent 40 μm. (D) Flow cytometric analysis of annexinV-633 staining was used to measure the percentages of apoptotic OGD-treated HUVECs (n = 3 per group) after miR-148b-3p…

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  Figure 6. Continued.

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  Figure 6. The effects of NEAT1 in OGD-HUVECs and reversed the miR-148b-3p-mediated on the proliferation and apoptosis of OGD-induced HUVECs. (A) MiR-148b-3p expression in OGD-treated HUVECs (n = 3 per group) after transfection with sh-NEAT1. Data represent the mean ± SD, **P < 0.01 vs sh-NC group. (B) qRT-PCR and WB analyses were performed to measure the expression of ICAM-1 mRNA and protein, respectively (n = 3 per group) in OGD-induced HUVECs after transfection with sh-NEAT1. Data represent the mean ± SD, **P <0.01 vs sh-NC group. (C) CCK-8 assays were performed to measure the effect of NEAT1 knockdown on the proliferation of OGD-induced HUVECs (n = 3 per group). Data represent the mean ± SD, **P <0.01 vs sh-NC group. (D) EdU assays were performed to measure the effect of NEAT1 knockdown on the proliferation of OGD-induced HUVECs. Data represent the mean ± SD, **P <0.01 vs sh-NC group. Scale bars represent 40 μm. (E) Flow cytometric analysis of annexinV-633 staining was used to measure the percentages of apoptotic OGD-treated HUVECs after NEAT1 knockdown. Data represent the mean ± SD (n = 3 per group). **P < 0.01 vs sh-NC group. (F) The predicted miR-148b-3p binding site in the NEAT1…

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  Figure 7. The schematic diagram of the mechanism of NEAT1/miR-148b-3p/ICAM-1 axis in CSF disease.