Silencing lncRNA AK136714 reduces endothelial cell damage and inhibits atherosclerosis

Figures

• 

  Figure 1. AK136714 is reduced in plaque tissue and blood of atherosclerotic mice. (A) GEO database analysis identified dysregulated lncRNAs in plaques from a mouse atherosclerosis model. (B) Volcano map showing the gene distribution. (C) Detection of AK136714 expression in plaques of atherosclerotic mice using qPCR. (D) Detection of AK136714 expression in the blood of atherosclerotic mice using qPCR. (n=8, *P < 0.05).

• 

  Figure 2. Overexpression of AK136714 inhibits atherosclerosis. (A) qPCR was used to detect the expression of AK136714 in vascular tissue of each group. (B) Vascular oil red O staining was performed to detect the degree of atherosclerosis. (C) ELISA was used to assess inflammatory factor expression. (D) Oil red O staining was performed to detect the degree of vascular injury. (E) Immunofluorescence detection showed that AK136714 is mainly in the cytoplasm of endothelial cells and colocalizes with the endothelial marker CD31. (F) Immunofluorescence staining of CD31 showed that high fat damages endothelial cells, and AK136714 knockdown can reverse endothelial cell damage. (n = 8, *P < 0.05, **P < 0.01).

• 

  Figure 3. Overexpression of AK136714 inhibits endothelial cell apoptosis. (A) Flow cytometry was used to detect the apoptosis of vascular endothelial cells in ox-LDL-induced injury models. (B) TUNEL assay was performed to detect changes in apoptosis. (C) Western blot was used to detect the changes in expression of apoptosis-related proteins. (n=6, **P < 0.01).

• 

  Figure 4. Overexpression of AK136714 protects the endothelial barrier and inhibits the inflammatory response of endothelial cells. (A) Detection of endothelial cell permeability. (B, C) Western blot and immunofluorescence were performed to detect endothelium tight junction biomarkers. (D) ELISA detection of inflammatory factor expression. (E) qPCR detection of inflammatory factor expression. (n=6, *P < 0.05).

• 

  Figure 5. AK136714 binds HuR and FOXO3. (A) RNA pull-down, silver staining, and mass spectrometry analysis were performed to identify specific proteins that bind with AK136714. (B) RNA pull-down was used to verify that AK136714 bind to HuR. (C, D) RIP experiments verified that AK136714 binds HuR. (E) qPCR was used to detect nuclear AK136714 expression. (F) Bioinformatics analysis was performed to predict the possibility of the interaction between HuR and AK136714. (G) Immunofluorescence was performed to detect the colocalization of AK136714 and HuR. (H) Detection of inflammatory factors (TNF-α, IL-1β and IL-16) by RT-PCR after AK136714 overexpression or knockdown. (I) After inhibiting transcription with actinomycin D, the mRNA expression of each inflammatory factor was detected at different times by qPCR. (J) RNA pull-down was used to verify that AK136714 binds to FOXO3. (K, L) RIP experiments verified that AK136714 binds with FOXO3. (M) Luciferase assay was carried out to detect whether AK136714 silencing or overexpression altered the stimulating effect of FOXO3 on Bim. (N) qPCR was used to assess Bim levels after overexpression or knockdown of AK136714. (n=6, *P < 0.05, **P < 0.01).

• 

  Figure 6. AK136714 inhibits endothelial cell apoptosis by binding to FOXO3. (A) Flow cytometry was used to analyze the apoptosis of endothelial cells. (B) TUNEL staining was used to analyze the apoptosis of endothelial cells. (C) Western blot was used to detect Bim expression in each group. (D) qPCR was performed to evaluate the expression of bax. (E) Western blot for detection of apoptotic protein expression in each group (n=6, *P < 0.05, **P < 0.01).