Upregulation of long non-coding RNA SNHG16 promotes diabetes-related RMEC dysfunction via activating NF-κB and PI3K/AKT pathways

Figure 1. SNHG16 expression is upregulated in hRMECs under high-glucose (HG) condition (A) SNHG16 expression in hRMECs cultured under different conditions was detected using quantitative real-time PCR, showing the upregulation of SNHG16 in hRMECs stimulated with HG (25 mM D-glucose) in comparison with low-glucose (LG; 5 mM D-glucose) or osmotic control (Osm; 25 mM L-glucose) groups. n = 3 in each group. (B and C) Multiple trials of quantitative real-time PCR showed that the SNHG16 level in hRMECs was increased in a glucose dose-dependent pattern (treated for 48 h) and in a culturing time-dependent pattern (25 mM). n = 3 in each group. (D) SNHG16 expression in the cytoplasm and nucleus of hRMEC using quantitative real-time PCR following hRMEC subcellular fractionation. GAPDH and U6 served as cytoplasmic and nuclear markers, respectively. n = 3 in each group. (E) SNHG16 subcellular distribution in hRMECs under LG or HG condition for 48 h was visualized using FISH (scale bars, 20 μm), in which data quantification was recorded as mean fluorescence intensity of SNHG16 probes accordingly. n = 3 in each group. All data were acquired from three independent experiments and presented as the mean ±…

Figure 2. SNHG16 positively regulates proliferation, migration, and angiogenesis of hRMECs Experiments were conducted in cells treated with LG (5 mM) or HG (25 mM) for 48 h. (A) Efficiency of SNHG16 overexpression in LG-treated hRMECs and of SNHG16 knockdown in HG-treated hRMECs was assessed using quantitative real-time PCR. Two shRNAs for SNHG16 with relatively higher knockdown capacity were selected. n = 3 in each group. (B) CCK-8 assay was performed to assess the influence of SNHG16 overexpression or knockdown on hRMEC proliferation. The shRNA for SNHG16 with the strongest effect was adopted for all following experiments. n = 3 in each group. (C) Cell proliferation ratio of each group was illustrated through calculating the percentage of EdU-positive cells using the EdU assay (scale bars, 200 μm). n = 3 in each group. (D) Wound-healing assay illustrated the effect of SNHG16 overexpression or knockdown on hRMEC migration through measuring the wound-healing distance (scale bars, 100 μm). n = 3 in each group. (E) Migration cell number of each group was counted using Transwell assay. n = 3 in each group. (F) quantitative real-time PCR analysis of VEGF mRNA level influenced by SNHG16…

Figure 3. SNHG16 is associated with NF-κB and PI3K/AKT pathways Experiments were conducted in cells treated with LG (5 mM) or HG (25 mM) for 48 h. (A) The downstream signaling pathway of SNHG16 was evaluated using Cignal Reporter Assay to detect the activities of a series of transcription factors in hRMECs with SNHG16 knockdown. The transcription factors listed above represent endoplasmic reticulum (ER) stress, hypoxia, MAPK/ERK, MAPK/c-Jun N-terminal kinase (JNK), NF-κB, Notch, PI3K/AKT, STAT3, TGF-β, and Wnt signaling pathways. n = 3 in each group. (B) Cignal Reporter Assay was performed to explore the signaling activity of NF-κB and PI3K/AKT in SNHG16-overexpressed hRMECs. n = 3 in each group. (C) Western blot analysis of p-IκBα and IκBα illustrated the effect of SNHG16 overexpression or knockdown on p-IκBα. n = 3 in each group. (D) Western blot analysis of nuclear and cytoplasmic p65 following subcellular fractionation illustrated the effect of SNHG16 overexpression or knockdown on p65 nuclear translocation. Histone H3: nuclear control; β-actin: cytoplasmic control. n = 3 in each group. (E) p65 activity in hRMECs with SNHG16 overexpression or knockdown was detected using ELISA-based…

Figure 4. SNHG16 directly interacts with miR-146a-5p and miR-7-5p Experiments were conducted in cells treated with LG (5 mM) or HG (25 mM) for 48 h. (A) ENCORI prediction showed the binding sites of miR-146a-5p and miR-7-5p in the sequence of SNHG16. The sequences highlighted in red stand for the mutant (Mut) binding sites of miR-146a-5p and miR-7-5p for luciferase reporter assay. (B) miR-146a-5p and miR-7-5p expression in hRMECs cultured with HG, LG, or Osm was detected using quantitative real-time PCR. n = 3 in each group. (C) Quantitative real-time PCR showed the levels of miR-146a-5p and miR-7-5p influenced by SNHG16 overexpression or knockdown. n = 3 in each group. (D) RIP assay with Ago2 antibody or IgG as a control, followed by quantitative real-time PCR, demonstrated the enrichment of SNHG16, miR-146a-5p, and miR-7-5p in RISCs. n = 3 in each group. (E) RNA pull-down assay, followed by quantitative real-time PCR, was performed to detect the level of SNHG16 interacting with miR-146a-5p or miR-7-5p. n = 3 in each group. (F) Luciferase reporter assay was performed in hRMECs co-transfected with SNHG16-WT or SNHG16-Mut1 luciferase reporters and miR-146a-5p mimics or NC mimics, as well as…

Figure 5. miR-146a-5p and miR-7-5p inhibitors reverse the effects generated by SNHG16 knockdown Experiments were conducted in cells treated with HG (25 mM) for 48 h. (A) Quantitative real-time PCR showed that the enhanced levels of miR-146a-5p and miR-7-5p in hRMECs after SNHG16 knockdown were suppressed by transfection of miR-146a-5p and miR-7-5p inhibitors, respectively. n = 3 in each group. (B and C) CCK-8 assay and EdU assay illustrated the effect of miR-146a-5p and/or miR-7-5p inhibitors on cell proliferation in hRMECs with SNHG16 knockdown. n = 3 in each group. (D and E) Wound-healing assay and Transwell assay illustrated the effect of miR-146a-5p and/or miR-7-5p inhibitors on cell migration in hRMECs with SNHG16 knockdown. n = 3 in each group. (F and G) Quantitative real-time PCR and ELISA showed the influence of miR-146a-5p and/or miR-7-5p inhibitors on VEGF level. n = 3 in each group. (H) Tube formation assay showed the influence of miR-146a-5p and/or miR-7-5p inhibitors on angiogenesis. n = 3 in each group. All data were acquired from three independent experiments and presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01.

Figure 6. Interleukin-1 receptor-associated kinase 1 (IRAK1) and IRS1 are, respectively, targeted by miR-146a-5p and miR-7-5p Experiments were conducted in cells treated with LG (5 mM) or HG (25 mM) for 48 h. (A) The levels of miR-146a-5p downstream genes, IRAK1, caspase recruitment domain family, member 10 (CARD10), and tumor necrosis factor receptor-associated factor 6 (TRAF6), were assessed using quantitative real-time PCR to detect the influence of SNHG16 knockdown on their expression. n = 3 in each group. (B) ENCORI prediction showed the binding sites of miR-146a-5p and miR-7-5p in the sequences of IRAK1 and IRS1 3′ UTRs. The sequences highlighted in red stand for the Mut binding sites for luciferase reporter assay. (C) RIP assay followed by quantitative real-time PCR demonstrated the enrichment of SNHG16, miR-146a-5p, miR-7-5p, and mRNAs of IRAK1 and IRS1 in RISCs. n = 3 in each group. (D) RNA pull-down assay followed by quantitative real-time PCR was performed to detect the level of IRAK1 or IRS1 mRNA interacting with miR-146a-5p or miR-7-5p. n = 3 in each group. (E) Luciferase reporter assay elucidated the interaction between IRAK1 or IRS1 mRNA and miR-146a-5p or miR-7-5p, as well…

Figure 7. SNHG16 promotes hRMEC dysfunction via the NF-κB pathway activation through IRAK1 Experiments were conducted in cells treated with LG (5 mM) for 48 h. (A) Quantitative real-time PCR showed the change of IRAK1 level in SNHG16-overexpressed hRMECs by transfecting shRNAs for IRAK1. Two shRNAs with relatively higher knockdown capacity were selected. n = 3 in each group. (B and C) CCK-8 assay and EdU assay showed the effects of IRAK1 shRNAs and BAY 11-7082 on cell proliferation in SNHG16-overexpressed hRMECs. The shRNA with the highest knockdown capacity was adopted. n = 3 in each group. (D and E) Wound-healing assay and Transwell assay showed the effects of sh-IRAK1#1 transfection and BAY 11-7082 addition on cell migration in hRMECs after SNHG16 overexpression. n = 3 in each group. (F and G) Quantitative real-time PCR and ELISA showed the effects of sh-IRAK1#1 transfection and BAY 11-7082 addition on VEGF level in hRMECs after SNHG16 overexpression. n = 3 in each group. (H) Tube formation assay showed the effects of sh-IRAK1#1 transfection and BAY 11-7082 addition on angiogenesis in SNHG16-overexpressed hRMECs. n = 3 in each group. (I) ELISA-based NF-κB activity assay showed the…

Figure 8. SNHG16 promotes hRMEC dysfunction via the PI3K/AKT pathway activation through IRS1 Experiments were conducted in cells treated with LG (5 mM) for 48 h. (A) Quantitative real-time PCR showed the change of IRS1 level in SNHG16-overexpressed hRMECs by transfecting shRNAs for IRS1. Two shRNAs with relatively higher knockdown capacity were selected. n = 3 in each group. (B and C) CCK-8 assay and EdU assay showed the effects of IRS1 shRNAs and LY294002 on cell proliferation in SNHG16-overexpressed hRMECs. The shRNA with the highest knockdown capacity was adopted. n = 3 in each group. (D and E) Wound-healing assay and Transwell assay showed the effects of sh-IRS1#1 transfection and LY294002 addition on cell migration in hRMECs after SNHG16 overexpression. n = 3 in each group. (F and G) Quantitative real-time PCR and ELISA showed the effects of sh-IRS1#1 transfection and LY294002 addition on VEGF level in hRMECs after SNHG16 overexpression. n = 3 in each group. (H) Tube formation assay showed the effects of sh-IRS1#1 transfection and LY294002 addition on angiogenesis in SNHG16-overexpressed hRMECs. n = 3 in each group. All data were acquired from three independent experiments and…