LncRNA XIST promotes neovascularization in diabetic retinopathy by regulating miR-101-3p/VEGFA

Figures

• 

  Figure 1. Low expression of XIST inhibits angiogenesis and inflammatory response in HG-treated HRMECs. (A) qRT-PCR was used to detect XIST expression in patients with diabetes or DR (n = 20). HRMECs were treated with HG and transfected with sh-NC or sh-XIST. Next, (B) qRT-PCR was performed to detect XIST expression, (C) MTT assay to detect cell proliferation, (D) transwell assay to detect cell migration, (E) tube formation assay to detect angiogenesis, and (F) ELISA to detect levels of inflammatory factors in culture supernatant. The data were expressed as mean ± standard deviation. Each cell experiment was repeated thrice. *P < 0.05, compared with the NDR or control group; #P < 0.05, compared with the sh-NC group.

• 

  Figure 2. XIST targets miR-101-3p in DR. (A) The binding sites and corresponding mutations of miR-101-3p and XIST. qRT-PCR was used to detect miR-101-3p expression in patients with DR (B) and HG-treated HRMECs (C). (D) Pearson's correlation analysis of the expression of XIST and miR-101-3p in patients with DR. Dual-luciferase reporter (E) and RIP (F) assays were performed to verify the binding of miR-101-3p and XIST. (G) qRT-PCR was used to detect the expression of miR-101-3p in HRMECs transfected with oe-NC, oe-XIST, sh-NC, or sh-XIST. The data were expressed as mean ± standard deviation. The clinical sample size was 20. Each cell experiment was repeated thrice. *P < 0.05, compared with the NDR or control group; #P < 0.05, compared with the mi-NC, oe-NC, or IgG group; &P < 0.05, compared with the sh-NC group.

• 

  Figure 3. Downregulation of miR-101-3p reverses anti-angiogenic and anti-inflammatory effects of XIST knockdown on HG-treated HRMECs. HRMECs were treated with HG and transfected with sh-NC + in-NC, sh-XIST + in-NC, or sh-XIST + in-miR-101-3p. Next, (A) qRT-PCR was performed to detect miR-101-3p expression, (B) MTT assay to detect cell proliferation, (C) transwell assay to detect cell migration, (D) tube formation assay to detect angiogenesis, and (E) ELISA to detect levels of inflammatory factors in culture supernatant. The data were expressed as mean ± standard deviation. Each cell experiment was repeated thrice. *P < 0.05, compared with the sh-NC+in-NC group; #P < 0.05, compared with the sh-XIST+in-NC group.

• 

  Figure 4. miR-101-3p targets VEGFA in DR. (A) The miR-101-3p-binding site in VEGFA and corresponding mutations. qRT-PCR and western blotting were used to detect the mRNA expression and protein level of VEGFA in patients with DR (B, C) and HG-treated HRMECs (D, E). (F) Pearson's correlation analysis of the expression of VEGFA and miR-101-3p in patients with DR. RNA pull-down (G) and dual-luciferase reporter (H) assays were performed to verify the binding of miR-101-3p and VEGFA. qRT-PCR (I) and western blotting (J) were used to detect the mRNA expression and protein level of VEGFA in HRMECs transfected with mi-NC, mi-miR-101-3p, in-NC, or in-miR-101-3p. The data were expressed as mean ± standard deviation. The clinical sample size was 20. Each cell experiment was repeated thrice. *P < 0.05, compared with the NDR, control, or mi-NC group; #P < 0.05, compared with the NC probe or in-NC group.

• 

  Figure 5. VFGFA overexpression reverses the anti-angiogenic and anti-inflammatory effects of XIST knockdown on HG-treated HRMECs. HRMECs were treated with HG and transfected with sh-NC + oe-NC, sh-XIST + oe-NC, or sh-XIST + oe-VFGFA. Next, (A) qRT-PCR was performed to detect VFGFA mRNA expression, (B) western blotting to detect VFGFA protein expression, (C) MTT assay to detect cell proliferation, (D) transwell assay to detect cell migration, (E) tube formation assay to detect angiogenesis, and (F) ELISA to detect levels of inflammatory factors in culture supernatant. The data were expressed as mean ± standard deviation. Each cell experiment was repeated thrice. *P < 0.05, compared with the sh-NC+oe-NC group; #P < 0.05, compared with the sh-XIST+oe-NC group.

• 

  Figure 6. XIST promotes retinal angiogenesis and inflammatory response in rats with DR through the miR-101-3p/VEGFA axis. DR model rats were injected with sh-NC + oe-NC, sh-XIST + oe-NC, or sh-XIST + oe-VFGFA. (A, B) qRT-PCR was used to detect the expression levels of XIST, miR-101-3p, and VEGFA mRNA in retinal tissue. (C) Western blotting was used to detect the expression of VEGFA protein in retinal tissue. (D) H&E staining was used to detect pathological changes in the retina. (E) ELISA was performed to detect the levels of TNF-α, IL-1β, and IL-6 in retinal tissue. (F) Western blotting was used to detect the expression levels of ZO-1, claudin-5, and occluding in retinal tissue. The data were represented by mean ± standard deviation. Each group had 6 rats. *P < 0.05, compared with the normal group; #P < 0.05, compared with the sh-NC+oe-NC group; &P < 0.05, compared with the sh-XIST+oe-NC group.