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Research Paper Volume 12, Issue 5 pp 4247-4267
Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling
Relevance score: 13.860927Wei Cai, Sanli Liu, Ziquan Liu, Shike Hou, Qi Lv, Huanhuan Cui, Xue Wang, Yuxin Zhang, Haojun Fan, Hui Ding
Keywords: hypoxic lung injury, high altitude, miRNA, miR-203a-3p, VEGF/Notch pathway
Published in Aging on February 29, 2020
Dynamic changes in hypoxic lung injury-related indicators. (A) H&E staining of rat lung tissue. Magnifications of the same section (200X and 400X) are shown. (B) Analysis of arterial blood gasses. (C, D) Measurements of total protein concentration in BALF and lung tissue W/D. (E) Immunohistochemical staining of occludin in rat lung tissue (400X). (F) ELISA analysis of IL-6 and TNF-α levels in serum and BALF. Data are mean ± SEM. **P < 0.01 compared with the normoxic control group; #P < 0.05, ##P < 0.01 compared with the 24-h hypoxia group; &P < 0.05, &&P < 0.01 compared with the 48-h hypoxia group.
Expression of VEGF/Notch pathway-related proteins and CD31 in rat lung tissues. (A) Immunohistochemical staining of Notch1, CD31, and VEGFR2 (400X), and VEGF-A and Hes-1 (200X). (B) Western blotting analysis of Hes-1, Notch1, VEGF-A and VEGFR2 expression in rat lung tissue. Data are mean ± SEM. *P<0.05, **P<0.01 compared with the normoxic control group; #P<0.05, ##P<0.01 compared with the 24-h hypoxia group. &&P<0.01 compared with the 48-h hypoxia group.
Screening and validation of miRNAs associated with hypoxic exposure. (A) Heatmap of miRNA microarray data. Unmonitored hierarchical clustering analysis was conducted for differentially expressed genes induced by hypoxia (24,48 and 72h) in the rat lung. A total of 57 miRNAs showed > 1.5 fold change relative to normoxic, control lung tissue samples; blue indicates downregulation. (B) Downregulation of miRNAs targeting Hes-1, Notch1, VEGF-A, VEGF-B, and VEGF-C by hypoxia. (C) Screening of 6 selected differentially expressed miRNAs associated with the VEGF/Notch pathway. (D) Verification of selected miRNAs expression in rat lung by qRT-PCR. Values are expressed as fold change ± SEM relative to control. *P < 0.05, **P < 0.01 compared with the normoxic control group; #P <0.05, ##P < 0.01 compared with the 24-h hypoxia group; &&P < 0.01 compared with the 48-h hypoxia group.
Analysis of hypoxia-induced changes in VEGF/Notch signaling effector molecules and related miRNAs in PMVECs. (A) PMVECs were cultured under hypoxia for 0, 24, 48, or 72 h. With prolongation of hypoxic exposure the cells lost their original morphology and cell population density decreased (200X). (B) Relative expression of miR-30b-5p, miR-532-5p, miR-203a-3p, miR-101a-5p, and miR-16-3p assessed by qRT-PCR. (C) Relative expression of Hes-1, Notch1, VEGF-A, and VEGFR2 mRNA measured by qRT-PCR. (D) Hes-1, Notch1, VEGF-A, and VEGFR2 expression assessed by western blotting. Data are mean ± SEM. *P < 0.05, **P < 0.01 compared with the normoxic control group; #P < 0.05, ##P < 0.01 compared with the 24-h hypoxia group; &P < 0.05, &&P < 0.01 compared with the 48-h hypoxia group.
miR-203a-3p mimics expression inhibits the expression of VEGF-A and downstream genes. (A) Strong GFP fluorescence in PMVECs transduced with miR-203a-3p mimics indicated significantly increased expression of miR-203a-3p, compared with the corresponding control (no transduction) (P < 0.01) (100X). (B) Morphological changes in PMVECs transduced with lentiviral vectors encoding miR-203a-3p-NC (negative miRNA-203a-3p mimics control) or miR-203a-mimics (100X). (C) Results of qRT-PCR analysis showing decreased expression of Hes-1, VEGF-A, and VEGFR2 in miR-203a-mimics-transduced PMVECs (P < 0.01). (D) Western blotting results showing reduction on Hes-1, VEGF-A and VEGFR2 expression in PMVECs transduced with miR-203a-3p-mimics. Data are mean ± SEM. **P < 0.01 compared with the control or normoxic control groups; ##P < 0.01 compared with miR-203a-3p-NC or 24-h hypoxia group; &P < 0.05, &&P < 0.01 compared with the 48-h hypoxia group.
miR-203a-3p mimics expression decreases survival and angiogenic activity and induces a pro-inflammatory response in PMVECs. (A) Apoptosis assay results showing increased apoptosis rate in PMVECs expressing miR-203a-3p mimics. (B) In vitro angiogenesis assay results. PMVECs transduced with miR-203a-3p mimics showed weak angiogenic ability, which improved however with prolonged hypoxic incubation time. (C) ELISA assay results showing increased IL-6, IL-10, and TNF-α secretion in PMVECs transfected with miR-203a-3p. (D, E) CCK8 assay results indicating decreased survival rate in PMVECs transfected with miR-203a-3p mimics. Data are mean ± SEM. *P < 0.05, **P < 0.01 compared with the control (no transduction) or normoxic control groups; #P < 0.05, ##P < 0.01 compared with the miR-203a-3p-NC (negative miRNA-203a-3p mimics control) or 24-h hypoxia groups; &P < 0.05, &&P < 0.01 compared with the 48-h hypoxia group.
VEGF-A is a direct target of miR-203a-3p. (A) Sequence information of miR-203 binding sites in the 3′-UTR region of the VEGF-A mRNA. (B) Dual luciferase assay indicating decreased fluorescence in PMVECs co-expressing pmirGLO/VEGF-UTR and miR-203-3p mimics. (C) Western blotting detection of target genes of miR-203a-3p. Compared with mimics-NC (negative miR-203a-3p mimics control), VEGF expression decreased after transfection with miR-203-mimics, and increased after transfection with miR-203-ASO. (D) qRT-PCR detection of target genes of miR-203a-3p. VEGF mRNA levels decreased after transfection with miR-203-mimics, and increased after transfection with miR-203-ASO. Data are mean ± SEM. *P < 0.05, **P < 0.01 compared with mimics-NC; ##P < 0.01 compared with miR-203a-3p-mimics; &&P < 0.01 compared with miR-203a-3p-ASO-NC (negative miR-203a-3p-ASO control).
VEGF-A expression is rescued by miR-203a-3p knockdown. (A) The validation of VEGF-A expression reduction in VEGF-shRNA-transduced cells by qRT-PCR. (B) Western blotting data showing reduced expression of VEGF-A in PMVECs transduced with VEGF-shRNA. (C) Morphological evaluation in PMVECs transduced with VEGF-shRNA+ASO-NC (negative VEGF-shRNA+ASO control) or VEGF-shRNA+ASO (miR-203a-3p knockdown). Note that inhibition of miR-203a-3p activity reversed morphological impairment caused by VEGF silencing. (D) Flow cytometry apoptosis assay indicates increased apoptosis in PMVECs expressing VEGF-shRNA, and reversal of the effect by ASO-mediated miR-203a-3p silencing. (E–F) CCK-8 assay showing improved viability in PMVECs expressing VEGF-shRNA+ASO, compared with the VEGF-shRNA+ASO-NC group. (G) qRT-PCR results showing reduced expression of VEGF-A and its downstream mediators, VEGFR2 and Hes-1, in cells transfected with VEGF-shRNA. The expression of these mRNAs increased instead in cells co-expressing miR-203a-3p-ASO. (H) Western blotting results showing protein expression changes consistent with the mRNA data showed in (G). Data are mean ± SEM. *P < 0.05, **P < 0.01 compared with VEGF-shRNA negative control (VEGF-shRNA-NC) and normoxia groups; #P < 0.05, ##P < 0.01 compared with the VEGF-shRNA or 24-h hypoxia group; &P < 0.05, &&P < 0.01 compared with the VEGF-shRNA+ASO-NC or 48-h hypoxia group.
Hypoxia stimulates VEGF/Notch signaling by downregulating miR-203a-3p expression. (A) Hypoxia upregulates the expression of VEGF-A and downregulates the expression of its negative regulator miR-203a-3p (red box). (B) VEGF-A binds and activates VEGFR2 in tip cells, leading to the activation of the Dll4 promoter (purple box). (C) Dll4 activates Notch1 receptor in neighboring stem cells. Gamma secretase (GS) cleaves the Notch1 receptor intracellular domain (NICD), which is transferred to the nucleus to enhance the expression of transcription factors (TFs) such as Hes-1. These TFs inhibit the expression of VEGFR2 in stem cells and promote the expression of downstream genes that regulate budding, proliferation, and differentiation of PMVECs (blue box).
Experimental hypoxia chamber. (A) The hypoxia chamber for animal experiments includes an external operation panel and an internal cabin; ① vacuum pump; ② fans; ③ monitor device; ④ monitor screen; ⑤ cooling hole. (B) Parameter settings. The red box indicates cabin’s air flow setup: cold air enters, air is discharged, and air is exchanged. (C) Animal modeling time and equipment running process. (D) Time-height curve, comprising a rising stage (t0), cultivated room running time (t1), and a constant speed drop phase (t2).