miRNA-142-3p aggravates hydrogen peroxide-induced human umbilical vein endothelial cell premature senescence by targeting SIRT1

Abstract

Vascular endothelial cell premature senescence plays an important part in stroke. Many microRNAs (miRNAs) are known to be involved in the pathological process of vascular endothelial cell premature senescence. The present study aimed to investigate the mechanism of hydrogen peroxide (H2O2)-induced premature senescence in human umbilical vein endothelial cells (HUVECs) and effect of miR-142-3p on hydrogen peroxide (H2O2)-induced premature senescence. HUVECs were exposed to H2O2 to establish a model premature senescence in endothelial cells. CCK-8 assay was performed to detect cell viability. Senescence-associated β-galactosidase staining assay and senescence-related proteins p16 and p21 were used to detect changes in the degree of cell senescence. RT-qPCR and Western blot were conducted to measure mRNA and protein levels, respectively. The scratch wound-healing assay, transwell assay, and EdU assay were performed to evaluate the ability of migration and proliferation, respectively. miRNA-142-3p and silencing information regulator 2 related enzyme 1 (SIRT1) binding was verified using Targetscan software and a dual-luciferase assay. We found that miRNA-142-3p is abnormally up-regulated in HUVECs treated with H2O2. Functionally, miRNA-142-3p inhibition may mitigate the degree of HUVEC senescence and improve HUVEC migration and proliferation. Mechanistically, SIRT1 was validated to be targeted by miRNA-142-3p in HUVECs. Moreover, SIRT1 inhibition reversed the effects of miRNA-142-3p inhibition on senescent HUVECs exposed to H2O2. To our knowledge, this is the first study to show that miRNA-142-3p ameliorates H2O2-induced HUVECs premature senescence by targeting SIRT1 and may shed light on the role of the miR-142-3p/SIRT1 axis in stroke treatment.

Keywords: Senescence; endothelial cells; miRNA-142-3p; oxidative stress; sirtuins; stroke.

Figure 1. H₂O₂ reduces cell viability and promotes premature senescence in HUVECs

(A) Different concentrations of H₂O₂ (0, 25, 50, 100, 200, and 400 μM) were applied to cultured HUVECs for 1 h. A CCK-8 assay was used to evaluate cell viability. (B,C) Representation and statistics of cell premature senescence in different H₂O₂-treated groups. Senescent cells become larger and stained blue. **P<0.01, ***P<0.001 vs. control; HUVEC, human umbilical vein endothelial cell.

Figure 2. H₂O₂ accelerates cellular premature and inhibits cell proliferation in HUVECs

(A) Determination of p16 and p21 protein expression by Western Blotting. (B,C) Densitometric quantification of p16 and p21 protein expression based on Western Blot assays. (D,E) Representation and statistics of cell proliferation in different H₂O₂ treated groups. *P<0.05, **P<0.01 vs Control, HUVEC, human umbilical vein endothelial cell; ns: no significance.

Figure 3. Relative expression of miR-142-3p and its effect on the cell viability of HUVECs

(A) miR-142-3p expression levels in control and 100 μM H₂O₂, revealed by RT-qPCR. (B) The relative expression level of miR-142-3p after knocking out miR-142-3p. (C) Cell viability of HUVECs between different treatment groups. **P<0.01, ***P<0.001, HUVEC, human umbilical vein endothelial cell; ns: no significance.

Figure 4. Knockdown of miR-142-3p mitigated the degree of premature senescent HUVECs induced by H₂O₂

(A–C) Representation and statistics of p16 and p21 protein relative expression in HUVECs with different miR-142-3p gene modifications. (D,E) Representation and statistics of cell proliferation in HUVECs with different miR-142-3p gene modifications. **P<0.01, ***P<0.001, HUVEC, human umbilical vein endothelial cell; ns: no significance.

Figure 5. Knockdown of miR-142-3p improved the migration and proliferation ability of premature senescent HUVECs

(A,B) Representation and statistics of cell migration in HUVECs with different miR-142-3p gene modifications in the scratch wound-healing experiment. (C,D) Representation and statistics of cell migration in HUVECs with different miR-142-3p gene modifications in the transwell experiment. (E,F) Representation and statistics of cell proliferation in HUVECs with different miR-142-3p gene modifications in the EdU staining assay. **P<0.01, ***P<0.001, HUVEC, human umbilical vein endothelial cell; ns: no significance.

Figure 6. SIRT1 was a direct target of miR-142-3p in HUVECs

(A) The binding sites between miR-142-3p and SIRT1 were predicted by TargetScan software (https://www.targetscan.org/cgi-bin/targetscan/vert_72/view_gene.cgi?rs=ENST00000212015.6&taxid=9606&members=miR-142-3p.2&showcnc=0&shownc=0&subset=1). (B) Luciferase assay confirmed the target relationship between miR‐142‐3p and SIRT1. SIRT1-WT binding with miR-142-3p.2 mimics significantly reduced the luciferase activity, while SIRT1-MUT could not bind with miR-142-3p and had no significant effect on luciferase activity. (C,D) Representation and statistics of SIRT1 protein expression in HUVECs between control and H₂O₂ treatment groups, revealed by Western Blot experiment; **P<0.01, ***P<0.001, HUVEC, human umbilical vein endothelial cell; ns: no significance.

Figure 7. miR-142-3p accelerates premature senescence by down-regulating SIRT1 expression in HUVECs

(A) The mRNA expression level of SIRT1 in the control and Si-SIRT1 groups was revealed by the RT-qPCR experiment. (B,C) Representation and statistics of SIRT1 protein relative expression level in control and Si-SIRT1 group revealed by Western Blot. (D–G) Representation and statistics of SIRT1, p16, and p21 protein relative expression levels between treatment groups revealed by Western Blot. (H,I) Representation and statistics of cell premature senescence between treatment groups revealed by SA β-gal staining assay. Senescent cells become larger and stained blue. *P<0.05, **P<0.01; ***P<0.001; HUVECs: human umbilical vein endothelial cells. SA β-gal staining assay: Senescence-associated β-galactosidase staining assay.

Figure 8. miR-142-3p inhibited the migration and proliferation ability of premature senescence by down-regulating SIRT1 expression in HUVECs

(A,B) Representation and statistics of cell migration in HUVECs between treatment groups in the scratch wound-healing experiment. (C,D) Representation and statistics of cell migration in HUVECs between treatment groups in the transwell experiment. (E,F) Representation and statistics of cell proliferation in HUVECs between treatment groups in the EdU staining assay; *P<0.05, **P<0.01, ***P<0.001, HUVEC, human umbilical vein endothelial cell; ns: no significance.