MALAT1 sponges miR-26a and miR-26b to regulate endothelial cell angiogenesis via PFKFB3-driven glycolysis in early-onset preeclampsia

Abstract

6-phosphofructo-2-kinase (PFKFB3) is a crucial regulator of glycolysis that has been implicated in angiogenesis and the development of diverse diseases. However, the functional role and regulatory mechanism of PFKFB3 in early-onset preeclampsia (EOPE) remain to be elucidated. According to previous studies, noncoding RNAs play crucial roles in EOPE pathogenesis. The goal of this study was to investigate the functional roles and co-regulatory mechanisms of the metastasis-associated lung adenocarcinoma transcript-1 (MALAT1)/microRNA (miR)-26/PFKFB3 axis in EOPE. In our study, decreased MALAT1 and PFKFB3 expression in EOPE tissues correlates with endothelial cell (EC) dysfunction. The results of in vitro assays revealed that PFKFB3 regulates the proliferation, migration, and tube formation of ECs by modulating glycolysis. Furthermore, MALAT1 regulates PFKFB3 expression by sponging miR-26a/26b. Finally, MALAT1 knockout reduces EC angiogenesis by inhibiting PFKFB3-mediated glycolysis flux, which is ameliorated by PFKFB3 overexpression. In conclusion, decreased MALAT1 expression in EOPE tissues reduces the glycolysis of ECs in a PFKFB3-dependent manner by sponging miR-26a/26b and inhibits EC proliferation, migration, and tube formation, which may contribute to abnormal angiogenesis in EOPE. Thus, strategies targeting PFKFB3-driven glycolysis may be a promising approach for the treatment of EOPE.

Keywords: PFKFB3; angiogenesis; early-onset preeclampsia; endothelial cells; glycolysis.

Graphical abstract

Figure 1

MALAT1 and PFKFB3 expression in tissues from patients with EOPE (A and B) The relative expression levels of the MALAT1 mRNA (A) and PFKFB3 mRNA (B) in 16 tissues from patients with EOPE and 16 tissues from normal controls were analyzed using quantitative real-time PCR. (C) Representative FISH images of MALAT1 expression in normal tissues and tissues from patients with EOPE. (D) Representative WB images of PFKFB3 levels in normal tissues and tissues from patients with EOPE. (E) Representative IHC images of PFKFB3 expression in normal tissues and tissues from patients with EOPE. All images were obtained at 200× magnification. The data are presented as the mean ± SD of three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 using Student’s t test.

Figure 2

PFKFB3 regulates glycolytic activity and EC angiogenesis Cells were transfected with the vector, PFKFB3 plasmid, si-NC, or si-PFKFB3. (A and B) The extracellular lactate production and the cellular levels of ATP, NADPH, and ROS were measured in ECs. (C and D) The proliferation of ECs was determined using CCK-8 assays. (E and F) The cell cycle phases of ECs were determined using flow cytometry. (G and H) The migration of ECs was determined using Transwell assays. (I and J) Tube formation of ECs was determined using tube-formation assays. (K and L) Filopodia (arrowheads) and lamellipodia (arrows) were measured in ECs stained with Alexa Fluor 488-labeled phalloidin. All images in (G) and (H) were obtained at 200× magnification, whereas images in (I) and (J) were obtained at 100× magnification, and images in (K) and (L) were obtained at 1,000× magnification. The data are presented as the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 using Student’s t test.

Figure 3

MALAT1 regulates PFKFB3 expression via miR-26a and miR-26b in ECs (A and B) After transfection with sh-NC and sh-MALAT1, the mRNA expression levels of MALAT1, miR-26a, miR-26b, and PFKFB3 (A) and the protein expression level of PFKFB3 (B) were measured in ECs. (C) PFKFB3 mRNA and protein expression in ECs transfected with the mimic-NC, miR-26a mimic, and miR-26b mimic. (D) Schematic representation of the putative miR-26a and miR-26b binding sites in MALAT1. (E) Dual-luciferase reporter assay in cells co-transfected with the MALAT1 WT or MUT reporter plasmid and miR-26a/26b. (F) RNA pull-down assays were performed using biotin-labeled sense or antisense MALAT1, and quantitative real-time PCR analyses for MALAT1 and miR-26a/26b expression were performed using the pull-down products. (G) Schematic representation of the putative miR-26a and miR-26b binding sites in PFKFB3. (H) Dual-luciferase reporter assay in cells co-transfected with the PFKFB3 WT or MUT reporter plasmid and miR-26a/26b. (I and J) After transfection with sh-NC, sh-MALAT1, the sh-NC + miR-26a inhibitor, and the sh-MALAT1 + miR-26a inhibitor, the mRNA expression levels of MALAT1, PFKFB3, and miR-26a (I) and the protein expression level of PFKFB3 (J) were measured in ECs. (K and L) After transfection with sh-NC, sh-MALAT1, the sh-NC + miR-26b inhibitor, and the sh-MALAT1 + miR-26b inhibitor, the mRNA expression levels of MALAT1, PFKFB3, and miR-26b (K) and the protein expression level of PFKFB3 (L) were measured in ECs. The data are presented as the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 using one-way ANOVA and Student’s t test. n.s., not significant.

Figure 4

MALAT1 regulates glycolytic activity and EC angiogenesis through PFKFB3 Cells were transfected with sh-NC, sh-MALAT1, the sh-NC + PFKFB3 plasmid, or the sh-MALAT1 + PFKFB3 plasmid. (A) MALAT1 and PFKFB3 mRNA expression in ECs. (B) PFKFB3 protein expression in ECs. (C) Extracellular lactate production and the cellular levels of ATP, NADPH, and ROS were measured in ECs. (D) The proliferation of ECs was determined using CCK-8 assays. (E) The cell cycle phase of ECs was determined using flow cytometry. (F) The migration of ECs was determined using Transwell assays. (G) Tube formation of ECs was determined by performing tube-formation assays. (H) Filopodia (arrowheads) and lamellipodia (arrows) were measured in ECs stained with Alexa Fluor 488-labeled phalloidin. All images in (F) were obtained at 200× magnification, whereas images in (G) were obtained at 100× magnification, and images in (H) were obtained at 1,000× magnification. The data are presented as the mean ± SD of three independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 using one-way ANOVA.

Figure 5

A schematic diagram depicting how MALAT1 competitively binds miR-26a and miR-26b to regulate EC angiogenesis by modulating PFKFB3-driven glycolysis in ECs