LncRNA MALAT1 modulates ox-LDL induced EndMT through the Wnt/β-catenin signaling pathway - PubMed (original) (raw)

LncRNA MALAT1 modulates ox-LDL induced EndMT through the Wnt/β-catenin signaling pathway

Hongrong Li et al. Lipids Health Dis. 2019.

Abstract

Background: Endothelial-to-mesenchymal transition (EndMT) plays significant roles in atherosclerosis, but the regulatory mechanisms involving lncRNAs remain to be elucidated. Here we sort to identify the role of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in ox-LDL-induced EndMT.

Methods: The atherosclerosis model was established by feeding ApoE-/- mice with high-fat diet, and the levels of lncRNA MALAT1 in mouse arterial tissue were detected by RT-qPCR. Cell model was established by treating human umbilical vein endothelial cells (HUVECs) with ox-LDL, and the levels of EndMT markers, such as CD31, vWF, α-SMA and Vimentin and lncRNA MALAT1 levels were detected and their correlations were analyzed. The role of MALAT1 in EndMT and its dependence on Wnt/β-catenin signaling pathway was further detected by knocking down or overexpressing MALAT1.

Results: MALAT1 was upregulated in high-fat food fed ApoE-/- mice. HUVECs treated with ox-LDL showed a significant decrease in expression of CD31 and vWF, a significant increase in expression of α-SMA and vimentin, and upregulated MALAT1. An increased MALAT1 level facilitated the nuclear translocation of β-catenin induced by ox-LDL. Inhibition of MALAT1 expression reversed nuclear translocation of β-catenin and EndMT. Moreover, overexpression of MALAT1 enhanced the effects of ox-LDL on HUVEC EndMT and Wnt/β-catenin signaling activation.

Conclusions: Our study revealed that the pathological EndMT required the activation of the MALAT1-dependent Wnt/β-catenin signaling pathway, which may be important for the onset of atherosclerosis.

Trial registration: Not applicable.

Keywords: Atherosclerosis; EndMT; MALAT1; Ox-LDL; Wnt/β-catenin.

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Conflict of interest statement

Competing interest

The authors declare that they have no competing interests.

All animal experiments were conducted according to the ethical guidelines of Animal Ethics Committee of Hebei Medical University.

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Figures

Fig. 1

Fig. 1

Changes of serum lipid, histology and MALAT1 in mice**.** _ApoE_−/− mice were fed with HFD for 16 weeks, and normally fed C57B/6 mice were used as control. a Serum lipid levels of the two groups were detected by automatic biochemical analyzer (n = 9, **P < 0.01, versus control group). b H&E staining of vessel wall. Magnification, 200x. c Level of MALAT1 in aortas tissues as determined by qRT-PCR (n = 3, **P < 0.01, versus control group)

Fig. 2

Fig. 2

Changes of cellular morphology, EndMT markers and MALAT1 in HUVECs. HUVECs were treated with ox-LDL at different concentrations (10, 20, 40 μg/ml) for 48 h. a Cellular F-actin was stained with rhodamine phalloidin. Nuclei were stained with DAPI Fluoromount-G®. Cells were observed under fluorescence microscopy. Magnification, 400x. b-d Endothelial markers (CD31, vWF) and mesenchymal markers (α-SMA, vimentin) were detected by qRT-PCR (B), Western blot (C) (n = 3, *P < 0.05, **P < 0.01, versus control group) and immunofluorescence analysis (D). Magnification, 400x. e MALAT1 expression was detected by qRT-PCR (n = 3, *P < 0.05, **P < 0.01 versus control group). The relative levels of α-SMA, vimentin, CD31, vWF and MALAT1 in ox-LDL groups were normalized to those in control group, respectively. f Pearson correlations between MALAT1 and α-SMA mRNA, vimentin mRNA, CD31 mRNA, vWF mRNA

Fig. 3

Fig. 3

Knockdown of MALAT1 attenuates ox-LDL-induced EndMT in HUVECs. a HUVECs were transfected with MALAT1-siRNA1, MALAT1-siRNA2, MALAT1-siRNA3 or scramble control (scr) for 24 h. The MALAT1 levels were determined by qRT-PCR (n = 3, *P < 0.05, versus negative control group). b The effect of MALAT1-siRNA on cell morphology induced by ox-LDL (40 μg/ml) was observed through rhodamine phalloidin-stained F-actin. Magnification, 400x. c-e The effect of MALAT1-siRNA3 on the expression of endothelial markers (CD31, vWF) and mesenchymal markers (α-SMA, vimentin) were detected by qRT-PCR (c), Western blot (d) (n = 3, *P < 0.05, **P < 0.01 versus control group, #P < 0.05, ##P < 0.01 versus ox-LDL group) and immunofluorescence analysis (e). Magnification, 400x

Fig. 4

Fig. 4

The effect of MALAT1 overexpression on EndMT of HUVECs. Full-length human MALAT1 cDNA plasmid (pcDNA-MALAT1) was constructed and transfected into HUVECs. The empty vector was used as negative control. a MALAT1 expression was detected by qRT-PCR (n = 3, *P < 0.05, versus pcDNA vector group). b The effect of MALAT1 overexpression on cell morphology induced by ox-LDL (40 μg/ml) was observed through rhodamine phalloidin-stained F-actin. Magnification, 400x. c-e The effect of MALAT1 overexpression on the expression of endothelial markers (CD31, vWF) and mesenchymal markers (α-SMA, vimentin) were detected by qRT-PCR (c), Western blot (d) (n = 3, *P < 0.05, **P < 0.01 versus pcDNA vector group) and immunofluorescence analysis (e). Magnification, 400x

Fig. 5

Fig. 5

Knockdown of MALAT1 inhibits ox-LDL-induced β-catenin translocation. HUVECs were transfected with MALAT1-siRNA or scramble control (scr) for 24 h. a The protein expression of β-catenin in nucleus (n = 3, *P < 0.05, versus control group, #P < 0.05, versus model group) was analyze by Western blot. b The location of β-catenin was detected by immunofluorescence analysis. Magnification, 400x

Fig. 6

Fig. 6

Overexpression of MALAT1 promotes activation of Wnt/β-catenin pathway. HUVECs were transfected with pcDNA-MALAT1 or pcDNA vector for 48 h. a The protein level of β-catenin in nucleus (n = 3, *P < 0.05, versus pcDNA vector group) was analyze by Western blot. b The location of β-catenin was detected by immunofluorescence analysis. Magnification, 400x

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