LncRNA MALAT1 modulates ox-LDL induced EndMT through the Wnt/β-catenin signaling pathway
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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.
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.
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.
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.
KeywordsAtherosclerosis EndMT Ox-LDL MALAT1 Wnt/β-catenin
Human umbilical vein endothelial cell line
Low-density lipoprotein cholesterol
Metastasis-associated lung adenocarcinoma transcript 1
Oxidized low-density lipoprotein
Atherosclerosis is a multistep cardiovascular disease promoted by several kinds of risk factors and is one of the most common causes of mortality in the world . To date, the pathological mechanism responsible for the development and progression of atherosclerosis is largely unrevealed. Endothelial-to-mesenchymal transition (EndMT), a special type of epithelial-to-mesenchymal (EMT) , is a complex biological process through which endothelial cells convert to mesenchymal or myofibroblastic cells. EndMT is characterized by the loss of specific endothelial markers, and acquisition of mesenchymal markers. Recent studies have demonstrated that EndMT might be closely related to the atherosclerosis progression [3, 4]. For example, EndMT-derived cells are common in atherosclerotic lesions in mice . The ‘transitioning’ cells co-expressing endothelial and fibroblast/ mesenchymal proteins are also detected in human plaques . Moreover, EndMT enhances plaque calcification to increase plaque burden , and the extent of EndMT is associated with plaque instability in the clinical events . More importantly, the canonical risk factors leading to atherosclerotic lesion formation, such as cytokines , reactive oxygen species (ROS) , and high glucose  are also found to trigger EndMT. As one of the causal risk factors for atherosclerosis, oxidized low-density lipoprotein (ox-LDL) activates the receptor of ox-LDL in endothelial cells  to up-regulate stimulators such as inflammatory cytokines , ROS  and TGF-β , which can further induce EndMT. However, the precise regulators of EndMT in response to ox-LDL are still poorly understood.
Metastasis associated lung adenocarcinoma transcript 1 (MALAT1), which is also known as NEAT2 (noncoding nuclear-enriched abundant transcript 2) and was first identified in 2003 in non-small cell lung cancer [14, 15], is one of the initially identified long non-coding RNAs associated with human diseases. MALAT1 is evolutionarily conserved among mammalians, and is abundantly expressed in many tissues [15, 16]. Mature MALAT1 is localized in nucleus with a cytoplasmic tRNA-like small RNA, known as mascRNA . When RNA polymerase II-dependent transcription is activated, MALAT1 becomes enriched in nuclear speckles-dynamic and irregularly shaped nuclear domains involved in pre-mRNA splicing and RNA transport in mammalian cells . MALAT1 has been implicated in functional regulation of a variety of cells involved in atherosclerosis, such as vascular endothelial cells [19, 20, 21], smooth muscle cells  and macrophages . It is reported that ox-LDL could induce MALAT1 transcription through NF-κB pathway in THP-1-derived macrophages , and MALAT1 can modulate TGF-β1-induced EndMT in endothelial progenitor cells .
Wnt/β-catenin pathway, the canonical Wnt signaling, is reported to be involved in multiple aspects of the development and progression of atherosclerosis , including endothelial dysfunction , macrophage activation [28, 29] and the proliferation and migration of vascular smooth muscle cells . On the other hand, the Wnt/β-catenin signaling pathway is reported to be critical in regulating EndMT . When the Wnt signaling pathway is activated, the phosphorylation of β-catenin is inhibited and free β-catenin accumulates and translocates into the nucleus to modulate expression of EndMT regulatory genes, such as Twist, Snail and Slug [32, 33, 34]. Wnt/β-catenin signaling pathway participates in the EndMT of adult valvular endothelial cells  and human renal glomerular endothelial cell line . It has also been shown that MALAT1 could promote the expression and nuclear accumulation of β-catenin in osteosarcoma , esophageal squamous cell carcinoma  and renal cell carcinoma .
In this study, we intended to elucidate whether MALAT1 participates in the regulation of ox-LDL-induced EndMT through the Wnt/β-catenin signaling pathway. We found that MALAT1 was dysregulated in arterial tissues from ApoE−/− mice with high-fat food-induced atherosclerosis and human umbilical vein endothelial cell line (HUVECs) stimulated with ox-LDL. Overexpression of MALAT1 had similar effect as ox-LDL on EndMT of HUVECs and nuclear translocation of β-catenin. MALAT1 knockdown rescued EndMT of HUVECs via partially reversing β-catenin nuclear accumulation triggered by ox-LDL. Collectively, these data highlight the importance of MALAT1 in the development and progression of atherosclerosis.
Materials and methods
Male C57BL/6 mice (8-week old) and ApoE−/− (B6/JNju-Apoeem1Cd82/Nju) mice were obtained from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). C57BL/6 mice were fed with standard chow diet and were used as control group. ApoE−/− mice were fed with high-fat diet (HFD) containing 21% fat, 1.25% cholesterol for 16 weeks and were used as atherosclerosis model group. All animals were kept in rooms with constant room temperature (25 °C) and with 12 h light-dark cycle (light on from 8:00 AM to 8:00 PM). The animal experiment was performed in accordance with the Animal Ethics Committee of Hebei Medical University.
The thoracic aorta was harvested from mice following 16 weeks of HFD (at 24 weeks of age). After humane euthanasia, the thorax was entered and the heart swiftly exposed. The left ventricle was punctured using a 20-gauge needle, and 1x PBS (20 ml) at 4 °C was infused at 2.5 ml/min. For H&E staining, the thoracic aorta was then fixed in situ by perfusing the heart with 20 ml of 4% paraformaldehyde in PBS. The thoracic aorta was then isolated and placed in 4% paraformaldehyde for 24 h. For real-time quantitative PCR, the thoracic aorta was harvested and preserved in liquid nitrogen. The blood was harvested from mice by removing the eyeball under anesthesia following 16 weeks of HFD (at 24 weeks of age). Serum was isolated from blood by centrifugation for 15 min at 3000 rpm/min at 4 °C and stored at − 80 °C until each assay was performed.
Serum lipid levels
The serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) levels were quantified by Hitachi 7080 automatic biochemical analyzer (Tokyo, Japan).
Hematoxylin and eosin (H&E) staining
After gradient dehydration and transparency, the thoracic aorta was embedded with paraffin and subsequently sectioned at 4 μm thickness. Then, the sections were stained with Hematoxylin and Eosin Staining Kit (H&E) (Beyotime, Shanghai, China), and then photo-graphed with Leica DM6000B fully automatic biological microscope (Wetzlar, Germany).
Cell culture and treatment
HUVECs were from Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and were cultured in Dulbecco’s modified Eagle medium (Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS (Gibco BRL, Gaithersburg, MD, USA), 2 mM glutamine, and 1% penicillin in a humidified atmosphere with 5% CO2 at 37 °C. HUVECs were treated with ox-LDL (Peking Union-Biology, Beijing, China) at different concentrations (10, 20, 40 μg/ml) for 48 h.
Plasmid construction and cell transfection
For MALAT1 overexpression, a pcDNA3.1 expression vector containing full-length human MALAT1 (pcDNA-MALAT1) was constructed by Shanghai Integrated Biotech Solutions (Shanghai, China). For MALAT1 knockdown, three MALAT1-targeting siRNAs (MALAT1-siRNA1, 5′-GGUGGUGG UAUUUAGAUAATTUUAUCUAAA UACCACCACCTT-3′; MALAT1-siRNA2, 5′-GCGUCAUUUAAAGCCUAGUTTA CUAGGCUUUAAAUGACGCTT3’; MALAT1-siRNA3, 5′-GGGCUGACAUUAAC UACAATTUUGUAGUUAAUGUCA GCCCTT-3′) and a scrambled siRNA (sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAA TT-3′) (Scramble) were designed and synthesized at GenePharma (Shanghai, China). Lipofectamine™ 3000 Transfection Reagent (Thermo Fisher, USA) was used. Real-time quantitative PCR was used to validate the efficiency of MALAT1 knockdown and overexpression. For knockdown of MALAT1, HUVECs were transfected with MALAT1 siRNA or negative control siRNA for 24 h before treatment with ox-LDL (40 μg/ml) for 48 h. For overexpression of MALAT1, HUVECs were transfected with MALAT1 pcDNA or MALAT1 expressing vector for 48 h.
Cell morphology analysis
For observing cell morphology, cellular F-actin was stained with rhodamine phalloidin (Thermo Fisher, USA), and nuclei were stained with DAPI Fluoromount-G® (Southern Biotech, Birmingham, USA). Then the cells were imaged using a confocal laser scanning microscopy (ZEISS, Oberkochen, Germany).
Real-time quantitative PCR
The sequences for the primers
Gene (Accession No.)
The total protein was extracted from cells with ice-cold lysis buffer supplemented with protease inhibitor cocktail (Merck, Germany) and PMSF. The nuclear protein was extracted with Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China). The protein concentration was determined with Detergent Compatible Bradford Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated on SDS-PAGE gels and transferred onto nitrocellulose membrane (Life Technologies, USA). After blocking with Odyssey® blocking buffer (PBS) (LI-COR, Lincoln, USA), the membranes were incubated overnight with the primary antibodies of CD31 (ab28364,1:500), vWF (ab6994,1:500), α-SMA (ab32575,1:1000), vimentin (ab92547,1:1000), Histone H3 (ab1791,1:1000), β-catenin (ab32572,1:5000) and GAPDH (ab181602,1:10000) (abcam, Cambridge, UK). After that, IRDye 800CW Goat anti-Rabbit IgG (H + L) Secondary Antibody (926–32,211, 1:10000), IRDye® 680LT Goat anti-Rabbit IgG (H + L) Secondary Antibody (926–68,021) (LI-COR, Lincoln, USA) were added and incubated for 1 h at room temperature. Then the band intensities were scanned by Odyssey (LI-COR, Lincoln, USA) and normalized to GAPDH or Histone H3.
After ox-LDL treatment or transfection, immunostaining of HUVECs was performed. Cells were fixed with 4% polyoxymethylene for 10 min at room temperature, 10 min at 4 °C and methanol for 5 min, then blocked with 1% BSA for 1 h at room temperature and incubated with primary antibodies of CD31 (ab28364,1:20), vWF (ab6994,1:400), α-SMA (ab32575,1:500), vimentin (ab92547,1:300), and β-catenin (ab32572,1:250) (Abcam, Cambridge, UK) overnight at 4 °C. After washing, Goat Anti-Rabbit IgG H&L (DyLight® 488) (ab96899, 1:200) was added and incubated for 1 h at room temperature, followed by nuclei staining with DAPI Fluoromount-G® (Southern Biotech, Birmingham, USA). Then the cells were imaged using a confocal laser scanning microscopy (ZEISS, Oberkochen, Germany).
All graphs were created using Graphpad Prism software. All data were analyzed using SPSS version 19.0 software (SPSS lnc) and presented as mean ± SD (standard deviation). The two-tailed Student’s t-test was used to evaluate differences between two groups. Results from multiple groups were compared using one-way ANOVA test. Correlation analyses between variables were performed using the Pearson rank correlation test. P < 0.05 was accepted as statistically significant.
MALAT1 is upregulated in mouse model of atherosclerosis
MALAT1 is upregulated during ox-LDL induced EndMT of HUVECs
Knockdown of MALAT1 attenuates ox-LDL-induced EndMT in HUVECs
Overexpression of MALAT1 promotes EndMT in HUVECs
Knockdown of MALAT1 inhibits ox-LDL-induced activation of Wnt/β-catenin pathway
Overexpression of MALAT1 promotes activation of Wnt/β-catenin pathway by ox-LDL
There is increasing evidence that EndMT plays important roles in the occurrence and development of atherosclerosis  and promoting the instability of plaques . When EndMT occurs, endothelial cells present the shape and properties of mesenchymal cells (smooth muscle cells, fibroblasts), including migration, proliferation, secretion of extracellular matrix (fibronectin, collagen, et al) and matrix metalloproteinases, and, expression of various leukocyte adhesion molecules , all of which are related to the formation, progression and instability of atherosclerotic plaques. Pathologic intimal thickening in human atherosclerosis is formed by dispersion of intimal smooth muscle cells and extracellular accumulation of plasma-derived lipids . EndMT results in a loss of barrier function, that is, increased permeability, which is conducive to the passage and deposition of lipids , and EndMT is one of the sources of vascular smooth muscle cells in vascular intima . The role of EndMT in atherosclerosis is very clear, but its mechanism remains to be further studied.
Extensive evidence shows that MALAT1 plays critical roles in coronary atherosclerotic heart disease . For instance, MALAT1 was upregulated in patients with unstable angina pectoris , but another study showed that MALAT1 was less expressed in atherosclerotic coronary artery plaques compared to the non-atherosclerotic internal artery, which is histopathologically similar to coronary artery in terms of structural layers but atherosclerosis-resistant . Moreover, rs619586AG/GG genotypes in MALAT1 were reported to protect against the occurrence of coronary atherosclerotic heart disease . In the present study, MALAT1 is upregulated in arterial tissues of in HFD-fed ApoE−/− mice. This is in line with previous studies, demonstrating that MALAT1 is upregulated in macrophages  and HUVECs  treated with ox-LDL, and in proliferative vascular smooth muscle cells , all of which are critical cells for atherosclerosis. Then, it has been shown that MALAT1 plays a significant role in the process of EMT and EndMT. MALAT1 promotes EMT of nasopharyngeal carcinoma cells through de-repressing Capn4 by sponging miR-124  and mediates TGF-β1-induced EndMT by regulating TGFBR2 and SMAD3 through miR-145 in endothelial progenitor cells (EPCs) . Thus, we performed further in vitro studies to elucidate the role of MALAT1 in EndMT process of atherosclerosis.
LDL-C, especially ox-LDL, plays a central role in the development of atherosclerosis, and subclinical atherosclerosis remains common in individuals with LDL-C < 70 mg/dl . Ox-LDL activates the receptor of ox-LDL in endothelial cells  to up-regulate stimulators of EndMT, such as inflammatory cytokines , ROS  and TGF-β . Moreover, ox-LDL can promote EMT of prostate cancer cells  and human renal proximal tubular epithelial cells . Recent study showed that ox-LDL induced EndMT by stabilizing Snail in human aortic endothelial cells . In the present study, the morphology of HUVECs changed from cobblestone-like shape to spindle shape after induction by ox-LDL, accompanying with a decrease of endothelial markers CD31 and vWF, and an increase of mesenchymal markers α-smooth muscle actin (α-SMA) and vimentin. These data suggest that ox-LDL could induce EndMT in HUVECs. Furthermore, there was a significant linear correlation between the change of MALAT1 and EndMT markers, suggesting a link between MALAT1 and EndMT of HUVECs induced by ox-LDL. Next, MALAT1 was knocked-down or overexpressed to detect whether MALAT1 participates in the process of ox-LDL-induced EndMT. As expected, MALAT1 silencing reversed the changes of morphology and EndMT markers in HUVECs treated with ox-LDL. In contrast, ectopic MALAT1 expression could induce the expression of CD31 and vWF, and inhibited the expression of α-SMA and Vimentin, with slightly changed morphology of HUVECs. These data suggested that MALAT1 was involved in ox-LDL-induced EndMT.
Wnt/β-catenin is one of the signaling pathways involved in EndMT . When Wnt/β-catenin signaling pathway is activated, β-catenin is translocated into the nuclei to interact with transcription factors from the T-cell factor / leucocyte enhancer factor family and to activate transcription of Wnt responsive genes, leading to the occurrence of EndMT . Here in this study, Wnt/β-catenin pathway was activated by ox-LDL, as demonstrated by increased protein expression of β-catenin in nuclei. The expression and nuclear transposition of β-catenin induced by ox-LDL was inhibited by MALAT1 knockdown while promoted by MALAT1 overexpression. So it can be concluded that MALAT1 may modulate ox-LDL-induced EndMT partially through the Wnt/β-catenin signaling pathway.
This study demonstrates that MALAT1 is dysregulated in arterial tissues from atherosclerotic mice and HUVECs treated with ox-LDL, and uncovers the important role of MALAT1 in promoting ox-LDL induced HUVEC EndMT in vitro, which is dependent on Wnt/β-catenin signaling pathway. Thus, these findings might connect the ox-LDL-induced EndMT to its notorious role in atherosclerosis and highlight the importance of MALAT1 in the development of atherosclerosis.
This work was supported by the National key research and development plan “Chinese medicine modernization research” key special project of China [grant number 2017YFC1700500].
Availability of data and materials
The data used to support the findings of this study are available from the corresponding author upon request.
LHR analyzed the data and wrote the manuscript; ZQF, YYJ and BHY analyzed the data; CLP, WC, WHT, LJQ and WYL revised the manuscript; LHR and CM designed the study; WYL contributed to the discussion and edited the manuscript as corresponding author.All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
All animal experiments were conducted according to the ethical guidelines of Animal Ethics Committee of Hebei Medical University.
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