Knockdown of the long non‑coding RNA MALAT1 ameliorates TNF‑α‑mediated endothelial cell pyroptosis via the miR‑30c‑5p/Cx43 axis
- Authors:
- Published online on: March 8, 2023 https://doi.org/10.3892/mmr.2023.12977
- Article Number: 90
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Copyright: © Yang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Atherosclerosis (AS) is a multifactorial chronic inflammatory disease with high morbidity and mortality rates worldwide (1). Cell death and inflammation are closely related to the pathogenesis mechanisms underlying AS (2,3). The former mainly occurs via apoptosis and necrosis, but a novel type of programmed cell death, that is, pyroptosis, was recently reported (4–6). The classical pyroptosis pathway depends on the activation of caspase-1. Cleaved caspase-1 is a cysteine-dependent protease that serves a vital role in the maturation and secretion of gasdermin D (GSDMD), IL-1β, IL-18 and apoptosis-associated speck-like protein containing a CARD (ASC) (7). In the late stage of pyroptosis, GSDMD is cleaved to generate the N-terminus of GSDMD (GSDMD-N) and then forms a transmembrane pore for IL-1β and IL-18 release (8). Numerous studies have reported that pyroptosis is related to the inflammatory process of AS (4,5,9). It has been reported that histone deacetylase 11 (HDAC11) promotes AS in high-fat diet (HFD)-fed ApoE-/- mice and that HDAC11 deletion alleviates tumor necrosis factor-α (TNF-α)-induced pyroptosis in human umbilical vein endothelial cells (HUVECs) by suppressing, NLR family pyrin domain containing 3 (NLRP3)/caspase-1/GSDMD and caspase-3/gasdermin E signaling pathways (10). Moreover, hydroxytyrosol acetate inhibits HFD-fed ApoE-/- mice AS by suppressing TNF-α-induced pyroptosis of HUVECs via negative regulation of the HDAC11 related signaling pathway (11). Another study reported that sirtuin 6 reduced TNF-α-induced pyroptosis of vascular endothelial cells by negatively regulating the Lin28b/let-7 signaling pathway in AS (12). Therefore, targeted regulation of pyroptotic endothelial cells is of significance to prevent AS.
Previous studies have reported that lncRNAs and micro RNAs (miRNAs) serve vital roles in cell pyroptosis (13). LncRNAs are composed of more than 200 nucleotides, and a member of the lncRNAs family, MALAT1, has been reported to be associated with pyroptosis in cardiovascular disease. For instance, MALAT1 promoted high glucose-induced pyroptosis of H9c2 cells by competitively binding to miR-141-3p (14). Moreover, up-regulation of MALAT1 aggravates NLRP3-mediated cell pyroptosis in diabetic atherosclerosis (15,16). MALAT1 knockdown significantly decreases high-glucose- induced endothelial and epithelial cell pyroptosis (16,17). However, the effect of MALAT1 on TNF-α-induced pyroptosis of rat aortic endothelial cells (RAOECs) and the underlying mechanisms have been inadequately investigated. miRNAs are composed of 20–24 nucleotides, and their main function is to participate in the regulation of post-transcriptional gene expression (18). Numerous studies have reported that dysregulation of microRNAs, such as miR-30c-5p, leads to the development of AS. Ceolotto et al (19) reported the decreased level of miR-30c-5p in microparticles accelerated AS. Moreover, Li et al (20) reported that a miR-30c-5p mimic decreased NLRP3-induced endothelial cells pyroptosis by inhibiting forkhead box O3 (FOXO3) expression. However, the effects and precise molecular mechanisms of the MALAT1/miR-30c-5p axis in TNF-α-mediated RAOEC pyroptosis remain to be elucidated.
Connexin 43 (Cx43) is an abundant component in numerous cells of the cardiovascular system. It has been reported that the development of AS is correlated with Cx43 expression (21). Furthermore, a previous study reported that miRNAs serve vital roles in the post-transcriptional regulation of Cx43 expression (22). Yang et al (23) reported that miR-1 aggravated arrhythmogenesis via regulation of Cx43. Interestingly, miR-130a also results in cardiac arrhythmias through the downregulation of Cx43 (24). Although MALAT1, miR-30c-5p and Cx43 have been reported to be involved in AS, the mechanisms and interactions still need to be elucidated.
The present study was designed to investigate the functional roles of the MALAT1/miR-30c-5p/Cx43 axis in TNF-α-induced RAOEC pyroptosis and its underlying mechanisms, which could provide new insights into AS treatment.
Materials and methods
Cell culture and treatment
RAOECs (Cell Applications Inc.) and 293T cells (American Type Culture Collection) were cultured in Dulbecco's Modified Eagle Medium (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.), penicillin (100 U/ml) and streptomycin (100 µg/ml) mixture (Beijing Solarbio Science & Technology Co., Ltd.) in a humidified incubator containing 5% CO2 at 37°C. RAOECs were exposed to TNF-α (PeproTech, Inc.) at 10 ng/ml for 24 h to induce cell pyroptosis in a humidified incubator containing 5% CO2 at 37°C.
The sense and anti-sense sequences for MALAT1, Cx43 and the negative controls were designed by Biomics Biopharma. The sequences for miR-30c-5p mimic (cat. no. miR10000804), miR-30c-5p inhibitor (cat. no. miR20000804), mimic negative control (NC) (cat. no. miR1N0000001-1-1) and inhibitor NC (cat. no. miR2N0000001-1-1) were designed and synthesized by Guangzhou RiboBio Co., Ltd. RAOECs were seeded in six-well plates with 1×106 cells/well and then incubated overnight in a humidified incubator containing 5% CO2 at 37°C. 50 nM short interfering RNA (siRNA), 30 nM miR-30c-5p mimic or 100 nM miR-30c-5p inhibitor and mimic NC or inhibitor NC were transfected into RAOECs using riboFECT™ CP Reagent (Guangzhou RiboBio Co., Ltd.) for 24 h followed by 10 ng/ml TNF-α for 24 h in a humidified incubator containing 5% CO2 at 37°C. The sequences of purchased siRNA, mimic and inhibitor were presented in Table I.
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was isolated from RAOECs using TRIzol® reagent (Takara Biotechnology Co., Ltd.). The thermocycling conditions used in RT-qPCR were set as follows: The cDNA synthesis reaction mix was incubated at 25°C for 5 min and 42°C for 30 min followed by 85°C for 5 sec. Then the reaction was terminated at 4°C. The qPCR reaction mix was incubated at 95°C for 30 sec for 1 cycle), at 95°C for 15 sec and at 60°C for 30 sec for 40 cycles), followed by dissociation. RT-qPCR was performed using Servicebio™ 2×SYBRGreen qPCR Master Mix (Wuhan Servicebio Technology Co., Ltd.) using a CFX Connect™ system (Bio-Rad Laboratories, Inc.). All experiments were performed three times, and the relative gene expression was calculated using the 2−ΔΔCq method (25). U6 was used as an internal control for miR-30c-5p, and GAPDH was used as the internal control for other targets. In the present study, the stem ring method was used for the design of primers for the detection of miR-30c-5p, as the length of mature miRNA is about 20 bp, and the appropriate detection length of RT-qPCR is 80–150 bp. Therefore, it was necessary to add stem-ring structures in the original sequence to extend the miRNA and the miRNA reverse primer was located in in the stem ring, not the miRNA. The primers for miR-30c-5p, MALAT1, U6 and GAPDH were designed and synthetized by Sangon Biotech Co., Ltd. The gap junction protein alpha 1 (GJA1) gene encodes the protein Cx43 and the primer for GJA1 was designed and synthetized by TsingKe Biological Technology. The sequences of the primers were presented in Table II.
Western blotting
Following experimental treatment, RAOECs were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology) mixed with phenylmethanesulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd.) at a ratio of 1:100. The protein concentration was assessed using a BCA protein assay kit (Applygen Technologies, Inc.). Protein samples of 20 µg/well were loaded and separated using 10 or 12% SDS-PAGE and then transferred onto PVDF membranes (MilliporeSigma). Subsequently, the membranes were blocked using 5% bovine serum albumin (Shanghai Yeasen Biotechnology Co., Ltd.) for 2 h at room temperature, and then incubated with primary antibodies against NLRP3 (1:500; cat. no. 27458-1-AP; Proteintech Group, Inc.), cleaved caspase-1 (1:500; cat. no. sc-398715; Santa Cruz Biotechnology, Inc.), IL-1β (1:500; cat. no. sc-515598; Santa Cruz Biotechnology, Inc.), Cx43 (1:1,000; cat. no. sc-271837; Santa Cruz Biotechnology, Inc.), GSDMD-N (1:1,000; cat. no. YT7991; ImmunoWay Biotechnology Company), ASC (1:1,000; cat. no. YT0365; Immunoway Biotechnology Company) and GAPDH (1:1,000 cat. no. TA802519; OriGene Technologies, Inc.) and β-actin (1:5,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) overnight at 4°C. After washing 3 times with TBST containing 0.05% Tween-20, the membranes were incubated with peroxidase-conjugated goat anti-rabbit IgG(H+L) (1:8,000; cat. no. 33101ES60; Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG(H+L) (1:8,000; cat. no. 33201ES60; Shanghai Yeasen Biotechnology Co., Ltd.) secondary antibodies for 2 h at room temperature. Protein signaling was detected using an enhanced chemiluminescence kit (Abbkine Scientific Co., Ltd.) and analyzed using ImageJ software v1.8.0 (National Institutes of Health). GAPDH was used as the internal control. Each experiment was performed three times.
Lactate dehydrogenase (LDH) release assay
An LDH assay kit (cat. no. C0017; Beyotime Institute of Biotechnology) was used to determine the LDH activity of RAOECs in each group according to the manufacturer's protocols. Briefly, 120 µl cell supernatant, collected by centrifugation at 400 × g for 5 min at room temperature and 60 µl reaction mixture (20 µl lactate, 20 µl 2-p-iodophenyl-3-nitrophenyl tetrazolium chloride and 20 µl diaphorase) were mixed and incubated for 0.5 h at room temperature. A Bio-Rad 680 spectrophotometric microplate reader (Bio-Rad Laboratories, Inc.) was used to quantify the absorbance at 490 nm.
Hoechst 33342/propidium iodide (PI) staining
RAOECs (1×105 cells/well in 12-well plates) were treated with TNF-α or transfected with different constructs. After washing with PBS, the cells were incubated with 2.5 µl Hoechst 33342 solution and PI (Beyotime Institute of Biotechnology) for 0.5 h in the dark at 4°C. After washing 3 times with PBS, the stained cells were imaged using a DMi8 inverted fluorescence microscope at 200× magnification (Leica Microsystems GmbH).
Dual-luciferase reporter assay
The targets of MALAT1 were predicted using the StarBase (http://starbase.sysu.edu.cn) and BiBiServ2 (http://bibiserv.techfak.uni-bielefeld.de/) databases. Briefly, the gene sequences of MALAT1 (human sequence used as no entry for rat on NCBI) and miR-30c-5p were acquired from NCBI (https://www.ncbi.nlm.nih.gov/) and searched using standard conditions on the BiBiServ2 website. The targets of miR-30c-5p in humans were searched using TargetScan8.0 online. The dual-luciferase reporter assay was performed to evaluate the binding of MALAT1 and miR-30c-5p as well as that of miR-30c-5p and Cx43. Partial sequences of MALAT1 or Cx43 carrying putative wild-type (WT) miR-30c-5p binding sites or mutant (MUT) sites were cloned into the GV272 luciferase vector (Shanghai GeneChem Co., Ltd.) and named MALAT1-WT, MALAT1-MUT, Cx43-WT and Cx43-MUT. Similarly, the CV045 vector containing the renilla luciferase reporter gene was also purchased from Shanghai GeneChem Co., Ltd. The 293T cell line, a powerful tool cell for expressing exogenous genes, is competent to replicate vectors carrying the SV40 region of replication, which has been widely used for retroviral production, gene expression and protein production (26). So 293T cells were co-transfected with 20 pmol miR-30c-5p mimic or mimic NC and the constructed vectors (0.5 µg of GV272 plasmid and 50 ng of CV045 plasmid) using Hieff Trans™ Liposomal Transfection Reagent (Shanghai Yeasen Biotechnology Co., Ltd.). After 48 h, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocols.
Statistical analysis
All the experiments in the present study were performed independently at least three times. The data were presented as the mean ± SD. GraphPad Prism software version 8.3.0 (GraphPad Software; Dotmatics) was used for statistical analysis. For comparisons between two groups, the unpaired Student's t-test was performed. For comparisons among multiple groups, one way analysis of variance with Tukey's post hoc test was used. P<0.05 was considered to indicate a statistically significant difference.
Results
Expression of MALAT1 mRNA and Cx43 protein are elevated but miR-30c-5p expression is decreased in TNF-α-mediated RAOEC pyroptosis
To evaluate whether MALAT1 mRNA, and miR-30c-5p and Cx43 protein expression were related to TNF-α-induced RAOEC pyroptosis, first, RAOECs were incubated with 10 ng/ml TNF-α for 0, 6, 12, 24 or 48 h. RAOEC morphology was characterized by large bubbles, which emerged from the plasma membrane at 12 h, and especially at 24 and 48 h (Fig. 1A). Consistently, western blotting results demonstrated that compared with control groups, the protein expression levels of GSDMD-N and ASC were significantly upregulated after RAOEC treatment with TNF-α for 24 and 48 h; however, there was no significant difference between the 24 and 48 h groups (P<0.05, Fig. 1B). Therefore, the RAOEC pyroptosis model which used 10 ng/ml TNF-α for 24 h was used in the subsequent experiments. Next, cell damage was evaluated by assessing LDH release, which indicated that TNF-α treatment significantly increased LDH release (t=6.031, P=0.0038, Fig. 1C). Furthermore, western blotting was performed to assess the protein expression levels of NLRP3, cleaved caspase-1 and IL-1β in TNF-α-stimulated RAOECs, and the results indicated that NLRP3 (t=7.967, P=0.0013), cleaved caspase-1 (t=6.482, P=0.0029) and IL-1β (t=5.149, P=0.0067) were all significantly upregulated compared with the control (Fig. 1D). Accordingly, it was demonstrated that the percentage of PI-positive cells was significantly elevated in TNF-α-treated RAOECs compared with the control (t=11.35, P=0.0003, Fig. 1E). Interestingly, it was also demonstrated that TNF-α treatment significantly increased the mRNA expression level of MALAT1 (t=5.801, P=0.0044, Fig. 1F) and protein expression level of Cx43 (t=6.512, P=0.0029, Fig. 1G), but significantly reduced the RNA expression level of miR-30c-5p (t=7.352, P=0.0018, Fig. 1H). The aforementioned results indicated that MALAT1, miR-30c-5p and Cx43 could be associated with TNF-α-induced RAOEC pyroptosis.
Knockdown of MALAT1 alleviates TNF-α-mediated RAOEC pyroptosis
To further assess the effect of MALAT1 on TNF-α-mediated RAOEC pyroptosis, three specific siRNAs targeting MALAT1 were designed, and the mRNA expression level of MALAT1 was assessed using RT-qPCR. The results demonstrated a significant reduction in MALAT1 expression in the siMALAT1-1 (P<0.0001) and siMALAT1-3 (P=0.0013) groups compared with the negative control, and siMALAT1-1 was considered the best and so used for subsequent experiments (Fig. 2A). Next, it was demonstrated that knockdown of MALAT1 significantly reversed the elevated mRNA expression level of MALAT1 caused by TNF-α treatment in RAOECs (Fig. 2B). Subsequently, the influence of silencing MALAT1 on TNF-α-induced pyroptosis was assessed using LDH release, pyroptosis-related protein expression levels and the percentage of PI-positive cell numbers. The results demonstrated that the increase in LDH release in TNF-α-stimulated RAOECs was significantly attenuated by MALAT1 knockdown (P=0.0007, Fig. 2C). Moreover, MALAT1 knockdown significantly reversed the up-regulation of NLRP3 (P=0.0161), GSDMD-N (P=0.0007), ASC (P=0.0159), cleaved caspase-1 (P=0.0396) and IL-1β (P=0.0267) protein expression levels in TNF-α-stimulated RAOECs compared with the TNF-α-stimulated negative control (Fig. 2D). Furthermore, the elevation of the number of PI-positive cells induced by TNF-α was significantly suppressed by MALAT1 knockdown (P=0.0135, Fig. 2E). These data indicated that MALAT1 knockdown was effective in inhibiting TNF-α-induced RAOEC pyroptosis.
miR-30c-5p overexpression attenuates TNF-α-mediated RAOEC pyroptosis
To elucidate the effect of miR-30c-5p in TNF-α-mediated RAOEC pyroptosis, miR-30c-5p overexpression was successfully established which significantly increased miR30c-5p RNA expression levels compared with the negative control (P<0.0001, Fig. 3A). In the present study, it was also demonstrated that the RNA expression level of miR-30c-5p in RAOECs transfected with 100 nM miR-30c-5p inhibitor for 48 h was significantly decreased (P=0.0030). Next, the cells were transfected with miR-30c-5p mimic before treatment with TNF-α. RT-qPCR analysis demonstrated that in the miR-30c-5p mimic + TNF-α group, the level of miR-30c-5p was significantly increased compared with the mimic NC + TNF-α group (P=0.0009, Fig. 3B). Furthermore, compared with the mimic NC + TNF-α group, the miR-30c-5p mimic significantly attenuated LDH release (P=0.0074, Fig. 3C), significantly reduced the increased protein expression levels of NLRP3 (P=0.0408), GSDMD-N (P=0.0016), ASC (P=0.0199), cleaved caspase-1 (P=0.0255) and IL-1β (P=0.0029) (Fig. 3D), and significantly reduced the proportion of PI-positive cells (P=0.0125, Fig. 3E). These results indicated that the overexpression of miR-30c-5p could inhibit TNF-α-mediated RAOEC pyroptosis.
miR-30c-5p is a target of MALAT1 and can target Cx43
Previous studies have reported that lncRNAs function as a competitive endogenous RNAs (ceRNAs) of certain miRNAs to regulate the expression and function of target mRNAs (27). To further elucidate the mechanism by which MALAT1 was involved in TNF-α-induced RAOEC pyroptosis a bioinformatic approach was used to screen the potential targets of MALAT1. Using the StarBase (http://starbase.sysu.edu.cn/) and BiBiServ (http://bibiserv.techfak.uni–bielefeld.de/) database, miR-30c-5p was identified as a potential target gene of MALAT1 (Fig. 4A). Next, to verify the predicted binding sites, a dual-luciferase reporter vector with either the wild-type MALAT1 fragment or the mutant MALAT1 fragment was constructed. The dual-luciferase reporter gene analysis results demonstrated that miR-30c-5p overexpression significantly attenuated the luciferase activity of the MALAT1-WT reporter (P=0.0047), whereas the luciferase activity of MALAT1-MUT reporter demonstrated no apparent change (Fig. 4B), which indicated a specific interaction between miR-30c-5p and MALAT1. Furthermore, the RNA expression level of miR-30c-5p was significantly elevated by MALAT1 knockdown compared with the negative control as shown using RT-qPCR analysis (P=0.0430, Fig. 4C). These demonstrated that miR-30c-5p was a target of MALAT1 and was negatively regulated by MALAT1.
Similarly, it was demonstrated that the 3′UTR of Cx43 contained certain miR-30c-5p binding sites according to the TargetScan online software (Fig. 4D). To further confirm the predicted binding sites, a dual-luciferase reporter vector with either the wild-type 3′UTR or mutant 3′UTR of Cx43 was constructed. The results demonstrated that the miR-30c-5p mimic significantly decreased the luciferase activity of the Cx43-WT reporter (P=0.0019) in 293T cells, but had no significant effect on the Cx43-MUT reporter (Fig. 4E). Moreover, compared with the mimic NC + TNF-α groups, the protein expression level of Cx43 was significantly decreased in the miR-30c-5p mimic + TNF-α groups in RAOECs (P=0.0099), whereas compared with the inhibitor NC + TNF-α groups, the protein expression level of Cx43 was significantly increased in the miR-30c-5p inhibitor + TNF-α group (P=0.0393, Fig. 4F). The above results indicated the negative regulatory effect of miR-30c-5p on Cx43 expression in TNF-α-treated RAOECs.
Cx43 downregulation attenuated TNF-α-mediated RAOEC pyroptosis
To further analyze the effect of Cx43 on TNF-α-induced RAOEC pyroptosis, RAOECs were transfected with 50 nM Cx43 siRNA and then stimulated using TNF-α. RT-qPCR and western blotting analysis demonstrated that Cx43 siRNA transfection significantly suppressed the mRNA (P=0.0149) and protein (P=0.0297) expression levels of Cx43 in TNF-α-treated RAOECs compared with the negative control (Fig. 5A and B). Moreover, compared with the siNC + TNF-α group, silencing of Cx43 significantly reduced LDH release (P=0.0265, Fig. 5C). Furthermore, the significantly increased levels of NLRP3 (P=0.0044), GSDMD-N (P=0.0310), ASC (P=0.0129), cleaved caspase-1 (P=0.0279) and IL-1β (P=0.0493) induced by TNF-α were significantly reduced by Cx43 knockdown (Fig. 5D). Downregulation of Cx43 significantly reduced the proportion of PI-positive cells stimulated by TNF-α compared with the negative control (P=0.0221, Fig. 5E). These results demonstrated that knockdown of Cx43 attenuated TNF-α-induced RAOEC pyroptosis.
MALAT1 knockdown inhibited TNF-α-induced RAOEC pyroptosis via miR-30c-5p/Cx43 axis
To further elucidate whether MALAT1 could inhibit TNF-α-induced RAOEC pyroptosis through the miR-30c-5p/Cx43 axis. RAOECs were transfected with siMALAT1 (or siNC) alone or together with miR-30c-5p inhibitor (or inhibitor NC) before treatment with TNF-α. Western blotting demonstrated that the protein expression level of Cx43 was significantly decreased by siMALAT1 transfection in TNF-α-induced RAOECs (P=0.0374), but this effect was significantly weakened by co-transfection of siMALAT1 with miR-30c-5p inhibitor (P=0.0456, Fig. 6A). Similarly, MALAT1 knockdown significantly decreased LDH release in TNF-α-induced RAOECs (P=0.0011), which was significantly reversed by co-transfection of siMALAT1 and miR-30c-5p inhibitor (P=0.0229, Fig. 6B). Furthermore, it was demonstrated that siMALAT1 significantly decreased the protein expression levels of NLRP3 (P=0.0100), GSDMD-N (P=0.0066), ASC (P=0.0115), cleaved caspase-1 (P=0.0041) and IL-1β (P=0.0304) (Fig. 6C), and the percentage of PI-positive cells (P=0.0008, Fig. 6D) compared with the negative control. However, co-transfection of siMALAT1 and miR-30c-5p inhibitor significantly attenuated the decrease in the protein expression levels of NLRP3 (P=0.0232), GSDMD-N (P=0.0421), ASC (P=0.0342), cleaved caspase-1 (P=0.0157) and IL-1β (P=0.0363), and the percentage of PI-positive cells (P=0.0174). The aforementioned results strongly indicated that miR-30c-5p inhibitor could reverse the inhibitory effects of siMALAT1 on TNF-α-induced RAOEC pyroptosis. Hence, it was concluded that MALAT1 served a vital role in TNF-α-induced RAOEC pyroptosis by the regulation of Cx43 expression by sponging miR-30c-5p.
Discussion
Endothelial dysfunction is considered to be an early step in the development of AS (28). In recent decades, anti-inflammatory agents have become an important therapeutic approach for AS (29–31). Pyroptosis is the result of inflammasome activation and has been reported to be closely involved in AS. However, the molecular mechanism has not yet been fully elucidated. Therefore, the present study assessed the upstream regulatory mechanism of pyroptosis. Previous studies have reported that cells incubated with certain concentrations of TNF-α for different time points will exhibit varied effects, including pyroptosis, apoptosis and exhaustion (32–34). Furthermore, accumulated evidence has indicated that TNF-α is a critical inflammatory factor that can increase pyroptosis-related protein expression (32). For instance, Wang et al (35) reported that a decrease in TNF-α could attenuate the process of pyroptosis. Moreover, ghrelin reduces pyroptosis, apoptosis and autophagy in human hepatocytes treated with TNF-α (32). In the present study, it was demonstrated that treatment with 10 ng/ml TNF-α for 24 h resulted in a typical pyroptotic morphology and increase in LDH release, pyroptosis-related protein expression and the proportion of PI-positive cells in RAOECs, which suggested that TNF-α induced RAOEC pyroptosis. However, whether treatment with 10 ng/ml TNF-α for 24 h impacts cells exhaustion and apoptosis requires further evaluation.
Recent studies have reported that MALAT1 is of significance in the regulation of pyroptosis in numerous cell types through direct or indirect pathways. For example, MALAT1 is considered a ceRNA that regulates the level of NLRP3 by downregulating miR-22 in high glucose-induced human endothelial cell pyroptosis (17). Similarly, knockdown of MALAT1 up-regulated miR-558 by decreasing GSDMD to inhibit inflammation, apoptosis and pyroptosis of chondrocytes in ankylosing spondylitis (36). Furthermore, MALAT1 knockdown significantly decreased the inflammation and pyroptosis of macrophages (37). In agreement with the aforementioned previous studies, in the present study, MALAT1 was significantly upregulated in TNF-α-induced RAOEC pyroptosis, whereas knockdown of MALAT1 significantly attenuated this effect. The demonstrated that MALAT1 was involved in TNF-α-induced RAOEC pyroptosis was an innovation of the present study. LncRNAs are reported to function as a ‘sponge’ or ‘ceRNA’, thus regulating miRNA expression (38). In the present study, it was demonstrated that TNF-α treatment significantly decreased the miR-30c-5p expression level in RAOECs. Previous studies have indicated that miR-30 family members, especially miR-30c-5p, act as protective factors in numerous solid tumors, such as non-small cell lung (39), pancreatic (40) and liver (41) tumors. Recently, miR-30c-5p has been reported to be closely associated with the development of cardiovascular diseases, including AS (19), diabetic nephropathy (42) and myocardial ischemia-reperfusion injury (43). Furthermore, Li et al (20) reported that the level of miR-30c-5p was decreased in ox-LDL-mediated endothelial cells, and that overexpression of miR-30c-5p suppressed the endothelial cell pyroptosis triggered by ox-LDL. Consistent with these findings, the present study also demonstrated that overexpression of miR-30c-5p attenuated TNF-α-induced RAOEC pyroptosis. Huntzinger et al (44) reported that microRNAs could induce the translational repression of targeted mRNA (relatively common in plants) or the degradation of targeted mRNA (relatively common in mammals). Subsequently, by performing a bioinformatic analysis, Pubmed search and dual-luciferase reporter assay, the potential interaction between MALAT1 and miR-30c-5p was demonstrated. Similarly, based on prediction using the TargetScan website, it was hypothesized that Cx43 might be a target gene for miR-30c-5p, which was involved in TNF-α-induced RAOEC pyroptosis. Furthermore, the dual-luciferase reporter assays significantly demonstrated that Cx43 was a direct target of miR-30c-5p, and was negatively regulated by miR-30c-5p, in accordance with the results that TNF-α treatment downregulated miR-30c-5p and upregulated Cx43 expression. Cx43 is a transmembrane protein whose main function is to form a gap junction that allows the exchange of molecules that are smaller than 1.2 kDa, such as small molecules (ATP, GTP, etc.), ions (K+, Ca2+, etc.) and secondary messengers (cAMP, cGMP, etc.) (45). Cx43 can function in various forms, namely, in gap junctions, in hemichannels or by itself, and it is involved in all cell cycle stages, such as growth, differentiation and apoptosis (46). Increasing evidence indicates that Cx43 is also closely associated with the development of cardiovascular diseases, especially AS (47,48). Nevertheless, the underlying mechanisms are not clearly defined. Morel et al (49) reported that decreased levels of Cx43 served a protective role in the development of atherosclerotic lesions in mice. Moreover, accumulating research has demonstrated that Cx43 is closely related to inflammasome activation and Cx43 gap junctions or hemichannels allowing communication between the intracellular and extracellular milieu (50,51). However, Cx43 can regulate immune cell activation, which is influenced by inflammasome activation (52). Similarly, Cx43 is associated with NLRP3 inflammasome activation in nerve pain (53), retinal disease (54) and chronic kidney disease (55). Zhang et al (56) reported that bioactive glass suppressed endothelial cell pyroptosis via decrease of the levels of Cx43 and reactive oxygen species. Blocking Cx43 alleviated renal fibrosis by decreasing the pyroptosis of macrophages (57). Consistently, the results of the present study demonstrated that Cx43 expression was markedly elevated in TNF-α-induced RAOECs and knockdown of Cx43 attenuated TNF-α-induced RAOEC pyroptosis, which indicated that Cx43 was related to the TNF-α-induced RAOEC pyroptosis.
The results of the present study demonstrated that MALAT1 could indirectly regulate the level of Cx43 by targeting miR-30c-5p. Western blotting analysis demonstrated that MALAT1 knockdown significantly reduced Cx43 expression, while miR-30c-5p inhibition significantly reversed this effect. Furthermore, miR-30c-5p inhibition suppressed the MALAT1 knockdown-induced inhibitory effect on pyroptosis in TNF-α-induced RAOEC. The present study demonstrated that MALAT1 could regulate Cx43 expression in TNF-α-induced RAOEC pyroptosis by sponging miR-30c-5p.
To the best of our knowledge, the present study is the first to demonstrate that MALAT1 regulates TNF-α-induced RAOEC pyroptosis via the miR-30c-5p/Cx43 axis, which further elucidates the pathogenesis of AS. However, certain limitations due to the complexity and diversity of the regulation between molecules in the cells used in the present study need to be taken into consideration in the future. For example, the effects of the MALAT1/miR-30c-5p/Cx43 axis in RAOEC pyroptosis in AS need to be further assessed in vivo. Secondly, in the present study, bioinformatic analyses demonstrated that miR-30c-5p has a broadly conserved binding site with lncRNA MALAT1, and it is known that miR-30c-5p is closely related to endothelial cells injury in AS (20). Furthermore, previous studies have reported that miR-30c-5p was a target gene of MALAT1 (58–60). However, the relationship between miRNA and lncRNA is very complex, and it is not currently possible to rule out other possible targets of MALAT1. Therefore, more molecules, such as miR-24, miR-204-5p and miR-26b-5p, need to be further evaluated in future. Finally, the present study was at the early stage and mainly assessed the role and underlying mechanisms of MALAT1 in TNF-α-mediated endothelial cell pyroptosis. Further studies are required to elucidate if manipulation of these genes impacted tube formation and angiogenesis of endothelial cells in vitro and in vivo.
In summary, MALAT1 was demonstrated to be significantly elevated in TNF-α-treated RAOECs and targeted miR-30c-5p expression. As a target gene of miR-30c-5p, Cx43 was significantly elevated in TNF-α-induced RAOECs. Knockdown of MALAT1 could inhibit the TNF-α-induced pyroptosis of RAOECs by indirectly downregulating Cx43 via the targeting of miR-30c-5p expression (Fig. 7), which suggested that MALAT1 may be a potential therapeutic target for the treatment of AS.
Acknowledgements
Not applicable.
Funding
This work was supported by the National Natural Science Foundation of China (grant no. 82160686), the Key Program of the Natural Science Foundation of Jiangxi Province (grant no. 20202ACB206001), the Key Research and Development Program of Jiangxi Province (grant no. 20192BBG70012), and the Research Fund for Key Laboratory of Drug Targets and Drug Screening of Jiangxi Province (grant no. 20171BCD40007).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Authors' contributions
ZJY and RL were responsible for conceptualization, the methodology, data curation and writing the original draft of the manuscript. XJH was responsible for conceptualization, formal analysis and reviewing the manuscript. CLQ and LHL were responsible for the methodology and data analysis. GLD, ZQL and YL were responsible for the methodology and data interpretation. LPJ was responsible for conceptualization, the methodology, formal analysis, reviewing the manuscript, funding acquisition and provided supervision. Z-JY and RL confirm the authenticity of all the raw data.
Ethics approval and consent for publication
Not applicable.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
LncRNAs |
long non-coding RNAs |
AS |
atherosclerosis |
MALAT1 |
metastasis associated lung adenocarcinoma transcript 1 |
TNF-α |
tumor necrosis factor-α |
RAOEC |
rat aorta endothelial cell |
miR |
microRNA |
Cx43 |
connexin 43 |
RT-qPCR |
reverse transcription-quantitative PCR |
GSDMD |
gasdermin D |
ASC |
apoptosis-associated speck-like protein containing a CARD |
GSDMD-N |
N-terminus of gasdermin D |
HDAC11 |
histone deacetylase 11 |
HFD |
high-fat diet |
HUVECs |
human umbilical vein endothelial cells |
NLRP3 |
NLR family pyrin domain containing 3 |
miRNAs |
micro RNAs |
FOXO3 |
forkhead box O3 |
siRNA |
short interfering RNA |
GJA1 |
gap junction protein alpha 1 |
PI |
propidium iodide |
NC |
negative control |
ceRNAs |
competitive endogenous RNAs |
LDH |
lactate dehydrogenase |
WT |
wild-type |
MUT |
mutant |
Cont |
control |
C-caspase-1 |
cleaved caspase-1 |
F |
forward |
R |
reverse |
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