NEAT1 contributes to ox-LDL-induced inflammation and oxidative stress in macrophages through inhibiting miR-128

Long noncoding RNAs (lncRNA) have been recognized as significant regulators in the progression of atherosclerosis (AS). Oxidized low-density lipoprotein (ox-LDL) can induce macrophage inflammation and oxidative stress, that serves important roles in AS. However, the exact function of lncRNA NEAT1 and its possible molecular mechanism in AS remain unclear. Here, we concentrated on the roles and molecular mechanisms of NEAT1 in AS development. In our current study, we observed that NEAT1 was elevated by ox-LDL in a dose-dependent and time-dependent manner. RAW264.7 cell survival was greatly enhanced, and cell apoptosis was significantly inhibited by LV-shNEAT1 transfection. In addition, knockdown of NEAT1 in RAW264.7 cells repressed CD36 expression and foam cell formation while NEAT1 overexpression shown an opposite process. Moreover, NEAT1 downregulation inhibited inflammation molecules including IL-6, IL-1β, and TNF-α. Meanwhile, silencing of NEAT1 can also suppress reactive oxygen species (ROS) and malondialdehyde (MDA) levels with an enhancement of superoxide dismutase (SOD) activity in RAW264.7 cells. MicroRNAs are some short RNAs, and they can regulate multiple biological functions in many diseases including AS. Here, we found that miR-128 expression was remarkably decreased in ox-LDL-incubated RAW264.7 cells. Interestingly, miR-128 mimics was able to reverse AS-correlated events induced by overexpression of NEAT1. By using bioinformatics analysis, miR-128 was predicted as a target of NEAT1 and the correlation between them was validated in our study. Taken these together, it was implied that NEAT1 participated in ox-LDL-induced inflammation and oxidative stress in AS development through sponging miR-128.     

Circ_0002984 induces proliferation,migration and inflammation response of VSMCs induced by ox-LDL through miR‑326-3p/VAMP3 axis in atherosclerosis

Atherosclerosis can result in multiple cardiovascular diseases. Circular RNAs (CircRNAs) have been reported as significant non-coding RNAs in atherosclerosis progression. Dysfunction of vascular smooth muscle cells (VSMCs) is involved in atherosclerosis. However, up to now, the effect of circ_0002984 in atherosclerosis is still unknown. Currently, we aimed to investigate the function of circ_0002984 in VSMCs incubated by oxidized low-density lipoprotein (ox-LDL). Firstly, our findings indicated that the expression levels of circ_0002984 were significantly up-regulated in the serum of atherosclerosis patients and ox-LDL-incubated VSMCs. Loss of circ_0002984 suppressed VSMC viability, cell cycle distribution and migration capacity. Then, we carried out ELISA assay to determine TNF-α and IL-6 levels. The data implied that lack of circ_0002984 obviously repressed ox-LDL–stimulated VSMC inflammation. Meanwhile, miR-326-3p, which was predicted as a target of circ_0002984, was obviously down-regulated in VSMCs treated by ox-LDL. Additionally, after overexpression circ_0002984 in VSMCs, a decrease in miR-326-3p was observed. Subsequently, miR-326-3p was demonstrated to target vesicle-associated membrane protein 3 (VAMP3). Therefore, we hypothesized that circ_0002984 could modulate expression of VAMP3 through sponging miR-326-3p. Furthermore, we confirmed that up-regulation of miR-326-3p rescued the circ_0002984 overexpressing-mediated effects on VMSC viability, migration and inflammation. Additionally, miR-326-3p inhibitor-mediated functions on VSMCs were reversed by knockdown of VAMP3. In conclusion, circ_0002984 mediated cell proliferation, migration and inflammation through modulating miR-326-3p and VAMP3 in VSMCs, which suggested that circ_0002984 might hold great promise as a therapeutic strategy for atherosclerosis.     

Long noncoding RNA NEAT1 sponges miR-495-3p to enhance myocardial ischemia-reperfusion injury via MAPK6 activation

The biological function of long noncoding RNA NEAT1 has been revealed in a lot of diseases. Nevertheless, it is still not yet clear whether NEAT1 can modulate the process of myocardial ischemia–reperfusion injury (M-I/R). Here, we reported that NEAT1 was able to sponge miR-495-3p to contribute to M-I/R injury through activating mitogen-activated protein kinase 6 (MAPK6). First, elevated expression of NEAT1 was revealed in M-I/R injury mice, meanwhile, lactate dehydrogenase (LDH) and creatine kinase-muscle/brain (CK-MB) were also upregulated in the serum. Meanwhile, as previously reported, miR-495 serves as a tumor suppressor or an oncogenic miRNA in different types of cancer. Currently, we found miR-495-3p was remarkably reduced in M-I/R mice. Additionally, NEAT1 was significantly induced whereas miR-495-3p was greatly reduced by H₂O₂ treatment in H9C2 cells. Moreover, loss of NEAT1 in H9C2 cells could repress the viability and proliferation of cells. For another, overexpression of NEAT1 exhibited an opposite phenomenon. Furthermore, LDH release and caspase-3 activity were obviously triggered by upregulation of NEAT1 while suppressed by NEAT1 knockdown. miR-495-3p was indicated and validated as a target of NEAT1 using the analysis of bioinformatics. Interestingly, we observed that miR-495-3p mimics repressed tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-18 protein expression while their levels were enhanced by the inhibition of miR-495-3p in H9c2 cells. Subsequently, it was manifested that MAPK6 was a target of miR-495-3p, which could exert a lot in the NEAT1/miR-495-3p-mediated M-I/R injury. Overall, our results implied that NEAT1 contributed to M-I/R injury via the modulation of miR-495-3p and MAPK6.     

LncRNA UCA1 sponges miR-206 to exacerbate oxidative stress and apoptosis induced by ox-LDL in human macrophages

Long noncoding RNA UCA1 has exerted a significant effect in cardiovascular disease. The biological role of UCA1 in atherosclerosis is unclear. Our study was to identify the potential mechanisms in the progression of atherosclerosis. Here, we observed that ox-LDL increased UCA1 expression greatly in THP-1 cells. Knockdown of UCA1 greatly inhibited CD36 expression, a crucial biomarker in atherosclerosis. Meanwhile, 20 μg/ml ox-LDL induced foam cell formation, which can be reversed by downregulation of UCA1. In addition, TC and TG levels induced by ox-LDL was rescued by UCA1 small interfering RNA. Accumulating studies have indicated that oxidative stress contributes to atherosclerosis progression. Here, we also found that reactive oxygen species, MDA, and THP-1 cell apoptosis were restrained by decreased of UCA1 with an increase of the superoxide dismutase activity. Moreover, miR-206 was predicted as a target of UCA1 and knockdown of UCA1 was able to repress miR-206 expression. Furthermore, overexpression of miR-206 inhibited oxidative stress process and it was reversed by UCA1 upregulation in vitro. In conclusion, we indicated that UCA1 sponged miR-206 to exacerbate atherosclerosis events induced by ox-LDL in THP-1 cells.     

MicroRNA-328 ameliorates oxidized low-density lipoprotein-induced endothelial cells injury through targeting HMGB1 in atherosclerosis

Atherosclerosis has been recognized as a chronic inflammatory disease, which can harden the vessel wall and narrow the arteries. MicroRNAs exhibit crucial roles in various diseases including atherosclerosis. However, so far, the role of miR-328 in atherosclerosis remains barely explored. Therefore, our study concentrated on the potential role of miR-328 in vascular endothelial cell injury during atherosclerosis. In our current study, we observed that oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptosis and inhibited cell viability dose-dependently and time-dependently. In addition, indicated dosage of ox-LDL obviously triggered HUVECs inflammation and oxidative stress process. Then, it was found that miR-328 in HUVECs was reduced by ox-LDL. HUVECs apoptosis was greatly repressed and cell survival was significantly upregulated by overexpression of miR-328. Furthermore, mimics of miR-328 rescued cell inflammation and oxidative stress process induced by ox-LDL. Oppositely, inhibitors of miR-328 strongly promoted ox-LDL-induced endothelial cells injury in HUVECs. By using bioinformatics analysis, high-mobility group box-1 (HMGB1) was predicted as a downstream target of miR-328. HMGB1 has been reported to be involved in atherosclerosis development. The correlation between miR-328 and HMGB1 was validated in our current study. Taken these together, it was implied that miR-328 ameliorated ox-LDL-induced endothelial cells injury through targeting HMGB1 in atherosclerosis.     

Nicotinate-Curcumin Impedes Foam Cell Formation from THP-1 Cells through Restoring Autophagy Flux

Our previous studies have indicated that a novel curcumin derivate nicotinate-curcumin (NC) has beneficial effects on the prevention of atherosclerosis, but the precise mechanisms are not fully understood. Given that autophagy regulates lipid metabolism, the present study was designed to investigate whether NC decreases foam cell formation through restoring autophagy flux in oxidized low-density lipoprotein (ox-LDL)-treated THP-1 cells. Our results showed that ox-LDL (100 μg/ml) was accumulated in THP-1 cells and impaired autophagy flux. Ox-LDL-induced impairment of autophagy was enhanced by treatment with the autophagy inhibitor chloroquine (CQ) and rescued by the autophagy inducer rapamycin. The aggregation of ox-LDL was increased by CQ, but decreased by rapamycin. In addition, colocalization of lipid droplets with LC3-II was remarkably reduced in ox-LDL group. In contrast, NC (10 μM) rescued the impaired autophagy flux by significantly increasing level of LC3-II, the number of autophagolysosomes, and the degradation of p62 in ox-LDL-treated THP-1 cells. Inhibition of the PI3K-Akt-mTOR signaling was required for NC-rescued autophagy flux. Notably, our results showed that NC remarkably promoted the colocalization of lipid droplets with autophagolysosomes, increased efflux of cholesterol, and reduced ox-LDL accumulation in THP-1 cells. However, treatment with 3-methyladenine (3-MA) or CQ reduced the protective effects of NC on lipid accumulation. Collectively, the findings suggest that NC decreases lipid accumulation in THP-1 cells through restoring autophagy flux, and further implicate that NC may be a potential therapeutic reagent to reverse atherosclerosis.     

Tanshinone IIA inhibits the adipogenesis and inflammatory response in ox-LDL-challenged human monocyte-derived macrophages via regulating miR-130b/WNT5A

Atherosclerosis is a kind of chronic cardiovascular disease, characterized by oxidized low-density lipoprotein (ox-LDL) accumulation in macrophage. Tanshinone IIA (Tan), a lipophilic pharmacologically activate compound from Salvia miltiorrhiza Bunge, has been indicated to exert cardioprotective roles. Nevertheless, the biological role of Tan and regulatory mechanism in atherosclerosis are not fully established. In present study, atherosclerosis model was established in THP-1-derived macrophages by treatment of ox-LDL. The adipogenesis was measured by Nile red staining. The expressions of inflammatory factors, microRNA-130b (miR-130b) and WNT5A were measured by quantitative real-time polymerase chain reaction or Western blot. The target association between miR-130b and WNT5A was explored via luciferase activity and RNA immunoprecipitation assay. The results showed that exposure of Tan inhibited ox-LDL-induced adipogenesis and expressions of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-alpha in THP-1-derived macrophages. miR-130b expression was decreased in THP-1-derived macrophages treated by ox-LDL and its overexpression attenuated adipogenesis as well as inflammatory response. miR-130b knockdown reversed the regulatory effect of Tan on adipogenesis and inflammatory response in THP-1-derived macrophages stimulated by ox-LDL. In addition, WNT5A acted as a functional target of miR-130b and inhibited by Tan and miR-130b. As a conclusion, Tan decreased the adipogenesis and inflammatory response by mediating miR-130b and WNT5A, providing a novel theoretical foundation for treatment of atherosclerosis.     

LncRNA NEAT1 promotes extracellular matrix accumulation and epithelial-to-mesenchymal transition by targeting miR-27b-3p and ZEB1 in diabetic nephropathy

Diabetic nephropathy (DN) is a kind of microvascular complications of diabetes. Long noncoding RNAs (lnRNAs) can participate in the development of various diseases, including DN. However, the function of lncRNA NEAT1 is unclear. In our present study, we reported that NEAT1 was significantly increased in streptozotocin-induced DN rat models and high-glucose-induced mice mesangial cells. We observed that knockdown of NEAT1 greatly inhibited renal injury of DN rats. Meanwhile, downregulation of NEAT1-modulated extracellular matrix (ECM) proteins (ASK1, fibronectin, and TGF-β1) expression and epithelial–mesenchymal transition (EMT) proteins (E-cadherin and N-cadherin) in vitro. Previously, miR-27b-3p has been reported to be involved in diabetes. Here, miR-27b-3p was decreased in DN rats and high-glucose-induced mice mesangial cells. The direct correlation between NEAT1 and miR-27b-3p was validated using the dual-luciferase reporter assay and RNA immunoprecipitation experiments. In addition, zinc finger E-box binding homeobox 1 (ZEB1), which has been identified in the process of EMT clearly contributes to EMT progression. ZEB1 was predicted as a target of miR-27b-3p and overexpression of miR-27b-3p dramatically repressed ZEB1 expression. Therefore, our data implied the potential role of NEAT1 in the fibrogenesis and EMT in DN via targeting miR-27b-3p and ZEB1.     

MicroRNA-181a regulates the activation of the NLRP3 inflammatory pathway by targeting MEK1 in THP-1 macrophages stimulated by ox-LDL

Atherosclerosis (AS) is a chronic inflammatory disease that is characterized by the deposition of lipids in the vascular wall and the formation of foam cells. Macrophages play a critical role in the development of this chronic inflammation. An increasing amount of research shows that microRNAs affect many steps of inflammation. The goal of our study was to investigate the regulatory effect of miR-181a on the NLRP3 inflammasome pathway and explore its possible mechanism. Compared with the control group, the expression of miR-181a was downregulated in the carotid tissue of AS group mice, while the expression of MEK1 and NLRP3-related proteins was upregulated significantly. In vitro, when THP-1 macrophages were stimulated with oxidized low-density lipoprotein (ox-LDL), the expression of miR-181a was decreased, the MEK/ERK/NF-κB inflammatory pathways were activated and the expression of NLRP3 inflammasome-related proteins was upregulated. Exogenous overexpression of miR-181a downregulated the activation of the MEK/ERK/NF-κB pathway and decreased the expression of NLRP3 inflammasome-related proteins (such as NLRP3, caspase-1, interleukin-18 [IL-18], IL-1β, etc). Exogenous miR-181a knockdown showed the opposite results to those of overexpression group. A luciferase reporter assay proved that miR-181a inhibited the expression of MEK1 by binding to its 3′-untranslated region. When we knocked down miR-181a and then treated cells with U0126 before ox-LDL stimulation, we found that U0126 reversed the increased activation of the MEK/ERK/NF-κB pathway and upregulation of NLRP3 inflammasome-related proteins (NLRP3, caspase-1, IL-18, IL-1β) that resulted from miR-181a knockdown. Our study suggests that miR-181a regulates the activation of the NLRP3 inflammatory pathway by altering the activity of the MEK/ERK/NF-κB pathway via targeting of MEK1.     

NEAT1 promotes colon cancer progression through sponging miR-495-3p and activating CDK6 in vitro and in vivo

Recently, increasing evidence has indicated lncRNAs are powerful regulators in the progression of multiple tumors. Dysregulation of lncRNA NEAT1 has been recognized in many cancer types. Meanwhile, the studies on NEAT1 function have suggested that NEAT1 can serve as a crucial oncogene. Nevertheless, the investigation of NEAT1 in colon cancer is still few. In our study, the function of NEAT1 was studied in colon cancer. As we observed, NEAT1 level was obviously elevated in colon cancer cells. Then, HCT-116 and SW620 cells were stably infected with shRNA-NEAT1 for 48 hr. As exhibited, silence of NEAT1 could greatly repress colon cancer cell progression. Apoptosis of colon cancer cells was triggered and the cell cycle progression was remarkably inhibited by downregulation of NEAT1. Interestingly, as exhibited, miR-495-3p was obviously decreased in colon cancer cells and it significantly suppressed colon cancer progression. Subsequently, miR-495-3p was predicted as a target of NEAT1. CDK6 was speculated as the target of miR-495-3p and miR-495-3p modulated its expression negatively. Finally, it was indicated that NEAT1 promoted colon cancer development through modulating miR-495-3p and CDK6 in vivo. Taken these together, we reported that NEAT1 could sponge miR-495-3p to contribute to colon cancer progression through activating CDK6.     

lncRNA NEAT1 facilitates melanoma cell proliferation,migration, and invasion via regulating miR-495-3p and E2F3

Melanoma contributes a lot to skin cancer-related deaths. lncRNAs are implicated in various diseases, including melanoma. lncRNA NEAT1 is frequently dysregulated and can play important roles in multiple cancers. Nevertheless, little has been studied about the function of NEAT1 in melanoma progression. In our present research, we displayed NEAT1 was overexpressed in melanoma cells. A series of functional assays showed that overexpression of NEAT1 promoted the proliferation, migration, and invasion of melanoma cells. By contrast, NEAT1 knockdown obviously restrained melanoma cell progression. Mechanistically, it was revealed that NEAT1 could directly bind with miR-495-3p, which led to a negative effect on miR-495-3p levels. In addition, miR-495-3p was significantly decreased in melanoma cells. Furthermore, E2F3 was postulated as the target of miR-495-3p and overexpression of this miR could suppress the levels of E2F3. Meanwhile, it was exhibited that melanoma cell proliferation, migration, and invasion induced by E2F3 silence was abrogated by miR-495-3p. Moreover, an in vivo xenograft nude mice model was established using A375 cells and it was indicated that NEAT1 promoted melanoma progression in vivo via regulating the miR-495-3p/E2F3 axis. In conclusion, we suggest that NEAT1 exerts an oncogenic effect on melanoma development via inhibition of miR-495-3p and induction of E2F3. NEAT1 might serve as a crucial prognostic biomarker of melanoma.     

Prdx2 Inhibits Macrophage Lipid Accumulation Through ROS-NF-κB-miR-33a-ABCA1 Pathway

Previous studies have unraveled that peroxiredoxin 2 (Prdx2) inhibits atherogenesis in mice, whereas its role in macrophage lipid accumulation or the underlying mechanisms remain unknown. THP-1 monocyte-derived foam cells were transfected with Prdx2-overexpressing plasmid vectors (pcDNA3.1-Prdx2) or Prdx2 siRNA. The expression of ABCA1, NF-κB p65 and miR-33a were detected by RT-PCR and Western blotting. Percentage of cholesterol efflux was evaluated by liquid scintillation counting. Cellular lipid droplets were assessed using Oil Red O staining. Intracellular cholesterol contents were measured using high performance liquid chromatography (HPLC). Furthermore, cells were pre-treated with NF-κB inhibitor PDTC and/or miR-33a inhibitor, followed by detection of the indices above. The results showed that overexpression of Prdx2 in THP-1 monocyte-derived foam cells significantly increased ABCA1 expression and the percentage of ［³H］-cholesterol efflux to apoA-1 (P＜0.05), whereas NF-κB p65 and miR-33a levels as well as lipid accumulation were decreased (P＜0.05). After pre-treatment with PDTC and/or miR-33a inhibitor, these effects were more obvious (P＜0.05). In contrast, silencing of Prdx2 significantly diminished ABCA1 expression and increased NF-κB p65 and miR-33a levels. At last, we found that Prdx2 overexpression obviously down-regulated the ROS level in THP-1 monocyte-derived foam cells. Altogether, Prdx2 promotes macrophage cholesterol efflux and inhibits intracellular lipid accumulation through the ROS-NF-κB-miR-33a-ABCA1 pathway.