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The role of the LncRNA-FA2H-2-MLKL pathway in atherosclerosis by regulation of autophagy flux and inflammation through mTOR-dependent signaling.

ABSTRACT: Atherosclerosis is a progressive, chronic inflammation in arterial walls. Long noncoding RNAs (lncRNAs) participate in inflammation, but the exact mechanism in atherosclerosis is unclear. Our microarray analyses revealed that the levels of lncRNA-FA2H-2 were significantly decreased by oxidized low-density lipoprotein (OX-LDL). Bioinformatics analyses indicated that mixed lineage kinase domain-like protein (MLKL) might be regulated by lncRNA-FA2H-2. In vitro experiments showed that lncRNA-FA2H-2 interacted with the promoter of the MLKL gene, downregulated MLKL expression, and the binding sites between -750 and 471 were necessary for lncRNA-FA2H-2 responsiveness to MLKL. Silencing lncRNA-FA2H-2 and overexpression of MLKL could activate inflammation and inhibited autophagy flux. Both lncRNA-FA2H-2 knockdown and overexpression of MLKL could significantly aggravate inflammatory responses induced by OX-LDL. We found that the 3-methyladenine (3-MA) and Atg7-shRNA enhanced inflammatory responses induced by knockdown of lncRNA-FA2H-2 and overexpression of MLKL. We demonstrated that the effects of MLKL on autophagy might be associated with a mechanistic target of rapamycin (mTOR)-dependent signaling pathways. In vivo experiments with apoE knockout mice fed a western diet demonstrated that LncRNA-FA2H-2 knockdown decreased microtubule-associated expression of microtubule-associated protein 1 light chain 3 II and lysosome-associated membrane protein 1, but increased expression of sequestosome 1 (p62), MLKL, vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, and interleukin-6 in atherosclerotic lesions. Our findings indicated that the lncRNA-FA2H-2-MLKL pathway is essential for regulation of autophagy and inflammation, and suggested that lncRNA-FA2H-2 and MLKL could act as potential therapeutic targets to ameliorate atherosclerosis-related diseases.

SUBMITTER: Guo FX

PROVIDER: S-EPMC6748100 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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The role of the LncRNA-FA2H-2-MLKL pathway in atherosclerosis by regulation of autophagy flux and inflammation through mTOR-dependent signaling.

Guo Feng-Xia FX Wu Qian Q Li Pan P Zheng Lei L Ye Shu S Dai Xiao-Yan XY Kang Chun-Min CM Lu Jing-Bo JB Xu Bang-Ming BM Xu Yuan-Jun YJ Xiao Lei L Lu Zhi-Feng ZF Bai Huan-Lan HL Hu Yan-Wei YW Wang Qian Q

Cell death and differentiation 20190125 9

Atherosclerosis is a progressive, chronic inflammation in arterial walls. Long noncoding RNAs (lncRNAs) participate in inflammation, but the exact mechanism in atherosclerosis is unclear. Our microarray analyses revealed that the levels of lncRNA-FA2H-2 were significantly decreased by oxidized low-density lipoprotein (OX-LDL). Bioinformatics analyses indicated that mixed lineage kinase domain-like protein (MLKL) might be regulated by lncRNA-FA2H-2. In vitro experiments showed that lncRNA-FA2H-2 ...[more]

PMID: 30683918

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Project description:BackgroundAtherosclerosis is one of the most common cardiovascular disorders. The dysfunction of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are 2 key factors in the formation of atherosclerosis. This study aims to find strategies to prevent VSMCs and ECs dysfunction for the treatment of atherosclerosis.MethodsWe investigated the expression patterns of long noncoding RNAs (lncRNAs) in 2 pairs of serum samples from both atherosclerosis patients and healthy volunteers through microarray analysis. Then we selected the most up-regulated lncRNA ENAST00113 (lnc00113) to further verify its roles in atherosclerosis. VSMCs, and human umbilical vein endothelial cells (HUVECs) transfected with small interfering RNA (siRNAs) (si-00113-1, si-00113-1) and a negative control (si-NC) were cultured. MTT assay, Caspase 3 enzyme-linked immunosorbent assay (ELISA) assay, and wound healing assay were performed to evaluate whether lnc00113 had an effect on proliferation, apoptosis, and migration ability. Further, the correlation between lnc00113 and PI3K/Akt/mTOR signaling pathway was explored.ResultsMicroarray results indicated that 243 lncRNAs were up-regulated and 187 lncRNAs were down-regulated. Therefore, we chose the most up-regulated lncRNA ENST (lnc00113) to further explore its roles in atherosclerosis. Real-time polymerase chain reaction (RT-qPCR) results showed that the expression of lnc00113 was highly increased in atherosclerosis patients. In vitro experiment demonstrated that lnc00113 down-regulation significantly suppressed VSMCs and HUVECs proliferation, survival, and migration. Furthermore, we found that the protein expressions of phosphorylated-PI3K (p-PI3K), phosphorylated-Akt (p-Akt), phosphorylated-mTOR (p-mTOR), and bcl-2 in HUVECs cells transfected with si-00113-1 or si-00113-2 were dramatically decreased compared with si-NC-transfected cells and control cells. However, the total- PI3K (t-PI3K), total-Akt (t-Akt), and total-mTOR (t-mTOR) protein expressions were not changed, indicating that lnc00113 could activate PI3K/Akt/mTOR signaling pathway in atherosclerosis.ConclusionsThis finding identified an important role of lnc00113 in VSMCs and HUVECs that promotes cell proliferation, survival, and migration by activating PI3K/Akt/mTOR signaling pathway, which could probably serve as a promising therapeutic target for atherosclerosis.

Project description:RationaleThe NLRP3 (NLR [NOD-like receptor] family, pyrin domain containing 3) inflammasome is an important driver of atherosclerosis. Our previous study shows that chaperone-mediated autophagy (CMA), one of the main lysosomal degradative process, has a regulatory role in lipid metabolism of macrophages. However, whether the NLRP3 inflammasome is regulated by CMA, and the role of CMA in atherosclerosis remains unclear.ObjectiveTo determine the role of CMA in the regulation of NLRP3 inflammasome and atherosclerosis.Methods and resultsThe expression of CMA marker, LAMP-2A (lysosome-associated membrane protein type 2A), was first analyzed in ApoE-/- mouse aortas and human coronary atherosclerotic plaques, and a significant downregulation of LAMP-2A in advanced atherosclerosis in both mice and humans was observed. To selectively block CMA, we generated macrophage-specific conditional LAMP-2A knockout mouse strains in C57BL/6 mice and ApoE-/- mice. Deletion of macrophage LAMP-2A accelerated atherosclerotic lesion formation in the aortic root and the whole aorta in ApoE-/- mice. Mechanistically, LAMP-2A deficiency promoted NLRP3 inflammasome activation and subsequent release of mature IL (interleukin)-1β in macrophages and atherosclerotic plaques. Furthermore, gain-of-function studies verified that restoration of LAMP-2A levels in LAMP-2A-deficient macrophages greatly attenuated NLRP3 inflammasome activation. Importantly, we identified the NLRP3 protein as a CMA substrate and demonstrated that LAMP-2A deficiency did not affect the NLRP3 mRNA levels but hindered degradation of the NLRP3 protein through CMA pathway.ConclusionsCMA function becomes impaired during the progression of atherosclerosis, which increases NLRP3 inflammasome activation and secretion of IL-1β, promoting vascular inflammation and atherosclerosis progression. Our study unveils a new mechanism by which NLRP3 inflammasome is regulated in macrophages and atherosclerosis, thus providing a new insight into the role of autophagy-lysosomal pathway in atherosclerosis. Pharmacological activation of CMA may provide a novel therapeutic strategy for atherosclerosis and other NLRP3 inflammasome/IL-1β-driven diseases.

Project description:Hepatocellular carcinoma (HCC) is a highly invasive malignancy. Recently, GATOR1 (Gap Activity TOward Rags 1) complexes have been shown to play an important role in regulating tumor growth. NPRL2 is a critical component of the GATOR1 complex. Therefore, this study used NPRL2 knockdown to investigate how GATORC1 regulates the prognosis and development of HCC via the mammalian target of rapamycin (mTOR) and autophagy signaling pathways. We established HepG2 cells with NPRL2 knockdown using small interfering RNA (siRNA) and short hairpin RNA (shRNA) systems. The siRNA-mediated and shRNA-mediated NPRL2 down-regulation significantly reduced the expression of NPRL2 and two other GATPOR1 complex components, NPRL3 and DEPDC5, in HepG2 cells; furthermore, the efficient down-regulation of NPRL2 protein expression by both the shRNA and siRNA systems enhanced the proliferation, migration, and colony formation in vitro. Additionally, the NPRL2 down-regulation significantly increased HCC growth in the subcutaneous and orthotopic xenograft mouse models. The NPRL2 down-regulation increased the Rag GTPases and mTOR activation and inhibited autophagy in vitro and in vivo. Moreover, the NPRL2 level in the tumors was significantly associated with mortality, recurrence, the serum alpha fetoprotein level, the tumor size, the American Joint Committee on Cancer stage, and the Barcelona Clinic Liver Cancer stage. Low NPRL2, NPRL3, DEPDC5, and LC3, and high p62 and mTOR protein expression in the tumors was significantly associated with disease-free survival and overall survival in 300 patients with HCC after surgical resection. Conclusion: The efficient down-regulation of NPRL2 significantly increased HCC proliferation, migration, and colony formation in vitro, and increased HCC growth in vivo. Low NPRL2 protein expression in the tumors was closely correlated with poorer clinical outcomes in patients with HCC. These results provide a mechanistic understanding of HCC and aid the development of treatments for HCC.

Dataset Information

The role of the LncRNA-FA2H-2-MLKL pathway in atherosclerosis by regulation of autophagy flux and inflammation through mTOR-dependent signaling.

Publications

The role of the LncRNA-FA2H-2-MLKL pathway in atherosclerosis by regulation of autophagy flux and inflammation through mTOR-dependent signaling.

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