Lipopolysaccharide Promotes Inflammatory Response via Enhancing IFIT1 Expression in Human Umbilical Vein Endothelial Cells.

Atherosclerosis is an immune inflammatory disease and a major cause of mortality and morbidity worldwide. It is generally considered that a number of potent proinflammatory cytokines have a great influence on its pathogenesis, including IL-1ß, IL-6, TNF-α, and NF-κB. A growing amount of empirical evidence indicates that the mechanism of cardiac dysfunction caused by lipopolysaccharide (LPS) is the activation of inflammation, but the exact mechanism in atherosclerosis is still unclear. Previous studies have shown that interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) participates in inflammation, but the effects and possible mechanism of action of IFIT1 on proinflammatory response remain largely unexplained. We found that LPS induced upregulation of IFIT1 expression in a time- and concentration-dependent manner in human umbilical vein endothelial cells (HUVECs). Overexpression of IFIT1 significantly upregulated LPS-induced expression of IL-1ß, IL-6, TNF-α, and NF-κB in HUVECs. IFIT1-siRNA treatment dramatically decreased LPS-induced expression of IL-1ß, IL-6, TNF-α, and NF-κB in HUVECs. The above results show that LPS induces expression of IL-1ß, IL-6, TNF-α, and NF-κB through upregulating IFIT1 expression in HUVECs, and suggested that IFIT1 could act as potential therapeutic target to ameliorate atherosclerosis-related diseases.

TTDA inhibited apoptosis by regulating the p53-Bax/Bcl2 axis in glioma.

The trichothiodystrophy group A protein (TTDA) functions in nucleotide excision repair and basal transcription. TTDA plays a role in cancers and serves as a prognostic and predictive factor in high-grade serous ovarian cancer; however, its role in human glioma remains unknown. Here, we found that TTDA was overexpressed in glioma tissues. In vitro experiments revealed that TTDA overexpression inhibited apoptosis of glioma cells and promoted cell growth, whereas knockdown of TTDA had the opposite effect. Increased TTDA expression significantly decreased the Bax/Bcl2 ratio and the level of cleaved-caspase3. TTDA interacted with the p53 gene at the -1959 bp and -1530 bp region and regulated its transcription, leading to inhibition of the p53-Bax/Bcl2 mitochondrial apoptosis pathway in glioma cells. These results indicate that TTDA is an upstream regulator of p53-mediated apoptosis and acts as an oncogene, suggesting its value as a potential molecular target for the diagnosis and treatment of glioma.

Atorvastatin inhibits pyroptosis through the lncRNA NEXN-AS1/NEXN pathway in human vascular endothelial cells.

BACKGROUND AND AIMS: Many clinical trials have demonstrated that statins convey protective effects against atherosclerosis independent of cholesterol-lowering capacities. Other evidence indicates that pyroptosis, a type of programmed cell death, is likely involved in atherosclerosis, but the effects and mechanisms of statins on pyroptosis must be further revealed. METHODS: Here, we explored the effects and mechanisms of atorvastatin on pyroptosis in human vascular endothelial cells by quantitative real-time polymerase chain reaction and Western blot analyses. RESULTS: Atorvastatin upregulated long non-coding RNA (lncRNA) NEXN-AS1 and the expression of NEXN at both the mRNA and protein levels in a concentration- and time-dependent manner. Atorvastatin inhibited pyroptosis by decreasing the expression levels of the canonical inflammasome pathway biomarkers NLRP3, caspase-1, GSDMD, IL-1ß, and IL-18 at both the mRNA and protein levels. The promotion effects of atorvastatin on NEXN-AS1 and NEXN expression could be significantly abolished by knockdown of lncRNA NEXN-AS1 or NEXN, and its inhibitory effects on pyroptosis were also markedly offset by knock-down of lncRNA NEXN-AS1 or interference of NEXN. CONCLUSIONS: These results demonstrated that atorvastatin regulated pyroptosis via the lncRNA NEXN-AS1-NEXN pathway, which provides a new insight into the mechanism of how atorvastatin promotes non-lipid-lower effects against the development of atherosclerosis and gives new directions on how to reverse atherosclerosis.

Microarray profiling analysis and validation of novel long noncoding RNAs and mRNAs as potential biomarkers and their functions in atherosclerosis.

Long noncoding (lnc)RNAs have been implicated in the development and progression of atherosclerosis. However, the expression and mechanism of action of lncRNAs in atherosclerosis are still unclear. We implemented microarray analysis in human advanced atherosclerotic plaques and normal arterial intimae to detect the lncRNA and mRNA expression profile. Gene Ontology functional enrichment and pathway analyses were applied to explore the potential functions and pathways involved in the pathogenesis of atherosclerosis. A total of 236 lncRNAs and 488 mRNAs were selected for further Ingenuity Pathway Analysis. Moreover, quantitative RT-PCR tests of most selected lncRNAs and mRNAs with high fold changes were consistent with the microarray data. We also performed ELISA to investigate the corresponding proteins levels of selected genes and showed that serum levels of SPP1, CD36, ATP6V0D2, CHI3L1, MYH11, and BDNF were differentially expressed in patients with coronary heart disease compared with healthy subjects. These proteins correlated with some biochemical parameters used in the diagnosis of cardiovascular diseases. Furthermore, receiver operating characteristic analysis showed a favorable diagnostic performance. The microarray profiling analysis and validation of differentially-expressed lncRNAs and mRNAs in atherosclerosis not only provide new insights into the pathogenesis of this disease but may also reveal new biomarkers for its diagnosis and treatment.

The binding of lncRNA RP11-732M18.3 with 14-3-3 ß/α accelerates p21 degradation and promotes glioma growth.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been identified as regulators of a number of developmental and tumorigenic processes. However, the functions of most lncRNAs in glioma remain unknown and the mechanisms governing the proliferation of tumor cells remain poorly defined. METHODS: Both in vitro and in vivo assays were performed to investigate the roles of lncRNAs in the pathophysiology of gliomas. lncRNA arrays were used to identify differentially expressed lncRNAs. Subcutaneous tumor formation and a brain orthotopic tumor model in nude mice were used to investigate the functions of lncRNAs in vivo. The in vitro functions of lncRNAs were analyzed by fluorescence-activated cell sorting, colony formation, and western blot analyses. RNA fluorescence in situ hybridization and immunoprecipitation were used to explore the underlying mechanisms. FINDINGS: Here, we describe the newly discovered noncoding RNA RP11-732M18.3, which is highly overexpressed in glioma cells and interacts with 14-3-3ß/α to promote glioma growth, acting as an oncogene. Overexpression of lncRNA RP11-732 M18.3 was associated with the proliferation of glioma cells and tumor growth in vitro and in vivo. Remarkably, lncRNA RP11-732M18.3 promoted cell proliferation and G1/S cell cycle transition. lncRNA RP11-732M18.3 is predominately localized in the cytoplasm. Mechanistically, the interaction of lncRNA RP11-732M18.3 with 14-3-3ß/α increases the degradation of the p21 protein. lncRNA RP11-732M18.3 promoted the recruitment of ubiquitin-conjugating enzyme E2 E1 to 14-3-3ß/α and the binding of 14-3-3ß/α with ubiquitin-conjugating enzyme E2 E1 (UBE2E1) promoted the degradation of p21. INTERPRETATION: Overall these data demonstrated that lncRNA RP11-732M18.3 regulates glioma growth through a newly described lncRNA-protein interaction mechanism. The inhibition of lncRNA RP11-732M18.3 could provide a novel therapeutic target for glioma treatment.

LncRNA AC096664.3/PPAR-γ/ABCG1-dependent signal transduction pathway contributes to the regulation of cholesterol homeostasis.

Atherosclerosis is a complex inflammatory disease that involves disrupted cellular cholesterol levels and formation of foam cells. Studies about long noncoding RNA (lncRNA) have revealed its function in the development of atherosclerosis, by mediating reverse cholesterol transport and formation of foam cells. In this study, we found that oxidized low-density lipoprotein (ox-LDL) markedly decreased lncRNA AC096664.3 in vascular smooth muscle cells (VSMCs) and THP-1 macrophages. We also found that ox-LDL reduced ATP-binding cassette (ABC) G1 through inhibiting lncRNA AC096664.3 in VSMCs. Further experiments showed that the downregulation of lncRNA AC096664.3 reduced ABCG1 expression through inhibiting the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and that ox-LDL reduced ABCG1 expression through inhibiting the expression of PPAR-γ. Furthermore, we discovered that ox-LDL inhibited ABCG1 via the lncRNA AC096664.3/PPAR-γ/ABCG1 pathway, which led to an increase in total and free cholesterol in VMSCs. Thus, we confirmed that ox-LDL induces cholesterol accumulation via the lncRNA AC096664.3/PPAR-γ/ABCG1 pathway in VSMCs, indicating a promising novel therapy in protecting against atherosclerosis.

Forkhead Box C2 Attenuates Lipopolysaccharide-Induced Cell Adhesion via Suppression of Intercellular Adhesion Molecule-1 Expression in Human Umbilical Vein Endothelial Cells.

Atherosclerosis is a chronic vascular inflammatory disease that involves diverse cell types and circulating regulatory factors, including intercellular adhesion molecule (ICAM)-1, a proinflammatory cytokine. Lipopolysaccharides (LPS) increase ICAM-1 expression and promote cell adhesion, but the mechanism is not clear. We found that LPS induced time- and dose-regulated upregulation of ICAM-1 expression and downregulation of forkhead box protein C2 (Foxc2) expression in human umbilical vein endothelial cells (HUVECs). Overexpression of Foxc2 significantly inhibited both LPS-induced ICAM-1 expression in HUVECs and LPS-induced adhesion of THP-1 cells to HUVECs. Foxc2 siRNA dramatically increased both LPS-induced ICAM-1 expression and LPS-induced adhesion of THP-1 human monocytes cells to HUVECs. We conclude that Foxc2 inhibited LPS-induced adhesion of THP-1 cells to HUVECs by suppressing ICAM-1 expression in HUVECs.

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

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.