Probes for LONG

Long noncoding RNAs (lncRNAs) are critical regulators of inflammation with great potential as new therapeutic targets. However, the role of lncRNAs in early atherosclerosis remains poorly characterized. This study aimed to identify the key lncRNA players in activated endothelial cells (ECs). The lncRNAs in response to pro-inflammatory factors in ECs were screened through RNA sequencing. ICAM-1-related non-coding RNA (ICR) was identified as the most potential candidate for early atherosclerosis. ICR is essential for intercellular adhesion molecule-1 (ICAM1) expression, EC adhesion and migration. In a high fat diet-induced atherosclerosis model in mice, ICR is upregulated in the development of atherosclerosis. After intravenous injection of adenovirus carrying shRNA for mouse ICR, the atherosclerotic plaque area was markedly reduced with the declined expression of ICR and ICAM1. Mechanistically, ICR stabilized the mRNA of ICAM1 in quiescent ECs; while under inflammatory stress, ICR upregulated ICAM1 in a nuclear factor kappa B (NF-κB) dependent manner. RNA-seq analysis showed pro-inflammatory targets of NF-κB were regulated by ICR. Furthermore, the chromatin immunoprecipitation assays showed that p65 binds to ICR promoter and facilitates its transcription. Interestingly, ICR, in turn, promotes p65 accumulation and activity, forming a positive feedback loop to amplify NF-κB signaling. Preventing the degradation of p65 using proteasome inhibitors rescued the expression of NF-κB targets suppressed by ICR. Taken together, ICR acts as an accelerator to amplify NF-κB signaling in activated ECs and suppressing ICR is a promising early intervention for atherosclerosis through ICR/p65 loop blockade.

BACKGROUND: Elucidation of the molecular mechanisms by which cancer cells overcome hypoxia is potentially important for targeted therapy. Complexation of hypoxia-inducible factors (HIFs) with aryl hydrocarbon receptor nuclear translocators can enhance gene expression and initiate cellular responses to hypoxia. However, multiple molecular mechanisms may be required for cancer cells to adapt to diverse microenvironments. We previously demonstrated that a physical interaction between the ubiquitously expressed transcription factor Sp1 and HIF2 is a major cause of FVII gene activation in poor prognostic ovarian clear cell carcinoma (CCC) cells under hypoxia. Furthermore, it was found that FVII activation is synergistically enhanced when serum-starved cells are cultured under hypoxic conditions. In this study, we investigated whether HIFs and transcription factor Sp1 cooperate to activate multiple genes in CCC cells under conditions of serum starvation and hypoxia (SSH) and then contribute to malignant phenotypes. METHODS: To identify genes activated under hypoxic conditions in an Sp1-dependent manner, we first performed cDNA microarray analyses. We further investigated the molecular mechanisms of synergistic gene activations including the associated serum factors by various experiments such as real-time RT-PCR, western blotting and chromatin immunoprecipitation. The study was further extended to animal experiments to investigate how it contributes to CCC progression in vivo. RESULTS: ICAM1 is one such gene dramatically induced by SSH and is highly induced by SSH and its synergistic activation involves both the mTOR and autonomously activated TNFα-NFκB axes. We identified long chain fatty acids (LCFA) as a major class of lipids that is associated with albumin, a serum factor responsible for synergistic gene activation under SSH. Furthermore, we found that ICAM1 can be induced in vivo to promote tumor growth. CONCLUSION: Sp1 and HIFs collaborate to activate genes required for the adaptation of CCC cells to severe microenvironments, such as LCFA starvation and hypoxia. This study highlights the importance of transcriptional regulation under lipid starvation and hypoxia in the promotion of CCC tumor growth.

Hypophosphatasia (HPP) is caused by loss-of-function mutations in the ALPL gene that encodes tissue-nonspecific alkaline phosphatase (TNAP), whose deficiency results in the accumulation of extracellular inorganic pyrophosphate (PPi ), a potent mineralization inhibitor. Skeletal and dental hypomineralization characterizes HPP, with disease severity varying from life-threatening perinatal or infantile forms to milder forms that manifest in adulthood or only affect the dentition. Enzyme replacement therapy (ERT) using mineral-targeted recombinant TNAP (Strensiq/asfotase alfa) markedly improves the life span, skeletal phenotype, motor function, and quality of life of patients with HPP, though limitations of ERT include frequent injections due to a short elimination half-life of 2.28 days and injection site reactions. We tested the efficacy of a single intramuscular administration of adeno-associated virus 8 (AAV8) encoding TNAP-D10 to increase the life span and improve the skeletal and dentoalveolar phenotypes in TNAP knockout (Alpl-/- ) mice, a murine model for severe infantile HPP. Alpl-/- mice received 3 × 1011 vector genomes/body of AAV8-TNAP-D10 within 5 days postnatal (dpn). AAV8-TNAP-D10 elevated serum ALP activity and suppressed plasma PPi . Treatment extended life span of Alpl-/- mice, and no ectopic calcifications were observed in the kidneys, aorta, coronary arteries, or brain in the 70 dpn observational window. Treated Alpl-/- mice did not show signs of rickets, including bowing of long bones, enlargement of epiphyses, or fractures. Bone microstructure of treated Alpl-/- mice was similar to wild type, with a few persistent small cortical and trabecular defects. Histology showed no measurable osteoid accumulation but reduced bone volume fraction in treated Alpl-/- mice versus controls. Treated Alpl-/- mice featured normal molar and incisor dentoalveolar tissues, with the exceptions of slightly reduced molar enamel and alveolar bone density. Histology showed the presence of cementum and normal periodontal ligament attachment. These results support gene therapy as a promising alternative to ERT for the treatment of HPP.