ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by aberrant alternative splicing (AS). Nuclear loss and cytoplasmic accumulation of the splicing factor TDP-43 in motor neurons (MN) are hallmarks of ALS at late stages of the disease. However, it is unknown if altered AS is present before TDP-43 pathology occurs. Here, we investigate altered AS and its origins in early stages of ALS using human induced pluripotent stem cell-derived motor neurons (MNs) from sporadic and familial ALS patients. We find high levels of the RNA-binding proteins NOVA1, NOVA2, and RBFOX2 in the insoluble protein fractions and observe that AS events in ALS-associated MNs are enriched for binding sites of these proteins. Our study points to an early disrupted function of NOVA1 that drives AS changes in a complex fashion, including events caused by a consistent loss of NOVA1 function. NOVA1 exhibits increased cytoplasmic protein levels in early stage MNs without TDP-43 pathology in ALS postmortem tissue. As nuclear TDP-43 protein level depletes, NOVA1 is reduced. Potential indications for a reduction of NOVA1 also came from mice over-expressing TDP-43 lacking its nuclear localization signal and iPSC-MN stressed with puromycin. This study highlights that additional RBP-RNA perturbations in ALS occur in parallel to TDP-43.
Subject(s)
Amyotrophic Lateral Sclerosis , DNA-Binding Proteins , Induced Pluripotent Stem Cells , Neuro-Oncological Ventral Antigen , Alternative Splicing/genetics , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Induced Pluripotent Stem Cells/metabolism , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuro-Oncological Ventral Antigen/genetics , Neuro-Oncological Ventral Antigen/metabolism , Nuclear Proteins/genetics , RNA Splicing Factors/genetics , RNA Splicing Factors/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Repressor Proteins/geneticsABSTRACT
Non-small cell lung cancer (NSCLC) is a serious threaten to human health globally. Circular RNAs (circRNAs) were testified to alter the progression of NSCLC. This work intended to investigate the functional role of circ_0016760 in NSCLC development and the potential mechanism. Expression of circ_0016760, microRNA (miR)-876-3p and NOVA alternative splicing regulator 2 (NOVA2) was determined via quantitative reverse transcription-PCT (qRT-PCR) or western blotting. Cell viability, clonogenicity and apoptosis were assessed by Cell Counting Kit-8 (CCK-8) assay, colony formation assay and flow cytometry, respectively. Transwell assay was performed to examine cell migration and invasion. Western blotting was also conducted to detect the levels of epithelial-to-mesenchymal transition (EMT)-related proteins. Role of circ_0016760 in vivo was evaluated via xenograft model assay. Moreover, the interaction between miR-876-3p and circ_0016760 or NOVA2 was verified by dual-luciferase reporter assay or RNA Immunoprecipitation (RIP) assay. Circ_0016760 and NOVA2 were upregulated, while miR-876-3p expression was decreased in NSCLC tissues and cells. Circ_0016760 depletion suppressed NSCLC cell proliferation and metastasis in vitro, as well as hampered tumor growth in vivo. Circ_0016760 acted as a sponge of miR-876-3p, and miR-876-3p could target NOVA2. Circ_0016760 might play vital roles in NSCLC by regulating miR-876-3p/NOVA2 axis. Circ_0016760 could promote the malignant development of NSCLC through miR-876-3p/NOVA2 axis, at least in part.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , MicroRNAs , Neuro-Oncological Ventral Antigen , RNA, Circular , Humans , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Lung Neoplasms/pathology , MicroRNAs/genetics , RNA, Circular/genetics , Neuro-Oncological Ventral Antigen/genetics , AnimalsABSTRACT
Blood-brain barrier dysfunction is recognized as a precursor of Alzheimer's disease development. Endothelial cells as structural basis of blood-brain barrier were observed tight junction failure in amyloid-ß(1-42)-stimulated environment. In this study, we found NOVA2, PPP2R3A were down-regulated while PART1, p-NFκB-p65 were up-regulated in amyloid-ß(1-42)-incubated endothelial cells. Knockdown of either NOVA2 or PPP2R3A and overexpression of PART1 all increased blood-brain barrier permeability. Lower blood-brain barrier permeability was observed in overexpression of NOVA2 and PPP2R3A and knockdown of PART1 and NFκB-p65. Same tendencies were found in the tight junction-related proteins expressions. Furthermore, overexpression and knockdown of NOVA2 and PART1 had no effect on cell viability. Mechanistically, NOVA2 overexpression was confirmed to reduce half-life of PART1. PART1 could destabilize PPP2R3A messenger RNA (mRNA) by interacting with STAU1. In addition, p-NFκB-p65 functioning as transcription factor reduced the expression of tight junction-related proteins, which was prompted by low protein level of PPP2R3A. Our study highlights the crucial role of NOVA2/PART1/PPP2R3A/p-NFκB-p65 pathway in amyloid-ß(1-42)-incubated endothelial cells to modulating blood-brain barrier permeability through STAU1-mediated messenger RNA degradation, implying a potential mechanism of lncRNA and protein interaction in pathogenesis of Alzheimer's disease.
