Project description:We evaluated the effect of NORAD (also known as LINC00657 or LOC647979) shRNA on TGF-beta induced changes in the gene expression in A549 cells by RNA-seq. Overall design: mRNA expression was determined in a lung adenocarcinoma cancer cell line A549 infected with NORAD shRNA-expressing lentiviral vector and treated with TGF-beta.
Project description:Substantial researches indicated that long non-coding RNAs (lncRNAs) exerted profound effects on chemo-resistance in cancer treatment. Nonetheless, the role of NORAD in non-small-cell lung cancer (NSCLC) remains unclear. In the present study, we chose NSCLC cell lines H446 and A549 to explore the function of non-coding RNA activated damage (NORAD) in response to cisplatin (DDP) resistance of NSCLC. Experimental data manifested that NORAD was up-regulated in DDP-resistant NSCLC tissues and cells. NSCLC patients with high NORAD expression suffered a poor prognosis. NORAD knockdown resensitized H446/DDP and A549/DDP to DDP. Besides, NORAD acted as a molecular sponge of miR-129-1-3p. MiR-129-1-3p showed a low level of expression in DDP-resistant NSCLC tissues. Moreover, miR-129-1-3p overexpression impaired DDP resistance in H446/DDP and A549/DDP cells. SOX4 was the downstream target of miR-129-1-3p. Especially, SOX4 overexpression offset the effects of NORAD silence on H446/DDP and A549/DDP cells resistance to DDP. NORAD knockdown resensitized H446/DDP and A549/DDP to DDP in NSCLC via targeting miR-129-1-3p/SOX4 axis, offering a brand-new target for NSCLC chemo-resistance.
Project description:<h4>Background</h4>To explore the role of non-coding RNA activated by DNA damage (NORAD), a long non-coding ribonucleic acid (lncRNA), in non-small cell lung cancer (NSCLC) and its possible mechanism.<h4>Methods</h4>Quantitative real-time polymerase chain reaction was adopted for the detection of the expression levels of NORAD, micro RNA (miR)-656-3p, and AKT serine/threonine kinase 1 (AKT1). The effects of NORAD, miR-656-3p, and AKT1 on cell proliferation and migration were examined through the Cell Counting Kit-8 (CCK-8) and Transwell assay. Subsequently, the binding relationships between miR-656-3p and AKT1 and between miR-656-3p and NORAD were verified by dual-luciferase reporter gene assay. Finally, the potential mechanisms of action of NORAD and miR-656-3p were explored through the torsion experiment.<h4>Results</h4>The lncRNA NORAD expression level in NSCLC patients was notably higher than that in people in control group, that in patients with metastasis was higher than that in patients without metastasis, and that in patients with NSCLC in stage III-IV was significantly higher than that in patients with NSCLC in stage I-II. Elevation of NORAD stimulated the proliferation and migration of NSCLC A549/H460 cells. According to the reporter gene assay, NORAD could bind to miR-656-3p. Besides, miR-656-3p was significantly under-expressed in cancer tissues of patients with NSCLC, and overexpression of miR-656-3p could block the proliferation and migration of A549/H460 cells and reversed promotion on cell proliferation and migration by NORAD. Furthermore, the reporter gene assay revealed that the overexpression of AKT1, a miR-656-3p target gene, could reverse miR-656-3p's inhibitory effect on the proliferation and migration of A549/H460 cells.<h4>Conclusion</h4>LncRNA NORAD is capable of promoting the proliferation and migration of NSCLC cells, and its mechanism may be that it increases the AKT1 expression by adsorbing miR-656-3p.
Project description:To find out genes regulated by TGF-β in A549 cells, we compared gene expression of cells treated with 1ng/ml TGF-β versus non-treated cells and find out that expression of one transmembrane protein, TM4SF20, is reduced by TGF-β. A549 cells were treated with or without 1ng/ml TGF-β for 12h. RNA was extracted and hybridized on Affymetrix microarrays
Project description:Although numerous long noncoding RNAs (lncRNAs) have been identified, our understanding of their roles in mammalian physiology remains limited. Here, we investigated the physiologic function of the conserved lncRNA Norad in vivo. Deletion of Norad in mice results in genomic instability and mitochondrial dysfunction, leading to a dramatic multi-system degenerative phenotype resembling premature aging. Loss of tissue homeostasis in Norad-deficient animals is attributable to augmented activity of PUMILIO proteins, which act as post-transcriptional repressors of target mRNAs to which they bind. Norad is the preferred RNA target of PUMILIO2 (PUM2) in mouse tissues and, upon loss of Norad, PUM2 hyperactively represses key genes required for mitosis and mitochondrial function. Accordingly, enforced Pum2 expression fully phenocopies Norad deletion, resulting in rapid-onset aging-associated phenotypes. These findings provide new insights and open new lines of investigation into the roles of noncoding RNAs and RNA binding proteins in normal physiology and aging.
Project description:Long noncoding RNAs (lncRNAs) have been shown to have several functional roles in tumor biology, and they are deregulated in many types of cancer. The role of a novel lncRNA, NORAD, in papillary thyroid carcinoma (PTC) is still unknown. In this study, we demonstrated that NORAD expression was upregulated in PTC cell lines and samples. Ectopic expression of NORAD promoted PTC cell growth, invasion and migration. Overexpression of NORAD promotes epithelial-to-mesenchymal transition (EMT) progression in the PTC cell. Furthermore, overexpression of NORAD suppressed miR-202-5p expression in PTC cells. The data suggested that miR-202-5p expression was downregulated in PTC cell lines and samples and was negatively correlated with NORAD expression in PTC tissues. Overexpression of miR-202-5p suppressed PTC cell growth, invasion and migration. In addition, we demonstrated that elevated expression of NORAD promoted PTC cell growth, invasion and migration by inhibiting miR-202-5p expression. These results suggested that the lncRNA NORAD acts as an oncogene in PTC progression, partly by regulating miR-202-5p expression.
Project description:Long noncoding RNAs (lncRNAs) are emerging as critical regulators in tumor initiation and progression. However, the biological mechanisms and potential clinical application of lncRNA NORAD in endometrial cancer (EC) remain unknown. Herein, we identified NORAD underwent promoter hypermethylation-associated downregulation in EC. Epigenetic inactivation of NORAD was correlated with EC progression (FIGO stage) and poor outcome. Overexpression of NORAD significantly inhibited cell growth and promoted apoptosis in EC cells. Mechanistic studies revealed that multiple regions of NORAD served as a platform for binding with the central domain of anti-apoptotic factor FUBP1. Our findings further indicated that the NORAD/FUBP1 interaction attenuated FUBP1 nuclear localization and thus impaired the occupancies of FUBP1 on its target pro-apoptotic gene promoters, resulting in apoptosis induction in EC. Moreover, knockdown of NORAD promoted tumor growth in the xenograft mice model. While, introduction of NORAD-4 fragment, which bound with FUBP1, successfully reversed tumor growth and apoptosis inhibition mediated by NORAD knockdown in vivo. Our findings provide mechanistic insight into the critical roles of NORAD as a tumor suppressor in EC progression. NORAD could possibly serve as a novel prognostic biomarker and provide the rationale for EC therapy.
Project description:Long noncoding RNAs (lncRNAs) have emerged as regulators of diverse biological processes. Here, we describe the initial functional analysis of a poorly characterized human lncRNA (LINC00657) that is induced after DNA damage, which we termed "noncoding RNA activated by DNA damage", or NORAD. NORAD is highly conserved and abundant, with expression levels of approximately 500-1,000 copies per cell. Remarkably, inactivation of NORAD triggers dramatic aneuploidy in previously karyotypically stable cell lines. NORAD maintains genomic stability by sequestering PUMILIO proteins, which repress the stability and translation of mRNAs to which they bind. In the absence of NORAD, PUMILIO proteins drive chromosomal instability by hyperactively repressing mitotic, DNA repair, and DNA replication factors. These findings introduce a mechanism that regulates the activity of a deeply conserved and highly dosage-sensitive family of RNA binding proteins and reveal unanticipated roles for a lncRNA and PUMILIO proteins in the maintenance of genomic stability.
Project description:Long noncoding RNAs (lncRNAs) play important roles in the development of vascular diseases. However, the effect of lncRNA NORAD on atherosclerosis remains unknown. This study aimed to investigate the effect NORAD on endothelial cell injury and atherosclerosis. Ox-LDL-treated human umbilical vein endothelial cells (HUVECs) and high-fat-diet (HFD)-fed ApoE-/- mice were used as in vitro and in vivo models. Results showed that NORAD-knockdown induced cell cycle arrest in G0/G1 phase, aggravated ox-LDL-induced cell viability reduction, cell apoptosis, and cell senescence along with the increased expression of Bax, P53, P21 and cleaved caspase-3 and the decreased expression of Bcl-2. The effect of NORAD on cell viability was further verified via NORAD-overexpression. NORAD- knockdown increased ox-LDL-induced reactive oxygen species, malondialdehyde, p-IKB? expression levels and NF-?B nuclear translocation. Proinflammatory molecules ICAM, VCAM, and IL-8 were also increased by NORAD- knockdown. Additionally, we identified the strong interaction of NORAD and IL-8 transcription repressor SFPQ in HUVECs. In ApoE-/- mice, NORAD-knockdown increased the lipid disorder and atherosclerotic lesions. The results have suggested that lncRNA NORAD attenuates endothelial cell senescence, endothelial cell apoptosis, and atherosclerosis via NF-?B and p53-p21 signaling pathways and IL-8, in which NORAD-mediated effect on IL-8 might through the direct interaction with SFPQ.