The role of long non-coding RNA NORAD in digestive system tumors.

In recent years, it has been discovered that the expression of long non-coding RNAs is highly deregulated in several types of cancer and contributes to its progression and development. Recently, it has been described that in tumors of the digestive system, such as colorectal cancer, pancreatic cancer, and gastric cancer, DNA damage-activated lncRNA (NORAD) was frequently up-regulated. The purpose of this review is to elucidate the functions of NORAD in tumors of the digestive system, emphasizing its involvement in important cellular processes such as invasion, metastasis, proliferation, and apoptosis. NORAD acts as a ceRNA (competitive endogenous RNA) that sponges microRNAs and regulates the expression of target genes involved in tumorigenesis. Thus, the mechanisms underlying the effects of NORAD are complex and involve multiple signaling pathways. This review consolidates current knowledge on the role of NORAD in digestive cancers and highlights the need for further research to explore its potential as a therapeutic target. Understanding the intricate functions of NORAD could elucidate the way for innovative approaches to cancer treatment.

Elevated circulating LncRNA NORAD fosters endothelial cell growth and averts ferroptosis by modulating the miR-106a/CCND1 axis in CAD patients.

Atherosclerosis is a leading cause of cardiovascular diseases, characterized by endothelial dysfunction and lipid accumulation. Long non-coding RNAs (lncRNAs) are emerging as key regulators of endothelial cell behavior. This study aimed to investigate the role of lncRNA NORAD in endothelial cell proliferation and as a potential therapeutic target for atherosclerosis. A total of 75 CAD patients and 76 controls were recruited, and plasma NORAD levels were measured using qRT-PCR. HUVECs were transfected with si-NORAD to evaluate its effects on cell cycle, proliferation, migration, and apoptosis. Plasma NORAD levels were significantly elevated in CAD patients. The NORAD-miRNA-mRNA ceRNA regulatory network was constructed based on GEO database, and G1/S-specific cyclin-D1 (CCND1) was identified as one of the hub factors. NORAD deficiency suppressed cell migration and induced G1 cell cycle arrest in HUVECs by downregulating CCND1 in vitro. NORAD upregulated CCND1 in HUVECs via sponging miR-106a that inhibited cell migration. The dual-luciferase assay confirmed the direct targeting of miR-106a by NORAD, and overexpression of miR-106a inhibited HUVEC proliferation and migration. Si-NORAD transfection resulted in induced early apoptosis, increased intracellular ROS levels, decreased GSH levels, and reduced mitochondrial membrane potential. Additionally, si-NORAD decreased the expression of GPX4, FTH1, KEAP1, NCOA4, and Nrf2, while increasing Xct levels, confirming the involvement of ferroptosis. Our findings reveal that NORAD plays a critical role in endothelial cell proliferation, migration, and apoptosis, and its silencing induces ferroptosis. The regulatory network involving NORAD, miR-106a, and their target genes provides a potential therapeutic avenue for atherosclerosis.

NORAD accelerates skin wound healing through extracellular vesicle transfer from hypoxic adipose derived stem cells: miR-524-5p pathway and Pumilio protein mechanism.

Skin wound healing is a multifaceted biological process that encompasses a variety of cell types and intricate signaling pathways. Recent research has uncovered that exosomes derived from adipose stem cells, commonly referred to as ADSC exosomes, play a crucial role in facilitating the healing process. Moreover, it has been demonstrated that an anoxic, or low-oxygen, environment significantly enhances the effectiveness of these exosomes in promoting skin repair. The primary objective of this study was to investigate the underlying mechanisms through which ADSC exosomes contribute to Skin wound healing, particularly by regulating the long non-coding RNA known as NORAD under hypoxic conditions. A significant focus of our research was to examine the interplay between the microRNA miR-524-5p and the Pumilio protein, as we aimed to understand how these molecular interactions might influence the overall healing process. In this study, ADSC exosomes were extracted by simulating hypoxia in vitro and their effects on the proliferation and migration of skin fibroblasts (FB) were evaluated. The expression levels of NORAD, miR-524-5p and Pumilio were analyzed by fluorescence quantitative PCR. Pumilio protein was silenced by siRNA technique to evaluate its role in ADSC exosome-mediated wound healing. The experimental results showed that under hypoxia conditions, NORAD levels in ADSC exosomes increased significantly and could effectively regulate the expression of miR-524-5p. After Pumilio protein silencing, the proliferation and migration ability of fibroblasts were significantly reduced, indicating that Pumilio protein played a role in the process of wound healing. By inhibiting miR-524-5p, the expression of Pumilio protein was restored, further confirming its regulatory mechanism.

Gain-Type Aneuploidies Influence the Burden of Selective Long Non-Coding Transcripts in Colorectal Cancer.

Chromosomal instability is a hallmark of colorectal carcinogenesis and produces an accumulation of different forms of aneuploidies or broad copy number aberrations. Colorectal cancer is characterized by gain-type broad copy number aberrations, specifically in Chr20, Chr8q, Chr13 and Chr7, but their roles and mechanisms in cancer progression are not fully understood. It has been suggested that broad copy number gains might contribute to tumor development through the so-called caricature transcriptomic effect. We intend to investigate the impact of broad copy number gains on long non-coding RNAs' expression in colorectal cancer, given their well-known role in oncogenesis. The influence of such chromosomal aberrations on lncRNAs' transcriptome profile was investigated by SNP and transcriptome arrays in our series of colorectal cancer samples and cell lines. The correlation between aneuploidies and transcriptomic profiles led us to obtain a class of Over-UpT lncRNAs, which are transcripts upregulated in CRC and further overexpressed in colon tumors bearing specific chromosomal aberrations. The identified lncRNAs can contribute to a wide interaction network to establish the cancer driving effect of gain-type aneuploidies.

Long non-coding RNA NORAD regulates megakaryocyte differentiation and proplatelet formation via the DUSP6/ERK signaling pathway.

Megakaryopoiesis and platelet production is a complex process that is underpotential regulation at multiple stages. Many long non-coding RNAs (lncRNAs) are distributed in hematopoietic stem cells and platelets. lncRNAs may play important roles as key epigenetic regulators in megakaryocyte differentiation and proplatelet formation. lncRNA NORAD can affect cell ploidy by sequestering PUMILIO proteins, although its direct effect on megakaryocyte differentiation and thrombopoiesis is still unknown. In this study, we demonstrate NORAD RNA is highly expressed in the cytoplasm during megakaryocyte differentiation. Interestingly, we identified for the first time that NORAD has a strong inhibitory effect on megakaryocyte differentiation and proplatelet formation from cultured megakaryocytes. DUSP6/ERK1/2 pathway is activated in response to NORAD knockdown during megakaryocytopoiesis, which is achieved by sequestering PUM2 proteins. Finally, compared with the wild-type control mice, NORAD knockout mice show a faster platelet recovery after severe thrombocytopenia induced by 6 Gy total body irradiation. These findings demonstrate lncRNA NORAD has a key role in regulating megakaryocyte differentiation and thrombopoiesis, which provides a promising molecular target for the treatment of platelet-related diseases such as severe thrombocytopenia.

lncRNA NORAD alleviates dysfunction of renal proximal tubular epithelial cells during the sepsis-associated acute kidney injury by modulating the miR-155-5p-PDK1 axis.

The sepsis-associated acute kidney injury (Sa-AKI) is closely related to high mortality rates worldwide. Injury to the renal proximal tubular epithelial cells (RPTECs), caused by pathological conditions, is a major cause of acute kidney injury (AKI). The lncRNA NORAD has been reported to be positively associated with kidney cancers. However, the biological roles and underlying mechanisms of NORAD in RPTECs during AKI are still unclear. In this study, we found that NORAD was significantly downregulated in RPTECs from AKI tissues. Overexpression of NORAD alleviated RPTECs injury induced by lipopolysaccharide (LPS). Additionally, glucose metabolism was significantly impaired during AKI, and LPS treatment inhibited glucose metabolism in RPTECs. We demonstrated that NORAD rescued the LPS-induced inhibition of glucose metabolism in RPTECs. Furthermore, miRNA-155-5p was significantly upregulated in RPTECs from AKI. Through bioinformatics analysis, RNA pull-down, RNA IP, and luciferase assays, we showed that NORAD directly associated with miR-155-5p to downregulate its expression. Moreover, overexpression of miR-155-5p inhibited glucose metabolism by directly targeting the 3'UTR of the glucose metabolism enzyme, pyruvate dehydrogenase kinase 1 (PDK1). Finally, rescue experiments validated that NORAD's protective effect on RPTECs injury was mediated through modulation of the miR-155-5p-PDK1-glucose metabolism pathway. In summary, these results reveal that lncRNA NORAD can alleviate RPTECs dysfunction by targeting the miR-155-5p-PDK1 axis, suggesting that NORAD has the potential to contribute to the development of therapeutic approaches against Sa-AKI.

METTL3-mediated m6A modification of NORAD inhibits the ferroptosis of vascular smooth muscle cells to attenuate the aortic dissection progression in an YTHDF2-dependent manner.

Ferroptosis of vascular smooth muscle cells (VSMCs) is related to the incidence of aortic dissection (AD). Long non-coding RNA (lncRNA) NORAD plays a crucial role in the progression of various diseases. The present study aimed to investigate the effects of NORAD on the ferroptosis of VSMCs and the molecular mechanisms. The expression of NORAD, HUR, and GPX4 was detected using quantitative real-time PCR (qPCR) or western blot. Ferroptosis was evaluated by detecting lactate dehydrogenase (LDH) activity, lipid reactive oxygen species (ROS), malonaldehyde (MDA) content, L-Glutathione (GSH) level, Fe2+ content, and ferroptosis-related protein levels. The molecular mechanism was assessed using RNA pull-down, RNA-binding protein immunoprecipitation (RIP), and luciferase reporter assay. The histology of aortic tissues was assessed using H&E, elastic Verhoeff-Van Gieson (EVG), and Masson staining assays. The data indicated that NORAD was downregulated in patients with AD and AngII-treated VSMCs. Overexpression of NORAD promoted VSMC growth and inhibited the ferroptosis induced by AngII. Mechanistically, NORAD interacted with HUR, which promoted GPX4 mRNA stability and elevated GPX4 levels. Knockdown of GPX4 abrogated the effects of NORAD on cell growth and ferroptosis of AngII-treated VSMCs. Moreover, METTL3 promoted m6A methylation of NORAD in an YTHDF2-dependent manner. In addition, NORAD attenuated AAD symptoms, incidence, histopathology, inflammation, and ferroptosis in AAD mice. In conclusion, METTL3-mediated NORAD inhibited ferroptosis of VSMCs via the HUR/GPX4 axis and decelerated AAD progression, suggesting that NORAD may be an AD therapeutic target.

NORAD-Regulated Signaling Pathways in Breast Cancer Progression.

Long non-coding RNA activated by DNA damage (NORAD) has recently been associated with pathologic mechanisms underlying cancer progression. Due to NORAD's extended range of interacting partners, there has been contradictory data on its oncogenic or tumor suppressor roles in BC. This review will summarize the function of NORAD in different BC subtypes and how NORAD impacts crucial signaling pathways in this pathology. Through the preferential binding to pumilio (PUM) proteins PUM1 and PUM2, NORAD has been shown to be involved in the control of cell cycle, angiogenesis, mitosis, DNA replication and transcription and protein translation. More recently, NORAD has been associated with PUM-independent roles, accomplished by interacting with other ncRNAs, mRNAs and proteins. The intricate network of NORAD-mediated signaling pathways may provide insights into the potential design of novel unexplored strategies to overcome chemotherapy resistance in BC treatment.

Long non-coding RNA (LncRNA) non-coding RNA activated by DNA damage (NORAD) knockdown alleviates airway remodeling in asthma via regulating miR-410-3p/RCC2 and inhibiting Wnt/ß-catenin pathway.

Background: Asthma is a chronic inflammatory disorder with high prevalence in childhood. Airway remodeling, an important structural change of the airways, is resulted from epithelial-mesenchymal transition. Long non-coding RNA non-coding RNA activated by DNA damage (NORAD) has been found to promote epithelial-mesenchymal transition in multiple cancers. This study aimed to analyze the role of NORAD in asthma, mainly focusing on epithelial-mesenchymal transition-mediated airway remodeling, and further explored the NORAD-miRNA-mRNA network. Methods: NORAD expression was analyzed in transforming growth factor-ß1-induced BEAS-2B human bronchial epithelial cells and ovalbumin-challenged asthmatic mice. The influences of NORAD on the epithelial-mesenchymal transition characteristics and Wnt/ß-catenin pathway activation were analyzed in vitro. The interactions between NORAD and miR-410-3p as well as miR-410-3p and regulator of chromosome condensation 2 were detected by dual-luciferase reporter assay and RNA pull-down assay. Rescue experiments using miR-410-3p antagonist and chromosome condensation 2 overexpression were used to confirm the mechanism of NORAD. Additionally, the role and mechanism of NORAD were further evaluated in asthmatic mice. Results: NORAD expression was elevated in both asthmatic models. Knockdown of NORAD impeded spindle-like morphology changes, elevated E-cadherin expression, decreased N-cadherin expression, suppressed cell migration, and inactivated the Wnt/ß-catenin pathway in transforming growth factor-ß1-stimulated BEAS-2B cells. NORAD acted as a sponge of miR-410-3p to regulate chromosome condensation 2 expression. Rescue assays demonstrated that silencing of NORAD ameliorated transforming growth factor-ß1-induced EMT via miR-410-3p/chromosome condensation 2/Wnt/ß-catenin axis. In vivo, knockdown of NORAD led to the reduction of inflammatory cell infiltration and collagen deposition, suppression of IL-4, IL-13, transforming growth factor-ß1 and immunoglobulin E production, decreasing of N-cadherin, chromosome condensation 2, ß-catenin and c-Myc expression, but increasing of E-cadherin and miR-410-3p expression. Conclusions: Silencing of NORAD alleviated epithelial-mesenchymal transition-mediated airway remodeling in asthma via mediating miR-410-3p/chromosome condensation 2/Wnt/ß-catenin pathway.

Diagnostic and prognostic potential of long non-coding RNA NORAD in patients with acute deep vein thrombosis and its role in endothelial cell function.

BACKGROUND: Deep venous thrombosis (DVT) is the common clinical cardiovascular disease, and easily develops into post-thrombotic syndrome (PTS). The study aimed to examine the clinical value of long non-coding RNA NORAD gene in the development of DVT and PTS. In vitro, the underlying mechanism was explored. METHODS: Serum levels of lncRNA NORAD gene in 85 DVT cases and 85 healthy individuals were tested. The role of lncRNA NORAD gene in human umbilical vein endothelial cells (HUVECs) proliferation, migration and inflammation was examined. The candidate downstream target gene was predicted via bioinformatic analysis. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were done for the function annotation and pathway enrichment. RESULTS: LncRNA NORAD gene was at high expression in the serum of DVT patients, it can distinguish DVT patients from healthy controls with the area under the curve of 0.919. Elevated expression of lncRNA NORAD gene in PTS patients was detected, DVT cases with high expression of lncRNA NORAD gene were more susceptible to PTS. LncRNA NORAD gene knockdown promoted HUVECs' proliferation, migration while suppressing cell apoptosis and inflammation. MiR-93-5p served as a target of lncRNA NORAD gene, and its overexpression reversed the role of lncRNA NORAD gene in the biological function of HUVECs. The target genes of miR-93-5p were enriched in HIF-1 signaling, TGF-beta signaling and PI3K-Akt signaling, protein-protein interaction (PPI) network indicated STAT3, MAPK1 to be the key targets. CONCLUSIONS: Upregulation of expression of lncRNA NORAD gene was a potential diagnostic biomarker for DVT and related to the development of PTS. LncRNA NORAD/miR-93-5p axis was involved in the progress of DVT through regulating endothelial cell function.

Clinical significance of long non-coding RNA NORAD in rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease that may cause joint deformities and seriously affect the normal life of the patients. In order to enable patients to receive timely attention and treatment, this study developed new diagnostic markers by exploring the expression and molecular mechanism of the long non-coding RNA NORAD (NORAD) in RA. METHODS: Participants including 77 RA patients and 52 healthy persons were enrolled, and the corresponding clinical data and serum samples were obtained. The NORAD and miR-204-5p expression were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The content of inflammatory cytokines (IL-6, TNF-α) were determined through enzyme-linked immunosorbent assay (ELISA). Luciferase activity reporter assay demonstrated the association between NORAD and miR-204-5p. In addition, receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy of NORAD, and Pearson's correlation analysis was applied for the correlation analysis. RESULTS: NORAD was enriched in RA serum with high diagnostic value. Simultaneously, IL-6 and TNF-α levels were also upregulated (P < 0.001). The C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR) and anti-cyclic citrullinated peptide antibody (Anti-CCP) levels in RA patients were generally elevated (P < 0.001). NORAD was positively correlated with the levels of clinical indicators and inflammatory factors (P < 0.0001). Mechanistically, NORAD may affect the progression of RA by targeting and negatively regulating miR-204-5p. CONCLUSIONS: There is a correlation between NORAD and the processes of RA, and NORAD has the potential to predict and diagnose the occurrence of RA.

Clinical significance of long non-coding RNA NORAD in rheumatoid arthritis

Abstract Background Rheumatoid arthritis (RA) is a chronic autoimmune disease that may cause joint deformities and seriously affect the normal life of the patients. In order to enable patients to receive timely attention and treatment, this study developed new diagnostic markers by exploring the expression and molecular mechanism of the long non-coding RNA NORAD (NORAD) in RA. Methods Participants including 77 RA patients and 52 healthy persons were enrolled, and the corresponding clinical data and serum samples were obtained. The NORAD and miR-204-5p expression were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The content of inflammatory cytokines (IL-6, TNF-α) were determined through enzyme-linked immunosorbent assay (ELISA). Luciferase activity reporter assay demonstrated the association between NORAD and miR-204-5p. In addition, receiver operating characteristic (ROC) curve was used to evaluate the diagnostic efficacy of NORAD, and Pearson's correlation analysis was applied for the correlation analysis. Results NORAD was enriched in RA serum with high diagnostic value. Simultaneously, IL-6 and TNF-α levels were also upregulated (P < 0.001). The C-reactive protein (CRP), rheumatoid factor (RF), erythrocyte sedimentation rate (ESR) and anti-cyclic citrullinated peptide antibody (Anti-CCP) levels in RA patients were generally elevated (P < 0.001). NORAD was positively correlated with the levels of clinical indicators and inflammatory factors (P < 0.0001). Mechanistically, NORAD may affect the progression of RA by targeting and negatively regulating miR-204-5p. Conclusions There is a correlation between NORAD and the processes of RA, and NORAD has the potential to predict and diagnose the occurrence of RA.

ARID3A and ARID3B exert direct regulatory control over the long non-coding RNAs (lncRNAs) MALAT1 and NORAD within the context of non-small cell lung cancer (NSCLC).

Lung cancer, known for its high mortality rates and poor prognosis, remains one of the most prevalent cancer types. Early detection and effective treatment methods are crucial for improving survival rates. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases. Long non-coding RNAs (lncRNAs), which play vital roles in various biological processes, have been implicated in the development of cancer and can impact key therapeutic targets in different cancer types. In NSCLC, the dysregulation of specific lncRNAs, such as MALAT1 and NORAD, has been associated with neoplastic initiation, progression, metastasis, tumor angiogenesis, chemoresistance, and genomic instability. Both MALAT1 and NORAD directly regulate the expression of the transcription factor E2F1, thereby influencing cell cycle progression. Additionally, MALAT1 has been reported to affect the expression of p53 target genes, leading to cell cycle progression through the repression of p53 promoter activity. NORAD, on the other hand, is indirectly regulated by p53. The AT-rich interaction domain (ARID) family of DNA-binding proteins, particularly ARID3A and ARID3B, are involved in various biological processes such as cell proliferation, differentiation, and development. They also play significant roles in E2F-dependent transcription and are transcriptional targets of p53. The intricate balance between promoting cellular proliferation through the pRB-E2F pathway and inducing growth arrest through the p53 pathway underscores the crucial regulatory role of ARID3A, ARID3B, and their interaction with lncRNAs MALAT1 and NORAD. In this study, we aimed to investigate the potential interactive and functional connections among ARID3A, ARID3B, MALAT1, and NORAD in NSCLC, considering their involvement in the pRB-E2F and p53 pathways. Our findings strongly suggest that ARID3A and ARID3B play a regulatory role in controlling MALAT1 and NORAD in NSCLC. Specifically, our study demonstrates that the activities of MALAT1 and NORAD were markedly increased upon the overexpression of ARID3A and ARID3B. Therefore, we can conclude that ARID3A and ARID3B likely contribute significantly to the oncogenic functions of MALAT1 and NORAD in NSCLC. Consequently, targeting ARID3A and ARID3B could hold promise as a therapeutic approach in NSCLC, given their direct control over the expression of MALAT1 and NORAD.

Norad Competently Binds with Pum2 to Regulate Neuronal Apoptosis and Play a Neuroprotective Role After SAH in Mice.

Subarachnoid Hemorrhage (SAH) is a cerebrovascular disorder that has been found to have severe consequences, including a high mortality and disability rate. Research has indicated that neuronal death, particularly apoptosis, plays a major role in the neurological impairment that follows SAH. RNA-binding protein Pum2 can interfere with translation or other biological functions by connecting to the UGUAHAUA sequence on RNA. Noncoding RNA activated by DNA damage (Norad) contains some Pum2 recognition sequences, which may bind to Pum2 protein and affect its capacity to attach to target mRNA. The time course expression of Norad and Pum2 after SAH is analyzed by establishing a mouse SAH model. Subsequently, the purpose of this study is to investigate the potential role and mechanism of the Norad-Pum2 axis after SAH using lentivirus overexpression of Pum2 and knockdown of Norad. Analysis of Pum2 and Norad levels reveal that the former is significantly reduce and the latter is significantly increased in the SAH group compared to the sham group. Subsequent overexpression of Pum2 and Norad knockdown is found to reduce SAH-induced oxidative stress, neuronal apoptosis, and ultimately improve behavioral and cognitive changes in SAH mice. Our study indicates that Norad-Pum2 acts as a neuromodulator in SAH, and that by increasing Pum2 and decreasing Norad levels, SAH-induced neuronal apoptosis can be reduced and neurological deficits alleviated. Consequently, Norad-Pum2 may be a promising therapeutic target for SAH.

Expression of NORAD correlates with breast cancer aggressiveness and protects breast cancer cells from chemotherapy.

The recently discovered human lncRNA NORAD is induced after DNA damage in a p53-dependent manner. It plays a critical role in the maintenance of genomic stability through interaction with Pumilio proteins, limiting the repression of their target mRNAs. Therefore, NORAD inactivation causes chromosomal instability and aneuploidy, which contributes to the accumulation of genetic abnormalities and tumorigenesis. NORAD has been detected in several types of cancer, including breast cancer, which is the most frequently diagnosed and the second-leading cause of cancer death in women. In the present study, we confirmed upregulated NORAD expression levels in a set of human epithelial breast cancer cell lines (MDA-MB-231, MDA-MB-436, and MDA-MB-468), which belong to the most aggressive subtypes (triple-negative breast cancer). These results are in line with previous data showing that high NORAD expression levels in basal-like tumors were associated with poor prognosis. Here, we demonstrate that NORAD downregulation sensitizes triple-negative breast cancer cells to chemotherapy, through a potential accumulation of genomic aberrations and an impaired capacity to signal DNA damage. These results show that NORAD may represent an unexploited neoadjuvant therapeutic target for chemotherapy-unresponsive breast cancer.

[Impacts of LncRNA NORAD on the Proliferation, Apoptosis, and Chemosensitivity of Non-small Cell Lung Cancer Cells by Regulating ZNF217 through MiR-199a-3p].

BACKGROUND: The treatment and diagnosis of non-small cell lung cancer (NSCLC) is still a difficult problem in the medical community, and exploring the molecular mechanism of the occurrence and development of NSCLC is a hot topic of the current research. Long non-coding RNA (lncRNA) NORAD is highly expressed in a variety of cancer cells. It may be a molecular target that promotes NSCLC. The aim of this study was to investigate the impacts of lncRNA NORAD on the proliferation, apoptosis, and chemosensitivity of NSCLC by regulating zinc finger protein 217 (ZNF217) through miR-199a-3p. METHODS: Real-time quantitative polymerase chain reaction (qRT-PCR) method was applied to detect the expressions of NORAD, miR-199a-3p and ZNF217 genes in normal lung epithelial cells BEAS-2B, lung cancer H460 cells, and Cisplatin (DDP) resistant cell lines H460/DDP. H460/DDP cells were devided into control group, si-NC group, si-NORAD group, miR-NC group, miR-199a-3p mimic group, si-NORAD+inhibitor NC group and si-NORAD+miR-199a-3p inhibitor group. Cell proliferation, apoptosis, the expression of NORAD, miR-199a-3p and ZNF217 genes of cells in each group were detected and the expression levels of Ki-67, caspase-9 and ZNF217 proteins in different cells were also observed. The relationship between miR-199a-3p, NORAD and ZNF217 was vefified. RESULTS: Compared with BEAS-2B cells, the expressions of NORAD, ZNF217 mRNA were significantly increased in H460 and H460/DDP cells (P<0.05) but the expression of miR-199a-3p was significantly reduced (P<0.05). Compared with H460 cells, the expression of NORAD and ZNF217 mRNA in H460/DDP cells was significantly increased (P<0.05) and the expression of miR-199a-3p was significantly reduced (P<0.05). Compared with the control group and si-NC group, the proliferation rate, NORAD and ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD group were significantly reduced (P<0.05), but the apoptosis rate, miR-199a-3p expression and caspase-9 expression were significantly increased (P<0.05). Compared with the miR-NC group, the proliferation rate, NORAD and ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the miR-199a-3p mimic group were significantly reduced (P<0.05), but the apoptosis rate, miR-199a-3p expression and caspase-9 expression were significantly increased (P<0.05). Compared with the si-NORAD+inhibitor NC group, the proliferation rate, ZNF217 mRNA expression, Ki-67 and ZNF217 protein expression of H460/DDP cells in the si-NORAD+miR-199a-3p inhibitor group were significantly increased (P<0.05), the apoptosis rate, miR-199a-3p expression and caspase-9 expression were obviously increased reduced (P<0.05). CONCLUSIONS: Down-regulating NORAD expression can enhance miR-199a-3p expression and inhibit ZNF217 expression, thereby inhibiting H460/DDP cell proliferation, promoting apoptosis and enhancing its DDP chemotherapy sensitivity.

LncRNA NORAD regulates the mechanism of the miR-532-3p/Nectin-4 axis in pancreatic cancer cell proliferation and angiogenesis.

Backgound: Pancreatic cancer (PC) is one of the deadliest cancers worldwide, and cell proliferation and angiogenesis play an important role in its occurrence and development. High levels of lncRNANORAD have been detected in many tumors, including PC, yet the effect and mechanism of lncRNA NORAD on PC cell angiogenesis are unexplored. Methods: qRT.PCR was applied to quantify lncRNA NORAD and miR-532-3p expression in PC cells, and a dual luciferase reporter gene was used to verify the targeting effects of NORAD, miR-532-3p and Nectin-4. Then, we regulated NORAD and miR-532-3p expression in PC cells and detected their effects on PC cell proliferation and angiogenesis using cloning experiments and HUVEC tube formation experiments. Results: LncRNA NORAD was upregulated and miR-532-3p was downregulated in PC cells compared with normal cells. Knockdown of NORAD inhibited PC cell proliferation and angiogenesis. LncRNA NORAD and miR-532-3p competitively bound to promote the expression of the miR-532-3p target gene Nectin-4, thereby promoting proliferation and angiogenesis of PC cells in vitro. Conclusion: LncRNA NORAD promotes the proliferation and angiogenesis of PC cells by regulating the miR-532-3p/Nectin-4 axis, which may be a potential biological target in the diagnosis and treatment of clinical PC.

LncRNA NORAD defects deteriorate the formation of age-related macular degeneration.

Long noncoding RNAs (lncRNAs) play important roles in the development of age-related macular degeneration (AMD). However, the effect of long non-coding RNA activated by DNA damage (NORAD) on AMD remains unknown. This study aimed to investigate the effect of NORAD on RPE cell senescence and degeneration. Irradiated adult retinal pigment epithelial cell line-19 (ARPE-19) and sodium iodate-treated mice were used as in vitro and in vivo AMD models. Results showed that irradiation-induced AMD characteristics of ARPE-19 and NORAD-knockdown aggravated cell cycle arrest in the G2/M phase, cell apoptosis and cell senescence along with the increased expression of phosphorylated P53 (p-P53) and P21. AMD factors C3, ICAM-1, APP, APOE, and VEGF-A were also increased by NORAD-knockdown. Moreover, NORAD-knockdown increased irradiation-induced reduction of mitochondrial homeostasis factors, (i.e., TFAM and POLG) and mitochondrial respiratory chain complex genes (i.e., ND1 and ND5) along with mitochondrial reactive oxygen species (ROS). We also identified a strong interaction of NORAD and PGC-1α and sirtuin 1 (SIRT1) in ARPE-19; that is, NORAD knockdown increases the acetylation of PGC-1α. In NORAD knockout mice, NORAD-knockout accelerated the sodium iodate-reduced retinal thickness reduction, function impairment and loss of retinal pigment in the fundus. Therefore, NORAD-knockdown accelerates retinal cell senescence, apoptosis, and AMD markers via PGC-1α acetylation, mitochondrial ROS, and the p-P53-P21signaling pathway, in which NORAD-mediated effect on PGC-1α acetylation might occur through the direct interaction with PGC-1α and SIRT1.

The effects and mechanism of LncRNA NORAD on doxorubicin-induced cardiotoxicity.

In recent years, the role and mechanism of long non-coding RNA (LncRNA) in cardiovascular diseases have received increasing attention. The chemotherapy agent, doxorubicin (DOX), is one of the most effective drugs for various cancers, but its efficacy is limited by its cardiotoxicity. Therefore, further exploration is required for the molecular mechanism of DOX-induced cardiotoxicity. This study intended to investigate the role of LncRNA Non-coding RNA activated by DNA damage (NORAD) in DOX-induced cardiotoxicity, for which we adopted the AC16 human cardiomyocyte cell line for the exploration. The results showed that LncRNA NORAD knockdown could increase DOX-induced cardiomyocyte apoptosis and mitochondrial ROS level. LncRNA NORAD overexpression obtained reverse results, which further validated its role in DOX-induced cardiomyocyte apoptosis and mitochondrial ROS level. Moreover, cardiotoxicity was induced in both LncRNA NORAD-knockout and wild-type mice with DOX, showing that gene knockout aggravated pathologic lesions in the myocardial tissues of mice. Taken together, LncRNA NORAD affected DOX-induced cardiotoxicity via mitochondrial apoptosis, fission (PUM-MFF), and autophagy (p53-Parkin) pathways both in vivo and in vitro.

lncRNA NORAD, soluble ICAM1 and their correlations may be related to the regulation of the tumor immune microenvironment in laryngeal squamous cell carcinoma (LSCC).

NORAD, non-coding RNA activated by DNA damage, is a Long non-coding RNA (lncRNA) transcript that modulates genome stability and has been reported to be dysregulated in different cancers. Although it has been reported to be upregulated in tumor cells mostly for solid organ cancers, it has also been reported to be downregulated in some cancers. Although the pathophysiological mechanism is not fully understood, a negative correlation between NORAD and intercellular cell adhesion molecule-1 (ICAM-1) has been shown in experimental models, but this situation has not been evaluated in terms of cancer. We aimed to evaluate the potential roles of these two biomarker candidates together and separately in the clinicopathological axis in Laryngeal squamous cell carcinoma (LSCC) in a case-control study setting. The interactions of NORAD and ICAM1 at the RNA level were evaluated interactively by the RIblast program. sICAM1 (soluble intercellular cell adhesion molecule-1) levels were determined by ELISA in one hundred and five individuals (forty-four LSCC, sixty-one control) and lncRNA NORAD expression in eighty-eight tissues (forty-four LSCC tumors, forty-four tumor-free surrounding tissues) was determined by Real-time PCR. While the energy treesholud was - 16 kcal/mol between NORAD and ICAM1, the total energy was 176.33 kcal/mol, and 9 base pair pairings from 4 critical points were detected. NORAD expression level was found to be higher in tumor surrounding tissue compared to tumor tissue, and sICAM1 was higher in the control group compared to LSCC (p = 0.004; p = 0.02). NORAD discreminte tumor surrounding tissue from tumor (AUC: 0.674; optimal sensitivity:87.50%; optimal specificity 54.55%; cut-off point as >1.58 fold change; P = 0.034). The sICAM1 level was found to be higher in the control (494,814 ± 93.64 ng/L) than LSCC (432.95 ± 93.64 ng/L) (p = 0.02). sICAM1 discreminte control group from LSCC (AUC: 0.624; optimal sensitivity 68,85%; optimal specificity 61,36%; cut-off point ≤115,0 ng/L; (p = 0.033). A very strong negative correlation was found between NORAD expression and patients' sICAM1 levels (r = -.967; n = 44; p = 0.033). sICAM1 levels were found to be 1.63 times higher in NORAD downregulated subjects compared to upregulated ones (p = 0.031). NORAD was 3.63 times higher in those with alcohol use, and sICAM 1 was 5.77 times higher in those without distant organ metastasis (p = 0.043; 0.004). The increased NORAD expression in the tumor microenvironment in LSCC, the activation of T cells via TCR signaling, and the decrease of sICAM in the control group in correlation with NORAD suggests that ICAM1 may be needed as a membrane protein in the tumor microenvironment. NORAD and ICAM1 may be functionally related to tumor microenvironment and immune control in LSCC.