Overexpression Of NEAT1 Research Articles

NEAT1 and CHROMR lncRNAs: a promising diagnostic tool for diffuse large B-cell lymphoma especially in elderly patients.

Background: Long non-coding (lnc) RNAs have crucial regulatory roles in molecular pathways, and their dysregulation is associated with the pathogenesis of malignancies such as Diffuse large B-cell lymphoma (DLBCL). Therefore, we aimed to study the NEAT1 and CHROMR expression in DLBCL and explore their association with clinicopathological characteristics.Methods & materials: DLBCL and non-tumor lymph node specimens were obtained to assess the expression levels.Results: NEAT1 and CHROMR expressions were significantly increased in DLBCL, and were linked with the age of DLBCL patients (aged >60). NEAT1 and CHROMR overexpression may serve as moderate-to-good diagnostic biomarkers, with NEAT1 and CHROMR exhibiting area under the curve values of 0.781 and 0.831, respectively.

Long non-coding RNA NEAT1 induced by BHLHE40 activates Wnt/β-catenin signaling and potentiates colorectal cancer progression

BackgroundNuclear-enriched abundant transcript 1 (NEAT1), a long noncoding RNA (lncRNA), has been implicated in the colorectal cancer (CRC) progression. However, its upstream mechanism has not been well studied. In the present study, the functions and mechanisms of NEAT1 in CRC were investigated.MethodsThe NEAT1 expression in CRC tissues and CRC cells was analyzed by RT-qPCR. The genes co-expressed with NEAT1 in CRC were obtained from UALCAN, which were intersected with the transcription factors targeting NEAT1 from hTFtarget. Dual-luciferase assay, RT-qPCR, and ChIP were conducted to analyze the transcriptional regulatory relationship between BHLHE40 and NEAT1. LoVo and HCT-15 cells knocking down BHLHE40 and overexpressing NEAT1 were subjected to MTT, Transwell, Western blot, and flow cytometry to examine the malignant aggressiveness of CRC cells. The effects of knocking down BHLHE40 and overexpressing NEAT1 on tumor and lung metastasis were investigated in mice using HE and immunohistochemical analyses.ResultsNEAT1 and BHLHE40 were significantly overexpressed in CRC tissues and cells. BHLHE40 has a binding relationship with the NEAT1 promoter. Knockdown of BHLHE40 resulted in a reverted malignant phenotype in vitro and slowed tumor growth and metastasis dissemination in vivo, which were reversed by NEAT1 overexpression. Overexpression of BHLHE40 increased Wnt/β-catenin pathway activity, but knockdown of NEAT1 decreased Wnt/β-catenin pathway activity.ConclusionsBHLHE40 mediates the transcriptional activation of NEAT1, which activates the Wnt/β-catenin pathway and promotes the CRC progression.

Long Non-coding RNA NEAT1 , NOD-Like Receptor Family Protein 3 Inflammasome, and Acute Kidney Injury.

Acute kidney injury (AKI) is common in hospitalized patients and is associated with high mortality. Inflammation plays a key role in the pathophysiology of AKI. Long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of the inflammatory and immune response, but its role in AKI remains unclear. We explored the role of lncRNA Neat1 in (1) a cross-sectional and a longitudinal cohort of AKI in human; (2) three murine models of septic and aseptic AKI and (3) cultured C1.1 mouse kidney tubular cells. In human, hospitalized patients with AKI (n=66) demonstrated significantly increased lncRNA Neat1 levels in urinary sediment cells and buffy coat versus control participants (n=152) from a primary care clinic; and among 6 kidney transplant recipients, Neat1 levels were highest immediately after transplant surgery followed by a prompt decline to normal levels in parallel with recovery of kidney function. In mice with AKI induced by sepsis (via LPS injection or cecal ligation and puncture) and renal ischemia-reperfusion, kidney tubular Neat1 was increased versus sham-operated mice. Knockdown of Neat1 in the kidney using short hairpin RNA preserved kidney function, suppressed overexpression of the AKI biomarker NGAL, leukocyte infiltration and both intrarenal and systemic inflammatory cytokines IL-6, CCL-2 and IL-1β. In LPS-treated C1.1 cells, Neat1 was overexpressed via TLR4/NF-κB signaling, and translocated from the cell nucleus into the cytoplasm where it promoted activation of NLRP3 inflammasomes via binding with the scaffold protein Rack1. Silencing Neat1 ameliorated LPS-induced cell inflammation, whereas its overexpression upregulated IL-6 and CCL-2 expression even without LPS stimulation. Our findings demonstrate a pathogenic role of Neat1 induction in human and mice during AKI with alleviation of kidney injury in 3 experimental models of septic and aseptic AKI after knockdown of Neat1. LPS/TLR4-induced Neat1 overexpression in tubular epithelial cells increases the inflammatory response by binding with the scaffold protein, Rack1, to activate NLRP3 inflammasomes.

Long Noncoding RNA NEAT1 Regulates Acute Graft-Versus-Host Disease By Promoting Macrophage M1 Polarization through JNK Pathway

Introduction Acute graft-versus-host disease (aGVHD) is a complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The function of macrophages in aGVHD is a hot point in current research. Long noncoding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) is closely related to immune-related diseases and affects the function of macrophages. However, the role of LncRNA NEAT1 in aGVHD is unclear in current study. Methods Peripheral blood mononuclear cells (PBMCs were collected from the patients with or without aGVHD after allo-HSCT. The expression of lncRNA NEAT1 in PBMCs was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic value of lncRNA NEAT1 in aGVHD was analyzed by receiver operating characteristic curve (ROC) and area under correlation curve (AUC). We transfected RAW264.7 cells and bone marrow-derived macrophages (BMDM) with NEAT1 lentiviral vector or NEAT1 small interfering RNA to change the expression level of NEAT1. The function of NEAT1 and its effect on JNK pathway were analyzed by flow cytometry, qRT-PCR, western blotting and ELISA. Finally, aGVHD mouse model was constructed to evaluate the function of JNK inhibitors in aGVHD. Results Compared with non-aGVHD patients, LncRNA NEAT1 was significantly up-regulated in PBMCs of aGVHD patients. ROC and AUC analysis confirmed that the expression level of lncRNA NEAT1 was correlated with the occurrence of aGVHD, which may have certain diagnostic value in aGVHD. We found the expressions of NEAT1 in BMDM and RAW264.7 were remarkably increased after being stimulated with lipopolysaccharide (100ng/ml). The overexpression of lncRNA NEAT1 in RAW264.7 could significantly up-regulate the expression level of iNOS, and induce the differentiation into M1 type. Knockdown of lncRNA NEAT1 in BMDM could significantly down-regulate the gene expressions (iNOS and NLRP3), reduce the protein levels (iNOS, NLRP3 and p-JNK), decrease the proportion of M1 macrophages and inflammatory cytokines secretion (TNF-α and IL-1β), and inhibit the JNK pathway. We found that JNK inhibitor could perform the same function as knocking down lncRNA NEAT1 in BMDM. In addition, JNK inhibitor could improve the survival and pathology, decrease the inflammatory cytokines secretion (TNF-α and IL-1β), and reduce the infiltration of M1 macrophages into the target organs in aGVHD mouse model. Conclusions Our results showed that lncRNA NEAT1 may regulate the M1 polarization of macrophages and the secretion of inflammatory cytokines by regulating the JNK pathway. JNK inhibitor may reduce the inflammation in aGVHD mouse models by blocking the function of NEAT1 in macrophages. This study provides a promising therapeutic target for aGVHD.

LncRNA NEAT1 suppresses cellular senescence in hepatocellular carcinoma via KIF11-dependent repression of CDKN2A.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Therapeutic options for advanced HCC are limited, which is due to a lack of full understanding of pathogenesis. Cellular senescence is a state of cell cycle arrest, which plays important roles in the pathogenesis of HCC. Mechanisms underlying hepatocellular senescence are not fully understood. LncRNA NEAT1 acts as an oncogene and contributes to the development of HCC. Whether NEAT1 modulates hepatocellular senescence in HCC is unknown. The role of NEAT1 and KIF11 in cellular senescence and tumor growth in HCC was assessed both in vitro and in vivo. RNA pulldown, mass spectrometry, Chromatin immunoprecipitation (ChIP), luciferase reporter assays, RNA FISH and immunofluorescence (IF) staining were used to explore the detailed molecular mechanism of NEAT1 and KIF11 in cellular senescence of HCC. We found that NEAT1 was upregulated in tumor tissues and hepatoma cells, which negatively correlated with a senescence biomarker CDKN2A encoding p16INK4a and p14ARF proteins. NEAT1 was reduced in senescent hepatoma cells induced by doxorubicin (DOXO) or serum starvation. Furthermore, NEAT1 deficiency caused senescence in cultured hepatoma cells, and protected against the progression of HCC in a mouse model. During senescence, NEAT1 translocated into cytosol and interacted with a motor protein KIF11, resulting in KIF11 protein degradation and subsequent increased expression of CDKN2A in cultured hepatoma cells. Furthermore, KIF11 knockdown caused senescence in cultured hepatoma cells. Genetic deletion of Kif11 in hepatocytes inhibited the development of HCC in a mouse model. Conclusively, NEAT1 overexpression reduces senescence and promotes tumor progression in HCC tissues and hepatoma cells, whereas NEAT1 deficiency causes senescence and inhibits tumor progression in HCC. This is associated with KIF11-dependent repression of CDKN2A. These findings lay the foundation to develop potential therapies for HCC by inhibiting NEAT1 and KIF11 or inducing senescence.

ZNF143 inhibits hepatocyte mitophagy and promotes non-alcoholic fatty liver disease by targeting increased lncRNA NEAT1 expression to activate ROCK2 pathway

ABSTRACT Background: Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorders worldwide. The mitophagy is suggested to be repressed in NAFLD, but the mechanism remains to be elucidated. Methods: NAFLD cell and mouse models were established by treating with free fatty acid (FFA) and feeding a high fat diet (HFD), respectively. QRT-PCR, Western blotting, or IHC measured the expression of ZNF143, lncRNA NEAT1, ROCK2, and lipid formation/mitophagy-related proteins. Cell viability and mitophagy were evaluated by MTT and immunofluorescence. The chloroform-methanol extraction method measured triglyceride and total cholesterol levels. ELISA detected ALT and AST levels. The interactions among ZNF143, lncRNA NEAT1 and SND1 were analysed by ChIP, dual-luciferase reporter, pull-down, and RIP. The lipid droplets were determined by Oil-red O and HE staining. Results: ZNF143 and lncRNA NEAT1 were upregulated in hepatic cells treated with FFA (p < 0.01 and p < 0.001). Knockdown of ZNF143 or lncRNA NEAT1 inhibited lipid droplets formation, while promoting mitophagy (p < 0.01 and p < 0.001). ZNF143 promoted lncRNA NEAT1 transcriptional expression through binding to its promoter. LncRNA NEAT1 increased ROCK2 mRNA stability by targeting SND1. LncRNA NEAT1 or ROCK2 overexpression reversed the effect of ZNF143 or lncRNA NEAT1 knockdown on hepatic steatosis and mitophagy (p < 0.01 and p < 0.001). ZNF143 or lncRNA NEAT1 knockdown inhibited HFD-induced steatosis and promoted mitophagy in vivo (p < 0.01 and p < 0.001). Conclusion: The upregulation of lncRNA NEAT1 caused by ZNF143 promoted NAFLD through inhibiting mitophagy via activating ROCK2 pathway by targeting SND1, providing potential targets for NAFLD therapy.

Neat1 lncRNA organizes the inflammatory gene expressions in the dorsal root ganglion in neuropathic pain caused by nerve injury.

Primary sensory neurons regulate inflammatory processes in innervated regions through neuro-immune communication. However, how their immune-modulating functions are regulated in concert remains largely unknown. Here, we show that Neat1 long non-coding RNA (lncRNA) organizes the proinflammatory gene expressions in the dorsal root ganglion (DRG) in chronic intractable neuropathic pain in rats. Neat1 was abundantly expressed in the DRG and was upregulated after peripheral nerve injury. Neat1 overexpression in primary sensory neurons caused mechanical and thermal hypersensitivity, whereas its knockdown alleviated neuropathic pain. Bioinformatics analysis of comprehensive transcriptome changes indicated the inflammatory response was the most relevant function of genes upregulated through Neat1. Consistent with this, upregulation of proinflammatory genes in the DRG following nerve injury was suppressed by Neat1 knockdown. Expression changes of these proinflammatory genes were regulated through Neat1-mRNA interaction-dependent and -independent mechanisms. Notably, Neat1 increased proinflammatory genes by stabilizing its interacting mRNAs in neuropathic pain. Finally, Neat1 in primary sensory neurons contributed to spinal inflammatory processes that mediated peripheral neuropathic pain. These findings demonstrate that Neat1 lncRNA is a key regulator of neuro-immune communication in neuropathic pain.

Expression of NEAT1 can be used as a predictor for Dex resistance in multiple myeloma patients

ObjectiveMultiple myeloma is a heterogeneous disorder and the intratumor genetic heterogeneity contributes to emergency of drug resistance. Dexamethasone has been used clinically for decades for MM. Nevertheless, their use is severely hampered by the risk of developing side effects and the occurrence of Dex resistance. LncRNA NEAT1 plays a oncogenic role and participates in drug resistance in many solid tumors. Therefore, we investigated a potential usefulness of this molecular as a biomarker for diagnosis of MM and possible correlations of NEAT1 expression with drug resistance and prognosis.MethodsBone marrow and peripheral blood mononuclear cells samples were collected from 60 newly diagnosed MM patients. The expression of NEAT1expression level were detected by quantitative real-time PCR analyses. The relationship about the expression levels of lncRNA with other clinical and cytogenetic features was analyzed. In addition, we measured to analysis the correlation between the expression of NEAT1 and Dex resistance in MM patients.ResultsIt was found that the expression of NEAT1 is significantly higher in multiple myeloma patients compared to controls and does not change with other clinical features and cytogenetic features. We further discovered that overexpression of NEAT1 was associated with Dex resistance and a poor prognosis in MM patients.ConclusionLncRNA NEAT1 has a significant value that might act as a promoting factor in the development of MM and may be severed as a diagnostic factor in MM. NEAT1 invovled in Dex resistance, which provide a new interpretation during the chemotherapy for MM.

LncRNA NEAT1 and MALAT1 are involved in polycystic ovary syndrome pathogenesis by functioning as competing endogenous RNAs to control the expression of PCOS-related target genes.

Accumulating evidence has shown an abnormal expression of several non-coding RNAs in ovarian tissues which might be closely linked with the pathogenesis of PCOS. The aim of this study was to identify competing endogenous (ce) RNA network: long non-coding RNA (lncRNA), microRNA (miRNA) and their target genes: androgen receptor (AR), follistatin (FST) and insulin receptor substrate-2 (IRS-2), which are relevant to PCOS, to underline the molecular pathogenesis of PCOS and assist in early diagnosis and treatment. Bioinformatic analysis was performed to retrieve a ceRNA network: [lncRNA (NEAT1 and MALAT1) - miRNA (miR-30a-5p and miR-30d-5p) - mRNA (AR, FST and IRS-2)] linked to PCOS. Expression of the selected RNAs was examined by qPCR in peripheral blood leukocytes obtained from 73 PCOS patients (41 obese and 32 non-obese) and 31 healthy controls. PCOS patients showed significantly higher expression levels of NEAT1, miR-30a-5p, AR, FST and IRS-2, with significantly lower expression levels of MALAT1 and miR-30d-5p relative to controls especially in obese versus non-obese patients. Receiver operating characteristic (ROC) curve analysis indicated that most of the selected RNAs could serve as potential early diagnostic markers for PCOS with the highest efficiency obtained upon combining NEAT1 and miR-30d-5p or MALAT1 and miR-30a-5p with either of PCOS target genes. Moreover, all addressed RNAs had been proved as potential predictors of PCOS. The obtained data of ceRNA network raised the possibility that NEAT1 overexpression may increase the expression levels of AR, FST and IRS-2 by sponging miR-30d-5p, while low expression of MALAT1 may allow higher expression of the above genes via increasing miR-30a-5p, suggesting their involvement in PCOS pathogenesis and promising role for future diagnosis and targeted therapy.

Long Non-Coding RNA Nuclear Paraspeckle Assembly Transcript 1: A Regulator of Trophoblast Cells in the Progression of Gestational Diabetes Mellitus

The potential function of NEAT1 in the progression of Gestational diabetes mellitus (GDM) and the molecular mechanisms are explored. NEAT1 levels in multiple types of cell lines and placental tissues collected from healthy, GDM and preeclampsia pregnancies were detected by quantitative real-time polymerase chain reaction (qRT-PCR). After intervening NEAT1 level in HTR-8/SVneo cells, proliferative, migratory and apoptotic potentials were examined by cell counting kit-8 (CCK-8), 5-Ethynyl-2′-deoxyuridine (EdU), transwell assay and flow cytometry. The regulatory effect of NEAT1 on the transcriptional activity of NSD1 was predicted by bioinformatic analysis and further confirmed by dual-luciferase reporter assay. In addition, histone modifications of NEAT1 on NSD1 transcription were examined by chromatin immunoprecipitation (ChIP). NEAT1 was upregulated in placental tissues collected from GDM patients. Overexpression of NEAT1 stimulated proliferative and migratory potentials, but inhibited apoptosis in HTR-8/SVneo cells. Knockdown of NEAT1 had the opposite outcomes. NEAT1 was able to regulate the transcriptional activity of NSD1 through histone modifications on H3K27Me3 and H3K27Cro. NEAT1 is upregulated in GDM cases, which triggers proliferative and migratory potentials in trophoblast cells mainly through regulating transcriptional activity of NSD1 via histone modification.

Evodiamine inhibits malignant progression of ovarian cancer cells by regulating lncRNA-NEAT1/miR-152-3p/CDK19 axis.

Evodiamine (EVO) has been demonstrated to promote apoptosis of ovarian cancer cells, and upregulate miR-152-3p level in colorectal cancer. Here, we explore part of the network mechanism of EVO and miR-152-3p in ovarian cancer. The bioinformatics website, dual luciferase reporter assay, and quantitative real-time polymerase chain reaction were applied to analyze the network among EVO, lncRNA, miR-152-3p, and mRNA. The effect and mechanism of EVO on ovarian cancer cells were determined using cell counting kit-8, flow cytometry, TUNEL, Western blot, and rescue experiments. As a result, EVO dose-dependently attenuated cell viability, induced G2/M phase arrest and apoptosis, promoted miR-152-3p level (4.5- or 2-fold changes), and inhibited expressions of NEAT1 (0.225- or 0.367-fold changes), CDK8 (0.625- or 0.571-fold changes), and CDK19 (0.25- or 0.147-fold changes) in OVCAR-3 and SKOV-3 cells. In addition, EVO decreased Bcl-2 expression, but increased the expressions of Bax and c-caspase-3. NEAT1 targeted miR-152-3p which bound to CDK19. The impacts of EVO on cell viability, cycle, apoptosis, and apoptosis-related proteins were partially reversed by miR-152-3p inhibitor, NEAT1 overexpression, or CDK19 overexpression. Furthermore, miR-152-3p mimic offset the effects of NEAT1 or CDK19 overexpression. The role of NEAT1 overexpression in the biological phenotype of ovarian cancer cells was counteracted by shCDK19. In conclusion, EVO attenuates ovarian cancer cell progression via the NEAT1-miR-152-3p-CDK19 axis.

Upregulation of HTRA1 mediated by the lncRNA NEAT1/miR-141-3p axis contributes to endometriosis development through activating NLRP3 inflammasome-mediated pyroptotic cell death and cellular inflammation

The present study identified a novel upstream long chain non-coding (lncRNA) NEAT1/miR-141-3p/HTRA1 axis that regulated the activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome to modulate endometriosis (EM) development. Specifically, clinical data suggested that the expression of NLRP3 and apoptosis-associated speck-like protein containing CARD (ASC), the cleavage of caspase-1 and gasdermin D (GSDMD), and the production of inflammatory cytokines (interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and IL-18) were all significantly increased in the ectopic endometrium (EE) tissues, compared to the normal endometrium (NE) tissues. Then, through analyzing the datasets from GEO database (GSE2339, GSE58178, and GSE7305) using the GEO2R bioinformatics tools, we verified that HtrA Serine Peptidase 1 (HTRA1) was especially enriched in the EE tissues compared to the NE tissues. To further confirm the biological functions of HTRA1, HTRA1 was overexpressed or downregulated in primary human endometrial stromal cells (hESCs) isolated from NE tissues or EE tissues, respectively. The results showed that upregulation of HTRA1 activated NLRP3 inflammasome-mediated pyroptotic cell death and cellular inflammation in NE-derived hESCs, whereas silencing of HTRA1 played an opposite role in EE-derived hESCs. In addition, the lncRNA NEAT1/miR-141-3p axis was screened as the upstream regulator of HTRA1. Mechanistically, lncRNA NEAT1 sponged miR-141-3p to positively regulate HTRA1 in a competing endogenous RNA (ceRNA) mechanisms-dependent manner. The recovery experiments in hESCs from NE and EE tissues confirmed that lncRNA NEAT1 overexpression promoted NLRP3 inflammasome-mediated pyroptotic cell death through regulating the miR-141-3p/HTRA1 axis. Taken together, this study firstly uncovered the underlying mechanisms by which a novel lncRNA NEAT1/miR-141-3p/HTRA1-NLRP3 pathway contributed to the development of EM, which provided novel diagnostic and therapeutic biomarkers for this disease.

Up-regulation of lncRNA NEAT1 in cerebral ischemic stroke promotes activation of astrocytes by modulation of miR-488-3p/RAC1

We aim to research the molecular mechanism of lncRNA NEAT1 in the activation of astrocytes in a cerebral ischemia-reperfusion injury model. Mouse model of cerebral ischemia-reperfusion injury was constructed, and shNEAT1 was transfected. The infarct area, brain water content, and neurological deficiency were detected. Immunofluorescence detection and fluorescence in situ hybridization (FISH) assay were processed to detect glial fibrillary acidic protein (GFAP) expression. Astrocyte cells were cultured for oxygen-glucose deprivation/re-oxygenation (OGD)/re-oxygenation model construction. After treatment by shNEAT1, miR-488-3p mimic, miR-488-3p inhibitor, Q-PCR assay, western blot and ELISA were undertaken to detect the expressions of NEAT1, miR-488-3p, RAC1, inflammatory cytokines, RAC1 and GFAP. Dual luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were used to verify the binding of NEAT1, miR-488-3p and RAC1. The expression of NEAT1 in brain tissue was significantly higher than that in Sham operation group. Knockdown of NEAT1 inhibited the brain damage caused by middle cerebral artery occlusion (MCAO) treatment, reduced the inflammatory response, and suppressed the activation of astrocytes. By constructing an in vitro OGD/R cell model, it was found that NEAT1 knockdown also inhibited the activation of astrocytes caused by OGD/R. Knockdown of NEAT1 caused the up-regulation of miR-488-3p and the down-regulation of RAC1. Knockdown of miR-488-3p or over-expression of RAC1 reversed the inhibitory effect of shNEAT1 on OGD/R-induced astrocyte activation. Over-expression of NEAT1 in cerebral ischemic stroke promotes activation of astrocytes by modulation miR-488-3p/RAC1, which is proved in vitro. Our study may provide a new idea for the diagnosis and treatment of MCAO.

The lncRNA NEAT1 Mediates Neuronal Cell Autophagy and Related Protein Expression After Cerebral Ischemia‒Reperfusion Injury.

The present study focuses on the role of the long noncoding RNA (lncRNA) NEAT1 in regulating autophagy during the ischemia‒reperfusion (I/R) injury process and its possible regulatory mechanism based on the results of laboratory experiments. Neuro-2a (N2a) cells and BV-2 microglial cells were cultured separately, and oxygen-glucose deprivation/reoxygenation (OGD/R) was induced in vitro to mimic cerebral I/R injury. The expression of lncRNA NEAT1 was measured after reoxygenation for different durations, and the results showed that NEAT1 expression was significantly different after OGD/R for 12h; thus, cell models of NEAT1 overexpression and knockdown were constructed. Knockdown of NEAT1 effectively relieved reperfusion injury. In an N2a and BV-2 cell coculture system, knockdown of NEAT1 reduced autophagic flow in neuronal cells after reperfusion. To clarify the mechanism of NEAT1 after neuronal I/R injury, label-free quantitative proteomics (LFQ) was used to identify the differentially expressed proteins (DEPs) in NEAT1 knockdown neurons after OGD/R for 12h. Additionally, Gene Ontology (GO) enrichment, protein‒protein interaction (PPI) network and parallel-reaction monitoring (PRM) quantitative analyses were carried out; the results showed that the expression levels of the autophagy-related proteins Gaa, Glb1, Prkaa1, Kif23, Sec24a and Vps25 were significantly reduced and that these proteins interact. In summary, this study shows that NEAT1 can regulate the interactions between autophagy-related proteins after neuronal I/R injury, reducing the level of autophagy and relieving neuronal reperfusion injury.

Epigenetic control of LncRNA NEAT1 enables cardiac fibroblast pyroptosis and cardiac fibrosis

Cardiac fibroblasts (CFs) drive extracellular matrix remodeling after inflammatory injury, leading to cardiac fibrosis and diastolic dysfunction. Recent studies described the role of epigenetics in cardiac fibrosis. Nevertheless, detailed reports on epigenetics regulating CFs pyroptosis and describing their implication in cardiac fibrosis are still unclear. Here, we found that DNMT3A reduces the expression of lncRNA Neat1 and promotes the NLRP3 axis leading to CFs pyroptosis, using cultured cells, animal models, and clinical samples to shed light on the underlying mechanism. We report that pyroptosis-related genes are increased explicitly in cardiac fibrosis tissue and LPS-treated CFs, while lncRNA Neat1 decreased. Mechanistically, we show that loss of DNMT3A or overexpression of lncRNA Neat1 in CFs after LPS treatment significantly enhances CFs pyroptosis and the production of pyroptosis-related markers in vitro. It has been demonstrated that DNMT3A can decrease lncRNA Neat1, promoting NLRP3 axis activation in CFs treated with LPS. In sum, this study is the first to identify that DNMT3A methylation decreases the expression of lncRNA Neat1 and promotes CFs pyroptosis and cardiac fibrosis, suggesting that DNMT3A and NEAT1 may function as an anti-fibrotic therapy target in cardiac fibrosis.

NEAT1 promotes the malignant development of bladder cancer by regulating the miR-101/VEGF-C pathway in vitro and in vivo

BackgroundNEAT1 has been shown to play an oncogenic role in many kinds of cancers. However, detailed roles of NEAT1 in bladder cancer are largely unknown.MethodsIn the present study, the expression of NEAT1, miR-101 and VEGF-C was detected in human bladder cancer samples. The relationship between NEAT1 and the prognosis of patients with bladder cancer was analysed. In vitro experiments explored the effects of NEAT1 on biological behaviours of bladder cancer T24 and 5637 cells. Bioinformatics prediction and luciferase assays were used to assay the regulatory mechanism of action of NEAT1 and miR-101. Loss and gain of the expression of miR-101 and VEGF-C were used to explore the effects of the NEAT1/miR-101/VEGF-C pathway on T24 and 5637 cells. The effect of NEAT1 on the growth of bladder cancer in vivo was explored using an orthotopic tumourigenesis model.ResultsNEAT1 and VEGF-C were significantly upregulated in bladder cancer samples, and miR-101 was significantly downregulated. NEAT1 upregulation was associated with poorer recurrence-free survival of patients with bladder cancer. Overexpression of NEAT1 promoted the proliferation, migration and invasion of bladder cancer cells. The results of the luciferase assay indicated that miR-101 was a target of NEAT1. The promoting effects of NEAT1 on bladder cancer cells were reversed by miR-101 upregulation, and inhibition of miR-101 enhanced the effects of NEAT1. Overexpression of VEGF-C had a clear synergistic effect with the action of NEAT1. Overexpression of NEAT1 increased tumour growth and induced the development of liver metastasis.ConclusionsIn conclusion, our data indicated that NEAT1 was expressed at high levels in bladder cancer patients and correlated with unfavourable prognosis. NEAT1 promoted malignant development of bladder cancer in vitro and in vivo by regulating the miR-101/VEGF-C pathway.

Activation of long non-coding RNA NEAT1 leads to survival advantage of multiple myeloma cells by supporting a positive regulatory loop with DNA repair proteins.

Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organelles and it has been found to be deregulated in multiple myeloma (MM) patients. Experimental evidence indicated that NEAT1 silencing negatively impacts proliferation and viability of MM cells, both in vitro and in vivo, suggesting a role in DNA damage repair (DDR). In order to elucidate the biological and molecular relevance of NEAT1 upregulation in MM disease we exploited the CRISPR/Cas9 synergistic activation mediator genome editing system to engineer the AMO-1 MM cell line and generate two clones that para-physiologically transactivate NEAT1 at different levels. NEAT1 overexpression is associated with oncogenic and prosurvival advantages in MM cells exposed to nutrient starvation or a hypoxic microenvironment, which are stressful conditions often associated with more aggressive disease phases. Furthermore, we highlighted the NEAT1 involvement in virtually all DDR processes through, at least, two different mechanisms. On one side NEAT1 positively regulates the posttranslational stabilization of essential PS proteins, which are involved in almost all DDR systems, thus increasing their availability within cells. On the other hand, NEAT1 plays a crucial role as a major regulator of a molecular axis that includes ATM and the catalytic subunit of DNA-PK kinase proteins, and their direct targets pRPA32 and pCHK2. Overall, we provided novel important insightsthe role of NEAT1 in supporting MM cells adaptation to stressful conditions by improving the maintenance of DNA integrity. Taken together, our results suggest that NEAT1, and probably PS organelles, could represent a potential therapeutic target for MM treatment.

LncRNA NEAT1-let 7b-P21 axis mediates the proliferation of neural stem cells cultured invitro promoted by radial extracorporeal shock wave.

In previous studies, we found radial extracorporeal shock wave (rESW), can promote the proliferation of neural stem cells (NSCs). Emerging evidence suggests that lncRNA NEAT1 can regulate NSCs proliferation. Whether lncRNA NEAT1 plays a role in the proliferation of NSC induced by shock waves is unclear. Cell Counting Kit-8（CCK 8) method was used to detect the proliferation of NSCs, and the relative protein and mRNA expression of related genes of Nestin, Cyclin D1 and P21 were detected by Western Blot and Quantitative real-time PCR (RT-qPCR) respectively. Immunofluorescence staining was used to observe the changes in the number of BrdU/nestin positive cells. Overexpression of NEAT1 and let 7 b in cells were used to explore whether rESW can rescue the decreased number of NSCs.We found that the optimal dose of R15 transmitter promoting NSCs proliferation is 1.5 bar, 500 pulse, 2 Hz. 1.2–1.5 bar showed a dose-dependent effect on the proliferation of NSCs, but it was negatively correlated with the proliferation effect of NSC when it was more than 1.5 bar. We revealed that let 7 b-P21 axis was involved in regulating the inhibition of NSC proliferation which was activated by NEAT1 in NSCs. In addition, we demonstrated that rESW treatment resulted in the decrease of NEAT1 expression, which was accompanied by the improved biological function including proliferation. Our results confirm that low-intensity rESW (1.5 bar，500 pulse，2 Hz) can promote the proliferation of NSCs through NEAT1-let 7 b-P21 axis.

P875: ACTIVATION OF LNCRNA NEAT1 LEADS TO SURVIVAL ADVANTAGE OF MULTIPLE MYELOMA CELLS BY SUPPORTING A REGULATORY LOOP WITH DNA REPAIR PROTEINS: RATIONAL BASES FOR NEAT1 THERAPEUTIC TARGETING IN THE DISEASE

Background: Multiple myeloma (MM) is a fatal malignant proliferation of antibody-secreting bone marrow plasma cells characterized by marked genomic instability. Long non-coding RNA (lncRNA) represents the largest class of non-protein coding genes in the human genome. Their crucial role in solid tumor and hematological malignances, including MM, is well documented. NEAT1 is a highly expressed nuclear lncRNA, representing the core structural component of paraspeckle organelles. We previously demonstrated that NEAT1 silencing negatively regulates proliferation and viability of MM cells, both in vitro and in vivo, highlighting its pivotal role in DNA integrity maintenance, by regulating the homologous recombination. Despite the increasing information obtained by loss-of-function approaches, there is still a lack of information regarding possible oncogenic benefits acquired by MM cells upon NEAT1 overexpression. Aims: Taking advantage of the use of the CRISPR/Cas9 SAM genome editing approach, the present study aimed to shed light on the biological and molecular implication of NEAT1 overexpression in MM. Methods: We adopted the CRISPR/Cas9 Synergistic Activation Mediator editing system to transactivate NEAT1 in AMO-1 MM cell line. LNA-gapmeR antisense oligonucleotide technology was used to silence NEAT1 in MM cells by gymnotic delivery. Hypoxia was induced by culturing the cells into a modular incubator chamber flushed with a mixture of 1% O2, 5% CO2 and 94%N2 at 37 °C. NEAT1 expression was assessed by qRT-PCR and RNA-FISH. Cell cycle phases distribution and apoptotic cells percentage were assessed by flow cytometry. Clonogenic potential was evaluated by methylcellulose assay. Protein expression was investigated by WB and IF. Cell morphological analysis was performed after May-Grunwald Giemsa staining. Transcriptomic analysis was performed by RNA sequencing, following the TruSeq Stranded Total RNA protocol. Libraries with optimal quality and quantity were run on NextSeq 500. Statistical significance was determined by Student t test analysis; differences were considered significant when P-value was <0.05. Results: We performed a transcriptomic analysis of AMO-1 cells either transactivated or silenced for NEAT1. Our data revealed a NEAT1 pivotal role in the maintenance of DNA integrity, showing a significant deregulation of almost all the crucial cellular DNA repair mechanisms for both single and double strand breaks. In particular, we found that NEAT1 transactivation affects DNA repair mechanisms through the activation of a molecular axis including two fundamental kinase proteins, i.e. ATM and DNA-PKcs, and the direct target pRPA32. Furthermore, our data strongly confirmed the activation of this NEAT1-orchestrated molecular axis whether MM cells are exposed to nutrient starvation and hypoxia, suggesting its implication in conferring oncogenic and pro-survival properties to MM cells. The disruption of this oncogenic loop leads to MM cell death and the loss of pro survival advantages, thus suggesting that NEAT1 should be considered a crucial factor for MM cells survival. Summary/Conclusion: Overall, we provided novel important insights into the role of NEAT1 in MM pathobiology, demonstrating that its deregulation strongly correlates with adaptation to stress condition, often associated with late stages of the disease. Taken together, our results suggest that NEAT1 could represent Achilles’ heel for MM cells and should be considered a potential therapeutic target for MM treatment.

LncRNA NEAT1 stabilized Wnt3a via U2AF2 and activated Wnt/β-catenin pathway to alleviate ischemia stroke induced injury

BackgroundIschaemic stroke is the leading cause of mortality and disability in the world. LncRNA NEAT1 has been shown to play an important role in ischaemic injury, but the molecular mechanism remains unclear. MethodsqRT–PCR was used to determine the expression of lncRNA NEAT1 in OGD/R-induced BV-2 cells. Cell viability was assessed by an MTT assay, and cell apoptosis was assessed by flow cytometry. The expression of related proteins was evaluated by Western blotting and ELISA. The interactions among lncRNA NEAT1, U2AF2 and Wnt3a mRNA was demonstrated by RIP and RNA pulldown assays. XAV-939 was used as an inhibitor of the Wnt/β-catenin pathway. ResultsLncRNA NEAT1 was found to be downregulated in OGD/R-induced BV-2 cells. Overexpression of lncRNA NEAT1 protected BV-2 cells against OGD/R-induced injury. LncRNA NEAT1 enhanced the stability of Wnt3a mRNA via U2AF2. Knockdown of Wnt3a or blockade of the Wnt/β-catenin pathway rescued the effect of lncRNA NEAT1. ConclusionsLncRNA NEAT1 protected cells against OGD/R-induced apoptosis and the inflammatory response by activating the Wnt/β-catenin pathway through upregulation of Wnt3a in a U2AF2-dependent manner. LncRNA NEAT1 could be a promising therapeutic candidate for ischaemic stroke treatment in the future.