Knockdown Of Nuclear Paraspeckle Assembly Transcript 1 Research Articles

MALAT1 and NEAT1 Are Neuroprotective During Hypoxic Preconditioning in the Mouse Hippocampus Possibly by Regulation of NR2B.

Wang L, Fu G, Han R, Fan P, Yang J, Gong K, Zhao Z, Zhang C, Sun K, Shao GMALAT1 and NEAT1 Are Neuroprotective during Hypoxic Preconditioning in the Mouse Hippocampus Possibly by Regulation of NR2B High Alt Med Biol. 00:000-000, 2024. Background: The regulation of noncoding ribonucleic acid (ncRNA) has been shown to be involved in cellular and molecular responses to hypoxic preconditioning (HPC), a situation created by the induction of sublethal hypoxia in the brain. The ncRNAs metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and nuclear paraspeckle assembly transcript 1 (NEAT1) are abundantly expressed in the brain, where they regulate the expression of various genes in nerve cells. However, the exact roles of MALAT1 and NEAT1 in HPC are not fully understood. Methods: A mouse model of acute repeated hypoxia was used as a model of HPC, and MALAT1 and NEAT1 levels in the hippocampus were measured using real-time polymerase chain reaction (PCR). The mRNA and protein levels of N-methyl-d-aspartate receptor subunit 2 B (NR2B) in the mouse hippocampus were measured using real-time PCR and western blotting, respectively. HT22 cells knocked-down for MALAT1 and NEAT1 were used for in vitro testing. Expression of NR2B, which is involved in nerve cell injury under ischemic and hypoxic conditions, was also evaluated. The levels of spectrin and cleaved caspase-3 in MALAT1 and NEAT1 knockdown HT22 cells under oxygen glucose deprivation/reperfusion (OGD/R) were determined by western blotting. Results: HPC increased the expression of MALAT1 and NEAT1 and decreased the expression of NR2B mRNA in the mouse hippocampus (p < 0.05). Knockdown of MALAT1 and NEAT1 increased both NR2B mRNA and protein levels nearly twofold and caused damage under OGD/R conditions in HT22 cells (p < 0.05). Conclusion: MALAT1 and NEAT1 exert neuroprotective effects by influencing the expression of NR2B.

Knockdown of NEAT1 prevents post-stroke lipid droplet agglomeration in microglia by regulating autophagy

BackgroundLipid droplets (LD), lipid-storing organelles containing neutral lipids like glycerolipids and cholesterol, are increasingly accepted as hallmarks of inflammation. The nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA with over 200 nucleotides, exerts an indispensable impact on regulating both LD agglomeration and autophagy in multiple neurological disorders. However, knowledge as to how NEAT1 modulates the formation of LD and associated signaling pathways is limited.MethodsIn this study, primary microglia were isolated from newborn mice and exposed to oxygen-glucose-deprivation/reoxygenation (OGD/R). To further explore NEAT1-dependent mechanisms, an antisense oligonucleotide (ASO) was adopted to silence NEAT1 under in vitro conditions. Studying NEAT1-dependent interactions with regard to autophagy and LD agglomeration under hypoxic conditions, the inhibitor and activator of autophagy 3-methyladenine (3-MA) and rapamycin (RAPA) were used, respectively. In a preclinical stroke model, mice received intraventricular injections of ASO NEAT1 or control vectors in order to yield NEAT1 knockdown. Analysis of readout parameters included qRT-PCR, immunofluorescence, western blot assays, and behavioral tests.ResultsMicroglia exposed to OGD/R displayed a temporal pattern of NEAT1 expression, peaking at four hours of hypoxia followed by six hours of reoxygenation. After effectively silencing NEAT1, LD formation and autophagy-related proteins were significantly repressed in hypoxic microglia. Stimulating autophagy in ASO NEAT1 microglia under OGD/R conditions by means of RAPA reversed the downregulation of LD agglomeration and perilipin 2 (PLIN2) expression. On the contrary, application of 3-MA promoted repression of both LD agglomeration and expression of the LD-associated protein PLIN2. Under in vivo conditions, NEAT1 was significantly increased in mice at 24 h post-stroke. Knockdown of NEAT1 significantly alleviated LD agglomeration and inhibited autophagy, resulting in improved cerebral perfusion, reduced brain injury and increased neurological recovery.ConclusionNEAT1 is a key player of LD agglomeration and autophagy stimulation, and NEAT1 knockdown provides a promising therapeutic value against stroke.Graphical abstract

Melatonin Attenuates H2O2-Induced Oxidative Injury by Upregulating LncRNA NEAT1 in HT22 Hippocampal Cells.

More research is required to understand how melatonin protects neurons. The study aimed to find out if and how long non-coding RNA (lncRNA) contributes to melatonin's ability to defend the hippocampus from H2O2-induced oxidative injury. LncRNAs related to oxidative injury were predicted by bioinformatics methods. Mouse hippocampus-derived neuronal HT22 cells were treated with H2O2 with or without melatonin. Viability and apoptosis were detected by Cell Counting Kit-8 and Hoechst33258. RNA and protein levels were measured by quantitative real-time PCR, Western blot, and immunofluorescence. Bioinformatics predicted that 38 lncRNAs were associated with oxidative injury in mouse neurons. LncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) was related to H2O2-induced oxidative injury and up-regulated by melatonin in HT22 cells. The knockdown of NEAT1 exacerbated H2O2-induced oxidative injury, weakened the moderating effect of melatonin, and abolished the increasing effect of melatonin on the mRNA and protein level of Slc38a2. Taken together, melatonin attenuates H2O2-induced oxidative injury by upregulating lncRNA NEAT1, which is essential for melatonin stabilizing the mRNA and protein level of Slc38a2 for the survival of HT22 cells. The research may assist in the treatment of oxidative injury-induced hippocampal degeneration associated with aging using melatonin and its target lncRNA NEAT1.

A novel ceRNA regulatory network involving the long noncoding NEAT1, miRNA-466f-3p and its mRNA target in osteoblast autophagy and osteoporosis.

Osteoporosis (OP) is a systemic metabolic disorder characterized by a reduction in bone tissue volume. LncRNAs have been reported to act as regulators of several human diseases. Specifically, lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in proliferation, differentiation and apoptosis in osteoclasts and bone marrow mesenchymal stem cells and regulates the occurrence and development of OP. However, the relationship between NEAT1 and osteoblast autophagy and its mechanism are still unclear. Western blotting of LC3 and P62 was used to evaluate the effect of fluid shear stress (FSS) on autophagy in MC3T3-E1 osteoblasts. Total transcriptome sequencing and bioinformatics analyses were performed on osteoblasts loaded with and without FSS. qPCR was performed to examine the expression of NEAT1 in OP bone tissues and osteoblasts. The RNA-FISH was performed to study the localization of lncRNA NEAT1 and miR-466f-3p in MC3T3-E1 osteoblasts. In vitro, western blotting, transmission electron microscopy (TEM), immunofluorescence (IF) staining and qPCR were performed to verify the biological functions of NEAT1, miR-466f-3p and HK2. Subsequently, we conducted bioinformatics analysis and dual luciferase reporter assays to identify the relationships among NEAT1, miR-466f-3p and HK2. Additionally, rescue assays were conducted on osteoblasts to clarify the regulatory network of the NEAT1/miR-466f-3p/HK2 signalling pathway. In vivo, the OVX mouse model was used to investigate the effects of si-NEAT1 on autophagy in OP mice. The distal femur and serum were collected for further micro-CT analysis, blood biochemistry, and haematoxylin-eosin and Alizarin red staining (ARS). Immunohistochemistry (IHC) was performed to assess the protein expression of LC3 and HK2. NEAT1 expression was upregulated in OP tissues and osteoblast lines exposed to FSS. Knockdown of NEAT1 inhibited autophagy in vitro and in vivo. Further studies demonstrated that NEAT1 positively regulated HK2 expression via its competing endogenous RNA effects on miR-466f-3p. Moreover, we found the NEAT1/miR-466f-3p/HK2 axis regulated autophagy in osteoblasts. Knocking down NEAT1 inhibited autophagy in osteoblasts via the miR-466f-3p/HK2 signalling pathway, which may provide new ideas for novel molecular therapeutic targets of postmenopausal OP. KEY MESSAGES: • Fluid shear stress (FSS) can promote autophagy of osteoblast and performed transcriptome sequencing. • NEAT1 is overexpressed in osteoporosis and could regulate osteoblast cells autophagy. • Knockdown of lncRNA NEAT1 inhibited osteoblast cells autophagy by sponging miRNA-466f-3p and targeting HK2 in osteoporosis.

LncRNA NEAT1 promotes hypoxia-induced inflammation and fibrosis of alveolar epithelial cells via targeting miR-29a/NFATc3 axis.

The objective of the present study was to explore the function and mechanism of long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) in pulmonary fibrosis (PF) progression. HPAEpic cells and A549 cells were exposed to hypoxic conditions to establish an in vitro model. Cell apoptosis was detected by TUNEL assay, and inflammatory cytokine levels were detected by ELISA. Gene and protein expression levels were identified by qRT-PCR and Western blot assays, respectively. The interaction among NEAT1, miR-29a, and NFATc3 was identified by dual-luciferase reporter and RNA pull-down assays. In hypoxia-treated cells, hypoxia markers (HIF-1α and HIF-2α), cytokines (TNF-α, IL-1β, and IL-6) and fibrotic markers (α-SMA, collagen I and collagen III) were significantly enhanced. Consistently, the expression levels of NEAT1 and NFATc3 were increased, but miR-29a was decreased in hypoxia-stimulated cells. Knockdown of NEAT1 significantly decreased cell apoptosis and the releases of TNF-α, IL-1β, and IL-6 as well as reduced the levels of α-SMA, collagen I, and collagen III. Moreover, NEAT1 positively regulated NFATc3 expression by directly targeting miR-29a. Functional experiments showed that the anti-apoptotic, anti-inflammatory, and anti-fibrotic effects mediated by NETA1 silencing were impeded by miR-29a inhibition or NFATc3 overexpression in hypoxia-stimulated HPAEpic and A549 cells. Collectively, these data demonstrated that NEAT1 knockdown inhibited hypoxia-induced cell apoptosis, inflammation, and fibrosis by targeting the miR-29a/NFATc3 axis in PF, suggesting that NEAT1 might be a potential therapeutic target for relieving PF progression.

Implication of long non-coding RNA NEAT1 in the pathogenesis of bacterial meningitis-induced blood-brain barrier damage.

Blood-brain barrier (BBB) damage is closely related to various neurological disorders, including bacterial meningitis (BM). Determining a reliable strategy to prevent BBB damage in the context of infection would be highly desirable. In the present study, we investigated the implications of the long non-coding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) in moderating BBB damage. In vitro BBB models were developed by co-culturing hCMEC/D3 cells with glioma cells, whereupon the glioma-exposed endothelial cells (GECs) were treated with a series of mimics, inhibitors, overexpression plasmids, and shRNAs for evaluating whether NEAT1, microRNA-135a (miR-135a) and hypoxia-inducible factor 1α (HIF1α) mediated BBB integrity and permeability. Furthermore, the in vivo biological function of NEAT1 was validated in a mouse model of BBB damage. NEAT1 and HIF1α were determined to be up-regulated, while miR-135a was under-expressed in GECs. As demonstrated by chromatin immunoprecipitation and dual-luciferase reporter assays, NEAT1 could bind to miR-135a, and HIF1α was confirmed as a target of miR-135a. Either overexpression of NEAT1 or depletion of miR-135a impaired the integrity and augmented the permeability of BBB. However, HIF1α silencing could reverse the BBB damage induced by NEAT1 overexpression or by inhibition of miR-135a. In vivo experiments substantiated that knockdown of NEAT1 could alleviate BBB damage in living mice. Hence, NEAT1 knockdown prevents BBB disruption and exerts promise as a potential target for BM treatment.

LncRNA NEAT1 promotes glioma cancer progression via regulation of miR-98-5p/BZW1.

Background: Glioma is the most common malignant tumor in the human central nervous system. Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) promotes oncogenesis in various tumors. In the present study, we aimed to examine the role of NEAT1 in altering the properties of gliomas.Methods: Quantitative real-time PCR technology was used to determine the expression levels of relevant genes in tumor tissues and cell lines. The protein expression levels were validated by Western blotting. Cell counting kit-8 (CCK-8) and colony formation assays were used to test the cell proliferation ability. A luciferase reporter assay was used to determine the interactions of the genes. Tumor xenografts were used to detect the role of NEAT1 in gliomas in vivo.Results: We demonstrated that NEAT1 up-regulated glioma cells and negatively correlated with miR-98-5p in glioma tissues. A potential binding region between NEAT1 and miR-98-5p was confirmed by dual-luciferase assays. NEAT1 knockdown inhibited glioma cell proliferation. The inhibition of miR-98-5p rescued the knockdown of NEAT1 in glioma cells. Basic leucine zipper and W2 domain containing protein 1 (BZW1) was identified as a direct target of miR-98-5p. We also identified that BZW1 was positively correlated with NEAT1 in glioma tissues. NEAT1 knockdown inhibited glioma cell proliferation in vivo via miR-98-5p/BZW1.Conclusion: Our results suggest that NEAT1 plays an oncogenic function in glioma progression. Targeting NEAT1/miR-98-5p/BZW1 may be a novel therapeutic treatment approach for glioma patients.

Long non‑coding RNA NEAT1 regulates glioma cell proliferation and apoptosis by competitively binding to microRNA‑324‑5p and upregulating KCTD20 expression.

Previous studies have demonstrated that long non-coding RNAs (lncRNAs) serve a key role in the development and progression of several types of cancer, including glioma. The lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) contributes to cancer growth through its effects on cell proliferation, migration, invasion and drug resistance. However, the exact regulatory mechanisms via which NEAT1 acts in glioma are unclear. In the present study, the expression levels and function of NEAT1 in glioma tissues and cell lines were examined in vitro and in vivo. By reverse transcription-quantitative PCR and fluorescence in situ hybridization analysis, NEAT1 expression was upregulated in glioma tissues compared with in adjacent normal brain tissues, and elevated NEAT1 levels were associated with poor prognosis. Cell Counting Kit-8, colony formation, ethynyldeoxyuridine, flow cytometry and western blotting assays were performed to detect the effects of NEAT1 on cell biological behavior. Knockdown of NEAT1 in glioma cell lines was associated with cell cycle arrest at the G0/G1 phase, decreased proliferation and elevated apoptosis in vitro, and resulted in reduced tumor growth and increased survival in a mouse xenograft model of glioma. Using bioinformatics analysis, RNA immunoprecipitation experiments and luciferase reporter assays, it was demonstrated that NEAT1 may competitively bind to microRNA (miR)-324-5p, thus blocking its interaction with target mRNAs. Potassium channel tetramerization protein domain containing 20 (KCTD20) was identified as a specific miR-324-5p target. Accordingly, the inhibition of NEAT1 resulted in the downregulation of KCTD20 through competitive binding with miR-324-5p, decreased cell proliferation and increased apoptosis. Concomitant NEAT1 knockdown and inhibition of miR-324-5p partially reversed the effects of NEAT1 knockdown on cell proliferation and apoptosis, and further regulated KCTD20 expression. Collectively, the present findings demonstrated that NEAT1 acted as a competing endogenous RNA for miR-324-5p, and identified the NEAT1/miR-324-5p/KCTD20 axis as a novel regulatory axis and a potential therapeutic target for human glioma.

Long non-coding RNA NEAT1/miR-320b/MSI2 axis regulates cisplatin resistance in ovarian cancer

IntroductionOvarian cancer (OC) frequently occurs in postmenopausal women and it has higher mortality rate. Accumulating researches proved that long non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) involved in the progression of chemoresistance in human OC. Here, the study aimed to investigate the partial molecular mechanism of OC chemoresistance.Material and methodsThe levels of NEAT1 and microRNA-320b (miR-320b) were measured by qRT-PCR. Western blot was carried out to determine the protein levels that used in this research. Cell viability was identified via Cell Counting Kit-8 (CCK-8). Transwell assay was employed to determine migration and invasion. The relationship between miR-320b and NEAT1 or MSI2 was clarified by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) and RNA pull down assay. Also, a murine xenograft assay was used to explore the effect of NEAT1 on cisplatin resistance in OC in vivo.ResultsThe level of NEAT1 was significantly increased in cisplatin resistant OC cell lines. Downregulation of NEAT1 enhanced cisplatin sensibility in OVCAR-3/DDP and HEY/DDP cells. Furthermore, miR-320b was a target of NEAT1, and the effects of knockdown of NEAT1 on the cell viability, IC50 of cisplatin, migration and invasion in OVCAR-3/DDP and HEY/DDP were restored by the inhibitor of miR-320. In addition, miR-320b directly targeted MSI2 to regulate cisplatin sensibility in cisplatin resistant OC cells. In addition, downregulation of NEAT1 decreased cisplatin resistance in OC in vivo.ConclusionsNEAT1 regulated cisplatin resistance through NEAT1/miR-320b/MSI2 axis in OC, which might offer a novel therapy target for the chemotherapy of OC.

Upregulation lnc-NEAT1 contributes to colorectal cancer progression through sponging miR-486-5p and activating NR4A1/Wnt/β-catenin pathway

Colorectal cancer is a major public health problem and fourth guiding cause of cancer-induced mortality worldwide. The five-year survival rate for patients with colorectal cancer remains poor, and almost half of colorectal cancer patients present recurrence and die within five years. The increasing studies showed that long non-coding RNA (lncRNA) was involved in colorectal cancer. Therefore, this study was used to explore molecular mechanisms of nuclear paraspeckle assembly transcript 1 (NEAT1) in colorectal cancer. The real-time quantitative polymerase chain reaction (RT-qPCR) was employed to estimate the expression levels of NEAT1, Nuclear receptor 4 A1 (NR4A1), and miR-486-5p in colorectal cancer tissues and cells. Kaplan-Meier curve was conducted to analyze relationship between survival time of colorectal cancer patients and level of NEAT1. The protein levels of NR4A1, β-catenin, c-Myc, and cyclinD1 were assessed with western blot assay. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT) and flow cytometry assays were performed to evaluate proliferation and apoptosis of colorectal cancer cells, respectively. The migration and invasion abilities of cells were examined by transwell assay. The relationship between miR-486-5p and NEAT1 or NR4A1 was confirmed by dual-luciferase reporter assay. We found NEAT1 and NR4A1 were highly expressed in colorectal cancer tissues and cell lines compared with controls. Loss-functional experiments revealed that knockdown of NEAT1 or NR4A1 repressed proliferation and motility, while inducing apoptosis of colorectal cancer cells. The gain of NR4A1 could abolish NEAT1 silencing-induced effects in colorectal cancer cells. In addition, NEAT1 contributed to colorectal cancer progression through mediating NR4A1/Wnt/β-catenin signaling pathway. In conclusion, NEAT1 stimulated colorectal cancer progression via acting as competing endogenous RNA to sponge miR-486-5p and regulate NR4A1/Wnt/β-catenin signaling pathway.

Knockdown of lncRNA NEAT1 expression inhibits cell migration, invasion and EMT by regulating the miR-24-3p/LRG1 axis in retinoblastoma cells.

Retinoblastoma (RB) is the most common primary intraocular cancer type that occurs during retinal development in childhood. Previous studies have reported that long non-coding RNAs (lncRNAs) are involved in the development of RB. Therefore, the aim of the present study was to investigate the effects and underlying regulatory mechanisms of nuclear paraspeckle assembly transcript 1 (NEAT1) in RB. The expression levels of NEAT1, microRNA (miR)-24-3p and leucine-rich-α-2-glycoprotein (LRG1) were detected using reverse transcription-quantitative PCR (RT-qPCR). Moreover, the protein expression levels of LRG1, matrix metalloproteinase 9, N-cadherin and E-cadherin were detected via western blotting. Furthermore, cell migration and invasion abilities were evaluated via Transwell assays. The targeting relationships between miR-24-3p and NEAT1 or LRG1 were predicted using online software and confirmed via dual-luciferase reporter assay. In the present study, NEAT1 and LRG1 were upregulated, and miR-24-3p was downregulated in RB tissues and cells compared with the corresponding healthy tissues and cells. Moreover, miR-24-3p was identified as a target of NEAT and LRG1 was demonstrated to be a direct target gene of miR-24-3p. Knockdown of NEAT1 or LRG1 significantly suppressed RB cell migration and invasion ability, while the effects were reversed by an miR-24-3p inhibitor. In addition, the downregulation of LRG1 caused by miR-24-3p was restored following the overexpression of NEAT1 in RB cells. It was also demonstrated that NEAT1 knockdown inhibited the epithelial-to-mesenchymal transition (EMT) pathway by inhibiting the expression of LRG via targeting miR-24-3p. In conclusion, the present results suggest that silencing of NEAT1 suppresses cell migration, invasion and the EMT process by downregulating LRG1 expression via sponging miR-24-3p in RB, thus indicating that NEAT1 may be a potential candidate for RB treatment.

LncRNA NEAT1 activates MyD88/NF-κB pathway in bronchopneumonia through targeting miR-155-5p

Background Bronchopneumonia is a disease of the respiratory tract. It leads to other complications and endangers life and health. Long non-coding RNA (lncRNA) participates in the occurrence and development of bronchopneumonia. Nuclear paraspeckle assembly transcript 1 (NEAT1) plays a key role in inflammatory diseases, but the function of NEAT1 in bronchopneumonia remains unclear. Methods RT-qPCR and Western blotting were performed to determine genes and proteins expressions. MTT was applied to test cell viability. Cell apoptosis was detected by flow cytometry. RIP was used to investigate the correlation between NEAT1 and miR-155-5p. The interaction between miR-155-5p and NEAT1 or MyD88 was evaluated by the dual-luciferase reporter gene. Results NEAT1 and MyD88 were upregulated in BEAS-2B cells by LPS, while miR-155-5p was downregulated. Knockdown of NEAT1 inhibited LPS-induced BEAS-2B cells growth inhibition by inhibiting the apoptosis. In addition, NEAT1 silencing suppressed LPS-induced inflammatory responses in BEAS-2B cells via suppression of TNF-α, IL-1β, IL-6, and IL-18. Meanwhile, NEAT1 is directly bound to miR-155-5p to regulate MyD88/NF-κB axis, and overexpression of miR-155-5p increased cell proliferation and suppressed inflammatory factors expression levels and cell apoptosis. Furthermore, sh-NEAT1-induced inhibition of BEAS-2B cells injury was partially reversed by miR-155-5p inhibitor or MyD88 overexpression. Conclusion NEAT1 silencing suppressed LPS-induced BEAS-2B cells injury and inflammation by the mediation of miR-155-5p/MyD88/NF-κB axis. Thus, our study might shed new light on exploring the new strategies for the treatment of bronchopneumonia.

LncRNA-NEAT1 promotes proliferation of T-ALL cells via miR-146b-5p/NOTCH1 signaling pathway

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) is a malignant tumor of the hematopoietic system, which can develop at any age, with the symptoms of weakness, fatigue, enlarged lymph nodes, or weight loss. Nuclear paraspeckle assembly transcript 1 (NEAT1) is involved in the process of T-ALL, but the regulatory mechanism is still not known clearly. MethodsThe expression levels of NEAT1 and miR-146b-5p in T-ALL cells were performed by qRT-PCR and NOTCH1 protein level- wwWwas determined by western blot assay. Dual-luciferase reporter assay was used to detect the interaction between NEAT1 and miR-146b-5p, as well as miR-146b-5p and NOTCH1. The cell proliferation was measured by using MTT assay and colony formation assay. ResultsThe expression levels of NEAT1 were markedly increased, but miR-146b-5p levels were reduced in T-ALL cells. Knockdown of NEAT1 or overexpression of miR-146b-5p decreased NOTCH1 expression, inhibited the proliferation of T-ALL cells. MiR-146b-5p bound both NEAT1 and NOTCH1 3′-UTR directly. Finally, inhibition of miR-146b-5p could abrogate the effects of NEAT1 knockdown on the proliferation of T-ALL cells. ConclusionNEAT1 promotes the proliferation of T-ALL cells by sponging miR-146b-5p to upregulate the expression of NOTCH1. The results of this study provide new insight into the action mechanism of NEAT1 modulating T-ALL progression.

NEAT1 Knockdown Suppresses the Cisplatin Resistance in Ovarian Cancer by Regulating miR-770-5p/PARP1 Axis.

BackgroundLong noncoding RNAs play essential roles in regulating drug resistance in cancers. However, how and whether lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) could mediate cisplatin resistance in ovarian cancer remain poorly understood.Patients and MethodsEighteen cisplatin-sensitive and 19 cisplatin-resistant patients with ovarian cancer were recruited. Cisplatin-resistant ovarian cancer cells were used for this study. The expression levels of NEAT1, microRNA (miR)-770-5p and poly adenosine diphosphate-ribose polymerase 1 (PARP1) were detected by quantitative real-time polymerase chain reaction or Western blot. Cisplatin resistance was assessed by the half-maximal inhibitory concentration (IC50) of cisplatin, cell viability and apoptosis using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, flow cytometry and Western blot, respectively. The target association between miR-770-5p and NEAT1 or PARP1 was investigated by dual-luciferase reporter assay. The xenograft model was used to investigate cisplatin resistance in vivo.ResultsNEAT1 expression is elevated in cisplatin-resistant ovarian cancer tissues and cells. Knockdown of NEAT1 repressed cisplatin resistance by decreasing the IC50 of cisplatin, cell viability and increasing apoptosis. MiR-770-5p was bound to NEAT1 and PARP1 was confirmed as a target of miR-770-5p. MiR-770-5p inhibition or PARP1 restoration could abate the effect of NEAT1 silencing on cisplatin resistance in cisplatin-resistant ovarian cancer cells. Moreover, NEAT1 knockdown reduced PARP1 expression by increasing miR-770-5p. Interference of NEAT1 decreased xenograft tumor growth by regulating miR-770-5p and PARP1.ConclusionKnockdown of NEAT1 inhibited cisplatin resistance in ovarian cancer cells by up-regulating miR-770-5p and down-regulating PARP1, providing a new target for improving the efficacy of cisplatin-based therapy in ovarian cancer.

Silencing of Long Non-Coding RNA (lncRNA) Nuclear Paraspeckle Assembly Transcript 1 (NEAT1) Protects PC-12 Cells from LPS-Induced Injury via Targeting miR-29a.

BackgroundSpinal cord injury (SCI) is a debilitating neuropathological condition that significantly affects the quality of life. The present study is basic research examining the underlying mechanisms of NEAT1 and miR-29a in regulating LPS-induced PC-12 cell injury.Material/MethodsThe model of cell injury was induced by the treatment of PC-12 cells with LPS. The expressions of NEAT1, miR-29a, and inflammatory cytokines were measured by real-time quantitative polymerase chain reactions (RT-qPCR). Cell proliferation and apoptosis were evaluated by CCK-8 and flow cytometry, respectively. Finally, the target between miR-29a and NEAT1 as well as miR-29a and BCL2L11 was investigated by luciferase and RNA pull-down assays.ResultsKnockdown of NEAT1 can inhibit inflammatory cytokine expression and PC-12 cell apoptosis and promote PC-12 cell proliferation by targeting miR-29a. However, the variation caused by NEAT1 knockdown can be reversed by the silencing of miR-29a and the overexpression of BCL2L11, which is the direct target gene of miR-29a.ConclusionsHigh NEAT1 levels can increase LPS-induced injury in PC-12 cells through the miR-29a/BCL2L11 pathway. lncRNA NEAT1 may, therefore, be a promising target for SCI treatment.

Knockdown of lncRNA NEAT1 suppresses hypoxia-induced migration, invasion and glycolysis in anaplastic thyroid carcinoma cells through regulation of miR-206 and miR-599

BackgroundAnaplastic thyroid carcinoma (ATC) is one of the most aggressive and lethal malignancies. Long non-coding RNAs (lncRNAs) are being found to play crucial roles in ATC progression. Herein, we focused on the role of nuclear paraspeckle assembly transcript 1 (NEAT1) on ATC progression under hypoxia and underlying mechanisms governing it.MethodsThe expression levels of NEAT1, miR-206 and miR-599 were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Cell migration and invasion abilities were detected using transwell assays. Glucose consumption and lactate production were determined using a corresponding commercial assay kit. Western blot was performed to evaluate the level of hexokinase 2 (HK2). The targeted interplays between NEAT1 and miR-206 or miR-599 were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. Xenograft model was established to observe the effect of NEAT1 on tumor growth in vivo.ResultsOur data indicated that NEAT1 was highly expressed in ATC tissues and cells, and hypoxia induced NEAT1 expression in ATC cells. NEAT1 depletion repressed ATC cell migration, invasion and glycolysis under hypoxia. Mechanistically, NEAT1 acted as a molecular sponge of miR-206 and miR-599. Moreover, the repressive effects of NEAT1 knockdown on ATC cell migration, invasion and glycolysis under hypoxia were mediated by miR-206 or miR-599. Additionally, NEAT1 knockdown weakened tumor growth in vivo.ConclusionIn conclusion, our study suggested that a low NEAT1 expression suppressed the migration, invasion, and glycolysis in ATC cells under hypoxia at least partially through modulating miR-206 and miR-599, providing new therapeutic strategies for ATC treatment.

Long Noncoding RNA NEAT1 Upregulates Survivin and Facilitates Gallbladder Cancer Progression by Sponging microRNA-335.

BackgroundGallbladder cancer (GBC) is the most common cancer of the biliary tract, but molecularly targeted therapies are not available for GBC. Loss of microRNA (miR)-335 expression may be a useful predictor of clinical outcomes and the reversal of its loss of expression may be a useful treatment strategy for GBC. In this study, we investigated whether a long noncoding RNA, nuclear paraspeckle assembly transcript 1 (NEAT1) sponges miR-335 in GBC cells.Materials and MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry were used to determine the expression of miR-335; NEAT1; survivin; and Ki67 in GBC cell lines (GBC-SD and SGC-996) and tissue samples from patients (n = 25). Cell Counting Kit-8, colony-formation, and Transwell migration and invasion assays were performed to measure cell proliferation, migration, and invasion. Bioinformatic analysis and dual-luciferase reporter assays were utilized to analyze correlativity.ResultsmiR-335 overexpression resulted in inhibition of GBC cell proliferation and invasion. In addition, knockdown of NEAT1 resulted in downregulation of survivin expression. As NEAT1 competitively “sponges” miR-335, NEAT1 knockdown resulted in inhibited GBC cell proliferation and invasion in vitro and GBC tumor growth in vivo. Furthermore, NEAT1 was found to be upregulated in GBC samples, and its expression was inversely correlated with miR-335 levels, but positively correlated with survivin levels.ConclusionThese findings indicate that NEAT1 promotes survivin expression by functioning as a competitive endogenous RNA for miR-335 in GBC cells; thus, we have identified a potential biomarker and target for GBC diagnosis and therapy.

ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1

N6-Methyladenosine (m6A) is the most common posttranscriptional modification of RNA and plays critical roles in cancer pathogenesis. However, the biological function of long noncoding RNA (lncRNA) methylation remains unclear. As a demethylase, ALKBH5 (alkylation repair homolog protein 5) is involved in mediating methylation reversal. The purpose of this study was to investigate lncRNA m6A modification and its role in gastric cancer (GC). Bioinformatics predicted interactions of ALKBH5 with lncRNAs. Five methods were employed to assess the function of nuclear paraspeckle assembly transcript 1 (NEAT1), including gene silencing, RT-PCR, separation of nuclear and cytoplasmic fractions, scrape motility assays, and transwell migration assays. Then, m6A RNA immunoprecipitation and immunofluorescence were used to detect methylated NEAT1 in GC cells. Rescue assays were performed to define the relationship between NEAT1 and ALKBH5. NEAT1 is a potential binding lncRNA of ALKBH5. NEAT1 was overexpressed in GC cells and tissue. Additional experiments confirmed that knockdown of NEAT1 significantly repressed invasion and metastasis of GC cells. ALKBH5 affected the m6A level of NEAT1. The binding of ALKBH5 and NEAT1 influences the expression of EZH2 (a subunit of the polycomb repressive complex) and thus affects GC invasion and metastasis. Our findings indicate a novel mechanism by which ALKBH5 promotes GC invasion and metastasis by demethylating the lncRNA NEAT1. They may be potential therapeutic targets for GC.

NEAT1 mediates paclitaxel-resistance of non-small cell of lung cancer through activation of Akt/mTOR signalling pathway

Development of paclitaxel-resistance is a main problem during non-small cell lung cancer (NSCLC) chemotherapy. Nuclear paraspeckle assembly transcript 1 (NEAT1) is an oncogenic long non-coding RNA (lncRNA) which has been proved to be aberrantly upregulated in many human malignancies. In this study, we investigated the mechanism by which NEAT1 contributed to paclitaxel-resistance in NSCLC. NEAT1 was upregulated significantly in paclitaxel-resistant NSCLC cell line, compared with other NSCLC cell lines and normal bronchial epithelial (BE) cell line. Knockdown of NEAT1 could reverse the paclitaxel-resistance through induction of apoptosis by increasing cleaved PARP and cleaved caspase-3 expression. Moreover, NEAT1 was associated with Akt/mTOR signalling pathway activation by increasing expression of p-Akt, p-mTOR, Bcl-2 and decreasing expression of Bax. In conclusion, these results demonstrated that NEAT1 underlay paclitaxel-resistance in NSCLC.

Long non-coding RNA NEAT1 confers oncogenic role in triple-negative breast cancer through modulating chemoresistance and cancer stemness

Triple-negative breast cancer (TNBC) is a malignant subtype of breast cancer with the absence of targeted therapy, resulting in poor prognosis in patients. Chemotherapy remains the mainstay of treatment for TNBC; however, development of drug resistance is the main obstacle for successful treatments. In recent years, long non-coding RNA (lncRNA) has been implicated in multiple biological functions in various diseases, particularly cancers. Accumulating evidence suggested that lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) expression is dysregulated in many human cancers and thus is a useful prognostic marker for cancer patients. Nevertheless, the mechanism of how NEAT1 confers drug resistance in TNBC is still largely unknown. We performed lncRNA profiling by the LncRNA Profiler qPCR Array Kit in normal control (NC) and breast cancers (BC) blood samples and further validated in a larger cohort of samples by qRT-PCR. Gene expression level and localization were investigated by qRT-PCR, western blotting, and immunofluorescence staining. Flow cytometric analysis was carried out to detect cancer stem cells. Functional studies were performed both in vitro and in vivo xenograft model. Among 90 lncRNAs, NEAT1 was highly expressed in the blood samples of breast cancer patients than in NC. In particular, the expression of NEAT1 was higher in TNBC tissues than other subgroups. Functional studies revealed that NEAT1 conferred oncogenic role by regulating apoptosis and cell cycle progression in TNBC cells. We identified that knockdown of NEAT1 sensitized cells to chemotherapy, indicating the involvement in chemoresistance. Importantly, shNEAT1 reduced stem cell populations such as CD44+/CD24−, ALDH+, and SOX2+, implicating that NEAT1 was closely related to cancer stemness in TNBC. Our data highlighted the roles of NEAT1 chemoresistance and cancer stemness, suggesting that it could be used as a new clinical therapeutic target for treating TNBC patients especially those with drug resistance.