NEAT1 Research Articles

MUC1-C regulates NEAT1 lncRNA expression and paraspeckle formation in cancer progression

The MUC1 gene evolved in mammals for adaptation of barrier tissues in response to infections and damage. Paraspeckles are nuclear bodies formed on the NEAT1 lncRNA in response to loss of homeostasis. There is no known intersection of MUC1 with NEAT1 or paraspeckles. Here, we demonstrate that the MUC1-C subunit plays an essential role in regulating NEAT1 expression. MUC1-C activates the NEAT1 gene with induction of the NEAT1_1 and NEAT1_2 isoforms by NF-κB- and MYC-mediated mechanisms. MUC1-C/MYC signaling also induces expression of the SFPQ, NONO and FUS RNA binding proteins (RBPs) that associate with NEAT1_2 and are necessary for paraspeckle formation. MUC1-C integrates activation of NEAT1 and RBP-encoding genes by recruiting the PBAF chromatin remodeling complex and increasing chromatin accessibility of their respective regulatory regions. We further demonstrate that MUC1-C and NEAT1 form an auto-inductive pathway that drives common sets of genes conferring responses to inflammation and loss of homeostasis. Of functional significance, we find that the MUC1-C/NEAT1 pathway is of importance for the cancer stem cell (CSC) state and anti-cancer drug resistance. These findings identify a previously unrecognized role for MUC1-C in the regulation of NEAT1, RBPs, and paraspeckles that has been co-opted in promoting cancer progression.

Identification of long noncoding RNA NEAT1 as a key gene involved in the extramedullary disease of multiple myeloma by bioinformatics analysis

ABSTRACTObjectiveLong non-coding RNAs (lncRNAs) are involved in tumorigenesis and play a key role in cancer progression. To determine whether lncRNAs are involved in extramedullary disease of multiple myeloma (EMD), we analyzed the expression profile of lncRNAs in EMD.MethodsThree pairs of EMD patients and their intramedullary MM cells were screened by microarray first. We extracted data from gene chips and made an identification of lncRNAs and mRNAs with significant differences between EMD group and non EMD group. WGCNA confirmed the EMD related gene module and drew a heat map to further determine the key gene lncRNA-NEAT1. In the meantime, bone marrow and extramedullary samples (hydrothorax and ascites) were collected from 2 MM patients and subjected to single-cell RNA-seq. Single cell Transcriptome analysis was conducted to verify the gene expression difference of malignant plasma cells derived from intramedullary and extramedullary. Then we verified high expression level of lncRNA-NEAT1 in EMD patients by using quantitative real-time PCR (qRT-PCR) and analyzed the correlation between expression patterns and survival and molecular genetics analysis of the LncRNA (NEAT1) involved in MM patients. At last, cell experiments were conducted to observe the effects of down-regulation of NEAT1on the proliferation, cell cycle and PTEN pathway related proteins of multiple myeloma cell lines U266 and RPMI8226.ResultsWe identified one of the EMD related key gene is lncRNA-NEAT1. Compared with patients without extramedullary lesions, intramedullary MM cells in EMD patients expressed NEAT1 highly. The outcome of parallel single-cell RNA sequencing (RNA-seq) revealed NEAT1 level of plasma cells came from pleural effusion /ascites increased significantly compared with myeloma-stricken bone marrow. By survival and molecular genetic analysis, NEAT1 gene expression was not associated with OS and PFS in MM patients. However, the expression of NEAT1 is related to adverse therapeutic reactions and the progression of MM. We found that the expressions of NEAT1 were negatively associated with albumin levels and were positively associated with gain of chromosome 1q, IGH-CCND1, IGH@-FGFR3/WHSC1,and IGH-MAF gene fusion, respectively. At the level of cell experiment, CCK-8, soft agar clone formation experiment and CFSE staining showed that down regulating NEAT1 could inhibit the proliferation of U266 and RPMI8226 cells; Cell cycle detection showed that down-regulation of NEAT1 would interfere with the cell cycle process, and RPMI 8226 cells were blocked in G1 phase. Western blot analysis showed that when the expression of NEAT1 was down regulated in U266 and RPMI 8226 cells, the expression of PTEN and p-PTEN (phosphorylated phosphatase and tensin homologue) was up-regulated, and the expression of PI3K, p-PI3K (human phosphorylated inositol 3 kinase), Akt, p-Akt (phosphorylated protein kinase B).Discuccion and conclusionThis study provides novel insights into the lncRNA-NEAT1 and reveals that NEAT1 maybe a potential lncRNA biomarkers in EMD.

Evaluation of Apoptosis, Cell Proliferation and Cell Cycle Progression by Inactivation of the NEAT1 Long Noncoding RNA in a Renal Carcinoma Cell Line Using CRISPR/Cas9.

Long noncoding RNAs (lncRNAs) play an important role in cellular mechanisms including transcription, translation, and apoptosis. NEAT1 is one of the essential types of lncRNAs in humans that can bind to active genes and modify their transcription. NEAT1 upregulation in various forms of cancer such as kidney cancer has been reported. Kidney cancer accounts for approximately 3% of all cancers worldwide and occurs almost twice as often in men as in women. This study has been performed to knockout the NEAT1 gene using the CRISPR/Cas9 technique in the Renal Cell Carcinoma ACHN cell line and to evaluate its effects on cancer progression and apoptosis. Two specific (single guide RNA (sgRNA) sequences for the NEAT1 gene were designed by CHOPCHOP software. These sequences were then cloned into plasmid pSpcas9, and recombinant vectors PX459-sgRNA1 and PX459-sgRNA2 were generated. ACHN cells were transfected using recombinant vectors carrying sgRNA1 and sgRNA2. The expression level of apoptosis-related genes was assessed by real-time PCR. Annexin, MTT and cell scratch tests were performed to evaluate the survival, proliferation, and migration of the knocked out cells, respectively. The results have shown successful knockout of the NEAT1 gene in the cells of the treatment group. Expressions of P53, BAK, BAX and FAS genes in the cells of the treatment group (NEAT1 knockout) showed significant increases in expression compared to the cells of the control group (P <0.01). Additionally, decreased expression of BCL2 and survivin genes was observed in knockout cells compared to the control group (p <0.05). In addition, in the cells of the treatment group compared to control cells, a significant decrease in cell viability, ability to migrate and cell growth and proliferation was observed. Inactivation of the NEAT1 gene in ACHN cell line using CRISPR/Cas9 technology elevated apoptosis and reduced cell survival and proliferation which makes it a novel target for kidney cancer therapeutics.

Achyranthes bidentata polysaccharides alleviate endoplasmic reticulum stress in osteoarthritis via lncRNA NEAT1/miR-377-3p pathway

Endoplasmic reticulum stress (ERS) has been identified to be an important factor leading to chondrocyte apoptosis in osteoarthritis (OA). Previous studies have confirmed that Achyranthes bidentata polysaccharides (ABPS) can inhibit chondrocyte apoptosis; however, the mechanism of action of ABPS on chondrocyte ERS remains unclear. Thus in this study, we aim to investigate whether ABPS could inhibit OA-associated chondrocyte apoptosis by regulating ERS, especially by observing the relationship between the lncRNA NEAT1 and miR-377-3p, to explore further the protective mechanism of ABPS in OA. In vitro and in vivo experiments showed that ABPS inhibited chondrocyte ERS by regulating the expression of lncRNA NEAT1 and miR-377-3p. Moreover, both lncRNA NEAT1 silencing and miR-377-3p inhibition could attenuate the therapeutic effect of ABPS on ERS. Dual-luciferase results indicated that miR-377-3p targets the lncRNA NEAT1 gene in mouse chondrocytes. Therefore, we concluded that ABPS could inhibit thapsigargin (TG)-induced chondrocyte ERS through the lncRNA NEAT1/miR-377-3p axis.

Association Between Genetic Polymorphisms of lncRNA NEAT1 and Pulmonary Tuberculosis Risk, Clinical Manifestations in a Chinese Population.

BackgroundRecent studies have shown that abnormal expression of lncRNA NEAT1 is associated with the progression of pulmonary tuberculosis (PTB). The aim of our study was to analyze the relationship between single nucleotide polymorphisms (SNPs) of NEAT1 gene and susceptibility to PTB.MethodsFour SNPs (rs2239895, rs3741384, rs3825071, rs512715) in NEAT1 gene were genotyped in 479 patients with PTB and 476 controls by improved multiple ligase detection reaction (iMLDR) in a Chinese population.ResultsWe found no significant differences in allele and genotype frequencies of NEAT1 gene rs2239895, rs3741384, rs3825071, rs512715 between PTB patients and controls (all P > 0.05). There was no statistically significant association between genotype frequency distribution of dominant model, as well as recessive model, and genetic susceptibility to PTB patients (all P > 0.05). The TT genotype, T allele frequencies of rs3825071 were significantly increased in sputum smear-positive PTB patients when compared to sputum smear-negative PTB patients (P = 0.010, P = 0.003, respectively). Haplotype analysis shown that NEAT1 haplotype frequency was not associated with PTB susceptibility.ConclusionNEAT1 gene polymorphisms were not associated with the risk of PTB in Chinese population, and rs3825071 polymorphism might be related to sputum smear-positive in PTB patients.

Expression and Clinical Significance of lncRNA NEAT1 in Patients with Spinal Tuberculosis.

Background Spinal tuberculosis (STB) often leads to irreversible neurological injury, resulting in serious social and economic problems. With the emergence of drug resistance, the management becomes even more challenging, given the treatment courses are generally longer for skeletal than pulmonary tuberculosis (PTB). The development and validation of nonsputum biomarkers for diagnosis and tailoring of treatment duration to enable personalized and evidence-based management of such diseases to improve treatment outcomes is being called for globally. Studies have demonstrated that lncRNA NEAT1 was highly expressed in pulmonary tuberculosis (TB) and was related to its progression and recovery. However, the expression and clinical significance of lncRNA NEAT1 in STB remains unclear. Methods The relative expression of lncRNA NEAT1 was quantified by relative real-time reverse transcription PCR (RT-PCR). The prognostic value was assessed by receiver-operating characteristic (ROC) curve analysis. Pearson and Spearman correlation coefficient and chi-square test were used to analyze the correlation between the lncRNA NEAT1 expression and the clinical characteristics. Univariate and multivariate logistic regression analyses were used to analyze independent predictors of STB recurrence. Results Compared with normal healthy individuals, the expression level of lncRNA NEAT1 in peripheral blood and granulomatous tissues of STB patients was significantly increased. The results of the in vitro Mycobacterium tuberculosis- (Mtb-) infected cell model showed that the expression level of lncRNA NEAT1 was significantly upregulated in macrophages infected with Mtb, and the difference was statistically significant compared with Mtb-uninfected group. The expression level of lncRNA NEAT1 in granulomatous tissue of STB was significantly higher than that in peripheral blood. The expression of lncRNA NEAT1 was related to segments of the lesions, paraspinal abscesses, anti-TB treatment, drug resistance, interleukin-6 (IL-6), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). Multivariate analysis results showed that relatively high expression of lncRNA NEAT1_1, the shorter transcript of the NEAT1 gene, was an independent prognostic factor of STB outcome. Conclusion LncRNA NEAT1 was highly expressed in peripheral blood mononuclear cells (PBMCs) and granulomatous tissue from patients with STB, as well as in Mtb-infected THP-1 cell lines. LncRNA NEAT1 expression was significantly associated with clinical characteristics (paraspinal abscesses, segments of the lesions and anti-TB treatment, IL-6, CRP, and ESR) of patients in STB. Increased expression of lncRNA NEAT1_1 predicted good prognosis of STB and might become a prognostic biomarker for STB.

HSF1-Activated Non-Coding Stress Response: Satellite lncRNAs and Beyond, an Emerging Story with a Complex Scenario.

In eukaryotes, the heat shock response is orchestrated by a transcription factor named Heat Shock Factor 1 (HSF1). HSF1 is mostly characterized for its role in activating the expression of a repertoire of protein-coding genes, including the heat shock protein (HSP) genes. Remarkably, a growing set of reports indicate that, upon heat shock, HSF1 also targets various non-coding regions of the genome. Focusing primarily on mammals, this review aims at reporting the identity of the non-coding genomic sites directly bound by HSF1, and at describing the molecular function of the long non-coding RNAs (lncRNAs) produced in response to HSF1 binding. The described non-coding genomic targets of HSF1 are pericentric Satellite DNA repeats, (sub)telomeric DNA repeats, Short Interspersed Nuclear Element (SINE) repeats, transcriptionally active enhancers and the NEAT1 gene. This diverse set of non-coding genomic sites, which already appears to be an integral part of the cellular response to stress, may only represent the first of many. Thus, the study of the evolutionary conserved heat stress response has the potential to emerge as a powerful cellular context to study lncRNAs, produced from repeated or unique DNA regions, with a regulatory function that is often well-documented but a mode of action that remains largely unknown.

Expression and functions of long non-coding RNA NEAT1 and isoforms in breast cancer.

NEAT1 is a highly abundant nuclear architectural long non-coding RNA. There are two overlapping NEAT1 isoforms, NEAT1_1 and NEAT1_2, of which the latter is an essential scaffold for the assembly of a class of nuclear ribonucleoprotein bodies called paraspeckles. Paraspeckle formation is elevated by a wide variety of cellular stressors and in certain developmental processes, either through transcriptional upregulation of the NEAT1 gene or through a switch from NEAT1_1 to NEAT1_2 isoform production. In such conditions, paraspeckles modulate cellular processes by sequestering proteins or RNA molecules. NEAT1 is abnormally expressed in many cancers and a growing body of evidence suggests that, in many cases, high NEAT1 levels are associated with therapy resistance and poor clinical outcome. Here we review the current knowledge of NEAT1 expression and functions in breast cancer, highlighting its established role in postnatal mammary gland development. We will discuss possible isoform-specific roles of NEAT1_1 and NEAT1_2 in different breast cancer subtypes, which critically needs to be considered when studying NEAT1 and breast cancer.

Therapeutic Efficacy Analysis of lncRNA NEAT1 Gene Knockout and Apoptosis Induction in Prostate Cancer Cell Line Using CRISPR/Cas9

Therapeutic Efficacy Analysis of lncRNA NEAT1 Gene Knockout and Apoptosis Induction in Prostate Cancer Cell Line Using CRISPR/Cas9

ENEMAL, an enhancer RNA transcribed from a distal MALAT1 enhancer, promotes NEAT1 long isoform expression.

Emerging evidence has shown that active enhancers are abundantly transcribed, generating long non-coding RNAs, called enhancer RNAs (eRNAs). While putative eRNAs are often observed from RNA sequencing, the roles of most eRNAs remain largely unknown. Previously, we identified putative enhancer regions at the MALAT1 locus that form chromatin-chromatin interactions under hypoxia, and one of these enhancers is located about 30 kb downstream of the NEAT1 gene and -20 kb upstream of the MALAT1 gene (MALAT1-20 kb enhancer). Here, we report that a novel eRNA, named eRNA of the NEAT1-MALAT1-Locus (eNEMAL), is transcribed from the MALAT1-20 kb enhancer and conserved in primates. We found that eNEMAL is upregulated in response to hypoxia in multiple breast cancer cell lines, but not in non-tumorigenic MCF10A cells. Overexpression and knockdown of eNEMAL revealed that alteration of eNEMAL level does not affect MALAT1 expression. Instead, we found that eNEMAL upregulates the long isoform of NEAT1 (NEAT1_2) without increasing the total NEAT1 transcript level in MCF7 breast cancer cells, suggesting that eNEMAL has a repressive effect on the 3'-end polyadenylation process required for generating the short isoform of NEAT1 (NEAT1_1). Altogether, we demonstrated that an eRNA transcribed from a MALAT1 enhancer regulates NEAT1 isoform expression, implicating the MALAT1-20 kb enhancer and its transcript eNEMAL in co-regulation of MALAT1 and NEAT1 in response to hypoxia in breast cancer cells.

Long non-coding RNA NEAT1 promotes tumor development and metastasis through targeting miR-224-5p in malignant melanoma.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Long non-coding RNA NEAT1 promotes tumor development and metastasis through targeting miR-224-5p in malignant melanoma, by J.-X. Zou, T.-W. Ge, published in Eur Rev Med Pharmacol Sci 2020; 24 (3): 1302-1308-DOI: 10.26355/eurrev_202002_20187-PMID: 32096166" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20187.

Long non-coding RNA NEAT1 regulates Hodgkin's lymphoma cell proliferation and invasion via miR-448 mediated regulation of DCLK1.

To explore whether long non-coding RNA nuclear enriched abundant transcript 1 (lncRNA NEAT1) could regulate Hodgkin's lymphoma (HL) cell proliferation and invasion through miR-448, which could target doublecortin like kinase 1 (DCLK1) and mediate DCLK1 expression. Expressions of NEAT1, miR-448 and DCLK1 were evaluated by qRT-PCR or Western blot assay. Cell Counting Kit-8 (CCK-8) and transwell assay were utilized to detect cell proliferation and invasion capability in L428 cells respectively. The target relationship between NEAT1, miR-448 and DCLK1 was confirmed by Luciferase reporter assay. QRT-PCR results showed that NEAT1 expressed higher in HL tissues and cell lines than that in controls. In vitro experiments, NEAT1 downregulation could decrease cell proliferation and invasion capability in L428 cells. NEAT1 directly interacted with miR-448 and negatively regulated it. Moreover, DCLK1 was confirmed as a target of miR-448. DCLK1 expression was increased in L428 cells and positively regulated by NEAT1. NEAT1 overexpression upregulated the protein level of DCLK1 in L428 cells according to Western blot analysis. Additionally, DCLK1 overexpression could reverse the suppression on cell proliferation and invasion capability induced by NEAT1 knockdown or miR-448 overexpression. NEAT1 might be contributed to HL progression by promoting cell proliferation and invasion capability via miR-448 mediated DCLK1 expression.

IncRNA NEAT1 aggravates cerebral ischemia/reperfusion injury by sponging miR-874-3p.

Many lncRNAs have been reported to affect cerebral ischemia/reperfusion (I/R) injury. The purpose of this study is to elucidate the role of lncRNA NEAT1 as well as the regulatory mechanism of lncRNA NEAT1/miR-874-3p in cerebral I/R injury. A cellular model of cerebral I/R injury was built. RT-qPCR was used to detect NEAT1 and miR-874-3p expression. Cell viability was detected by MTT assay. The expression of apoptosis-related proteins (Bcl-2 and Bax) was measured by Western blot analysis. The relationship between NEAT1 and miR-874-3p was confirmed by dual luciferase reporter assay. We found that LncRNA NEAT1 was upregulated in the PC12 cells treated by I/R and upregulation of lncRNA NEAT1 can aggravate I/R injury of PC12 cells. Additionally, lncRNA NEAT1 overexpression decreased cell viability and induced apoptosis in PC12 cells treated by I/R. Furthermore, miR-874-3p was confirmed to be a target of lncRNA NEAT1. mR-874-3p and NEAT1 expression are found to be reciprocally inhibited in PC12 cells. In summary, LncRNA NEAT1 aggravates cerebral I/R injury by suppressing miR-874-3p expression.

Overexpression of the long noncoding RNA NEAT1 protects against As2O3-induced injury of cardiomyocyte by inhibiting the miR-124/NF-κB signaling pathway.

Arsenic trioxide (As2O3) an evident effect in the treatment of acute promyelocytic leukemia and other malignant tumors in recent years. However, more and more studies have found that the cardiac toxicity of As2O3 was increased, limiting its wide clinical application. This study aims to explore the molecule mechanisms of As2O3 on cardiomyocyte injury. The cardiomyocyte injury under As2O3 was detected by MTT assay. The levels of NEAT1 and miR-124 were examined by RT-PCR. The functions of NEAT1 and miR-124 at H9c2 cell injury under As2O3 were detected by cell transfection of the overexpression or repression. The expression levels of inflammation factors, apoptosis genes and NF-κB signals were measured by Western blot in H9c2 cell lines under As2O3. The luciferase assay detected the direct interaction between NEAT1 and miR-124. The overexpression of NEAT1 decreased the H9c2 cells injury under As2O3. The levels of IL-1β, IL-6, TNF-α were upregulated after NEAT1 overexpression. Moreover, the luciferase assay results showed NEAT1 was directly interacting with miR-124. Silencing of miR-124 significantly increased the H9c2 cell survival under As2O3 by repressing NF-κB signaling pathway. Furthermore, the overexpression of NEAT1 markedly increased H9c2 cells survival under As2O3, while the miR-124 could reverse the effects. Finally, NEAT1 regulated the H9c2 cells As2O3 injury by repressing the miR-124, NF-kappa B expressions and inflammatory response. According to the results, we found that long non-coding RNA NEAT1 regulated the expression of inflammatory factors to protect cardiomyocytes from As2O3 damage by inhibiting miR-124/NF-kappa B signaling pathway. It provides a novel potential treatment strategy for As2O3 cardiomyocytes injury.

LncRNA NEAT1 regulates fibrosis and inflammatory response induced by nonalcoholic fatty liver by regulating miR-506/GLI3.

As one of the most common liver disorders worldwide, nonalcoholic fatty liver disease (NAFLD) begins with the abnormal accumulation of triglyceride (TG) in the liver and can lead to inflammation and fibrosis. Long noncoding RNA (lncRNA) NEAT1 was reported to promote NAFLD progress. However, its molecular mechanism in NAFLD was not fully clear. In vitro cellular model of NAFLD was established with BRL3A cell treated by free fatty acid (FFA). Cell Counting Kit-8 (CCK-8) assay was carried out to assess cell proliferation. The expression of mRNA and protein of inflammation and fibrosis in BRL3A cell was detected by qRT-PCR and Western blot. Bioinformatics and dual-luciferase reporter assays were used to predict and validate the interaction between NEAT1 and miR-506 as well as GLI3 and miR-506. NEAT1 was upregulated while miR-506 was downregulated in the progression of NAFLD. Meanwhile, NEAT1 and miR-506 were proved to regulate fibrosis, inflammatory response, and lipid metabolism. Knockdown of NEAT1 inhibited GLI3 expression and promoted miR-506 expression, Overexpression of miR-506 inhibited NEAT1 and GLI3 expression. Moreover, dual-luciferase reporter assays proved that miR-506 could bind to NEAT1 and GLI3, whereas NEAT1 could sponge miR-506 to regulate GLI3 expression. lncRNA NEAT1 could regulate fibrosis, inflammatory response, and lipid metabolism via the miR-506/GLI3 axis as a ceRNA, which is a novel mechanistic role in the regulation of NAFLD. These results provide a new potential treatment target for NAFLD.

Effect of long-chain noncoding RNA NEAT1 on the proliferation and invasion of renal cell carcinoma

Objective To observe the expression of long non-coding RNA (LncRNA-NEAT1) in renal cell carcinoma and cell lines, and to explore its influence on the malignant biological behavior of renal cell carcinoma and its possible molecular mechanism. Methods The expression of NEAT1 was detected by quantitative polymerase chain reaction (qPCR) in 10 cases of renal cell carcinoma and paracancerous tissues, and in 4 types of renal cell carcinoma cells and normal renal tubular epithelial cells. ACHN cells infected with negative control lentivirus (LV-NC) were used as control group, and ACHN cells infected with recombinant lentivirus carrying NEAT1 gene (LV-NEAT1) were used as experimental group. Bioinformatics predicts targeted miRNA and downstream genes of NEAT1. The effect of overexpression of NEAT1 on the mRNA expression of miR-342-3p and cell adhesion molecule 1 (CADM1) was detected by qPCR. The protein expression of CADM1 was detected by Western blot. The proliferation and invasion of ACHN cells were detected by methyl thiazolyl tetrazolium (MTT) method and Transwell invasion assay. Results The expression level of NEAT1 in renal cell carcinoma was lower than that in paracancerous tissue (P<0.01). The expression of NEAT1 in renal carcinoma cell lines was lower than that in renal tubular epithelial cells (P<0.01). After LV-NEAT1 infection, the expression of NEAT1 and CADM1 gene were significantly increased (P<0.01), while the expression of miR-342-3p decreased (P<0.01). Overexpression of NEAT1 significantly inhibited cell proliferation and invasion of ACHN cells (P<0.05). Conclusions LncRNA NEAT1 was down-regulated in renal cell carcinoma and cell lines. Overexpression of NEAT1 inhibited the proliferation and invasion of ACHN cells. The molecular mechanism may be that NEAT1 up-regulates the expression of CADM1 gene through complementary binding to miR-342-3p. Key words: RNA, long noncoding; Kidney neoplasms; Cell proliferation; Neoplasm invasiveness; In vitro

CRISPRa-mediated NEAT1 lncRNA upregulation induces formation of intact paraspeckles

Long noncoding RNAs (lncRNAs) are fundamental genomic regulatory factors under various physiological and pathological conditions. A class of lncRNAs termed architectural RNAs (arcRNAs) plays an essential scaffolding role in building nuclear bodies. NEAT1 arcRNA is an abundant, nuclear-retained lncRNA that constructs paraspeckle nuclear bodies. NEAT1 is upregulated in various developmental and disease conditions including cancer and virus infection. However, it remains unclear how elevated expression of NEAT1 influences such conditions. Here, we set up an experimental method to selectively increase NEAT1 expression. We applied the synergistic activation mediator (SAM) system using catalytically dead Cas9 (dCas9) proteins to activate transcription of the NEAT1 gene. We examined 10 pre-designed and 15 originally designed single-guide RNAs (sgRNAs) in the NEAT1 promoter region for CRISPR activation (CRISPRa). We validated several sgRNAs that we designed for the SAM system to strongly activate NEAT1 expression in two human cell lines and induced formation of paraspeckles with intact core-shell structures. Thus, this selective NEAT1 upregulation method using the SAM system would be useful for further functional analyses of NEAT1 lncRNA in both basic and applied research.

HIV-1 replication in CD4+ T cells exploits the down-regulation of antiviral NEAT1 long non-coding RNAs following T cell activation

The related NEAT1_1 and NEAT1_2 long noncoding RNAs (lnc RNAs) have been recently implicated in innate immunity against viral infection. We used CRISPR-Cas9 to generate Jurkat CD4+ T cell lines with a knockout (KO) of the NEAT1 gene. Viabilities of NEAT1 KO Jurkat lines were indistinguishable from parental Jurkat cells, as was the induction of CD69 after T cell activation. The KO lines were however more sensitive to the induction of apoptosis than parental Jurkat cells. HIV-1 replication was higher in the KO lines than parental Jurkat cells, demonstrating an anti-HIV function of NEAT1 lncRNAs. We observed a strong down-regulation of NEAT1 lncRNAs following activation of resting peripheral blood mononuclear cells and purified CD4+ T cells. These findings indicate that HIV-1 infection exploits the normal down-regulation of anti-viral NEAT1 lncRNAs in activated CD4+ T cells to enhance viral replication.

The effects and mechanism of long non-coding RNA NEAT1 on cell proliferation and apoptosis in gastric carcinoma cells

Objective To investigate the effect and mechanism of long non-coding RNA NEAT1 (lncRNA NEAT1) on proliferation and apoptosis of gastric carcinoma cells. Methods The expression level of lncRNA NEAT1 and normal gastric cell line GES-1 was measured by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR). The AGS cell line was divided into three groups, si-NEAT1 group, si-Ctrl group and Blank group. The proliferation ability was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and clone formation assay. Apoptosis ability was tested by flow cytometry. The expression level of Phosphor-protein KinaseB (p-Akt), B cell lymphoma/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) protein was measured by Western blotting. Results The expression level of lncRNA NEAT1 in AGS, BSG823, Hs746T and MGC-803 cell line was significantly higher than that in GES-1 (P=0.000). CCK-8 demonstrated that the A450 nm of si-NEAT1 group was significantly lower than that in si-Ctrl and Blank group after culturing for 72 and 96 h (P=0.001). The number of clone formation cells in si-NEAT1 group was significantly lower than that in si-Ctrl group, (31.43±3.25)% vs. (67.27±5.81)%, P=0.000. The apoptosis rate of si-NEAT1 group was significantly higher than that in si-Ctrl group, (19.5±1.2)% vs. (3.7±0.3)%, P=0.000. The expression level of p-Akt and bcl-2 protein in si-NEAT1 group was significantly up-regulated, while bax down-regulated. Conclusion LncRNA NEAT1 was over-expressed in gastric carcinoma cell lines. Knockdown of lncRNA NEAT1 could inhibit the proliferation and induce apoptosis, possibly through down-regulation of p-Akt and bcl-2 protien, and up-regulation of bax. Key words: Long non-coding RNA NEAT1; Gastric carcinoma; Proliferation; Apoptosis

Dysregulation of the BRCA1/long non-coding RNA NEAT1 signaling axis contributes to breast tumorigenesis.

Dysregulation of long non-codng RNA (lncRNA) expression has been found to contribute to tumorigenesis. However, the roles of lncRNAs in BRCA1-related breast cancer remain largely unknown. In this study, we delineate the role of the novel BRCA1/lncRNA NEAT1 signaling axis in breast tumorigenesis. BRCA1 inhibits NEAT1 expression potentially through binding to its genomic binding site upstream of the NEAT1 gene. BRCA1 deficiency in human normal/cancerous breast cells and mouse mammary glands leads to NEAT1 overexpression. Our studies show that NEAT1 upregulation resulting from BRCA1 deficiency stimulates in vitro and in vivo breast tumorigenicity. We have further identified molecular mediators downstream of the BRCA1/NEAT1 axis. NEAT1 epigenetically silences miR-129-5p expression by promoting the DNA methylation of the CpG island in the miR-129 gene. Silencing of miR-129-5p expression by NEAT1 results in upregulation of WNT4 expression, a target of miR-129-5p, which leads to activation of oncogenic WNT signaling. Our functional studies indicate that this NEAT1/miR-129-5p/WNT4 axis contributes to the tumorigenic effects of BRCA1 deficiency. Finally our in silico expression correlation analysis suggests the existence of the BRCA1/NEAT1/miR-129-5p axis in breast cancer. Our findings, taken together, suggest that the dysregulation of the BRCA1/NEAT1/miR-129-5p/WNT4 signaling axis is involved in promoting breast tumorigenesis.