Long Non-coding RNA Expression Research Articles

LncRNA HOTAIR regulates the PI3K/AKT pathway via the miR-126-3p/PIK3R2 axis to participate in synovial angiogenesis in rheumatoid arthritis.

The abnormal expression of long noncoding RNA (LncRNA) HOTAIR has been associated with synovial angiogenesis in rheumatoid arthritis (RA). The aim of this study is to investigate whether LncRNA HOTAIR plays a role in synovial angiogenesis in RA by regulating the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway through the miR-126-3p/PIK3R2 axis. In this study, we conducted in vitro experiments by designing overexpression plasmids and small interfering RNAs targeting LncRNA HOTAIR and then transfected them into rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). We then co-cultured the RA-FLS with human umbilical vein endothelial cells (HUVEC) to establish a RA-FLS-induced HUVEC model. We investigated the effects of LncRNA HOTAIR on the proliferation, migration, lumen forming ability of HUVEC, as well as the expression of synovial endothelial cell markers, angiogenic factors, and the PI3K/AKT pathway. To validate the interactions between LncRNA HOTAIR, miR-126-3p, and PIK3R2, we used bioinformatics and luciferase reporter experiments. We also employed real-time fluorescence quantitative, Western blotanalysis, and immunofluorescence techniques to analyze the target genes and proteins. The expression of LncRNA HOTAIR was upregulated in HUVEC induced by RA-FLS. The overexpression of LncRNA HOTAIR significantly increased the expression of vascular endothelial growth factor, basic fibroblast growth factor, CD34, and CD105 in HUVEC, promoting their proliferation, migration, and lumen formation. At the same time, the overexpression of LncRNA HOTAIR inhibited the expression of miR-126-3p, promoted the expression of PIK3R2, activated the PI3K/AKT pathway, and promoted the expression of PI3K, AKT and phosphorylated-AKT, while the silence of LncRNA HOTAIR reversed these expressions. Bioinformatics and double luciferase reporter gene experiments confirmed the targeting relationship among LncRNA HOTAIR, miR-126-3p, and PIK3R2. Finally, the rescue experiments showed that PI3K agonists could reverse the inhibitory effect of silent LncRNA HOTAIR on HUVEC. LncRNA HOTAIR has the potential to activate the PI3K/AKT pathway, likely through the regulatory axis involving miR-126-3p/PIK3R2, consequently contributing to synovial angiogenesis in RA.

Aerobic exercise improves cognitive impairment in mice with type 2 diabetes by regulating the MALAT1/miR-382-3p/BDNF signaling pathway in serum-exosomes

BackgroundIt has been documented that aerobic exercise (AE) has a positive effect on improving cognitive function in type 2 diabetes (T2DM) patients. Here, we tried to explore how AE regulates the expression of long non-coding RNA in serum-exosomes (Exos), thereby affecting cognitive impairment in T2DM mice as well as its potential molecular mechanism.MethodsT2DM mouse models were constructed, and serum-Exos were isolated for whole transcriptome sequencing to screen differentially expressed lncRNA and mRNA, followed by prediction of downstream target genes. The binding ability of miR-382-3p with a long non-coding RNA MALAT1 and brain-derived neurotrophic factor (BDNF) was explored. Then, primary mouse hippocampal neurons were collected for in vitro mechanism verification, as evidenced by the detection of hippocampal neurons' vitality, proliferation, and apoptosis capabilities, and insulin resistance. Finally, in vivo mechanism verification was performed to assess the effect of AE on insulin resistance and cognitive disorder.ResultsTranscriptome sequencing analysis showed that MALAT1 was lowly expressed and miR-382-3p was highly expressed in serum-Exos samples of T2DM mice. There were targeted binding sites between MALAT1 and miR-382-3p and between miR-382-3p and BDNF. In vitro experiments showed that MALAT1 upregulated BDNF expression by inhibiting miR-382-3p. Silencing MALAT1 or overexpressing miR-382-3p could reduce the expression of INSR, IRS-1, IRS-2, PI3K/AKT, and Ras/MAPK, inhibit neuronal proliferation, and promote apoptosis. In vivo experiments further confirmed that AE could increase the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thereby improving cognitive impairment in T2DM mice.ConclusionAE may upregulate the expression of MALAT1 in serum-Exos to competitively inhibit miR-382-3p and upregulate BDNF expression, thus improving cognitive impairment in T2DM mice.

DNA methylation-based depiction of the immune microenvironment and immune-associated long non-coding RNAs in oral cavity squamous cell carcinomas

Oral cavity squamous cell carcinoma (OSCC) is a complex and dynamic disease characterized by clinicopathological and molecular heterogeneity. Spatial and temporal heterogeneity of cell subpopulations has been associated with cancer progression and implicated in the prognosis and therapy response. Emerging evidence indicates that aberrant epigenetic profiles in OSCC may foster an immunosuppressive tumor microenvironment by modulating the expression of immune-related long non-coding RNAs (lncRNAs). DNA methylation analysis was performed in 46 matched OSCC and normal adjacent tissue samples using a genome-wide platform (Infinium HumanMethylation450 BeadChip). Reference-based computational deconvolution (MethylCIBERSORT) was applied to infer the immune cell composition of the bulk samples. The expression levels of genes encoding immune markers and differentially methylated lncRNAs were investigated using The Cancer Genome Atlas dataset. OSCC specimens presented distinct immune cell composition, including the enrichment of monocyte lineage cells, natural killer cells, cytotoxic T-lymphocytes, regulatory T-lymphocytes, and neutrophils. In contrast, B-lymphocytes, effector T-lymphocytes, and fibroblasts were diminished in tumor samples. The hypomethylation of three immune-associated lncRNAs (MEG3, MIR155HG, and WFDC21P) at individual CpG sites was confirmed by bisulfite-pyrosequencing. Also, the upregulation of a set of immune markers (FOXP3, GZMB, IL10, IL2RA, TGFB, IFNG, TDO2, IDO1, and HIF1A) was detected. The immune cell composition, immune markers alteration, and dysregulation of immune-associated lncRNAs reinforce the impact of the immune microenvironment in OSCC. These concurrent factors contribute to tumor heterogeneity, suggesting that epi-immunotherapy could be an efficient alternative to treat OSCC.

Insights into the involvement of long non-coding RNAs in doxorubicin resistance of cancer.

Doxorubicin is one of the most classical chemotherapeutic drugs for the treatment of cancer. However, resistance to the cytotoxic effects of doxorubicin in tumor cells remains a major obstacle. Aberrant expression of long non-coding RNAs (lncRNAs) has been associated with tumorigenesis and development via regulation of chromatin remodeling, transcription, and post-transcriptional processing. Emerging studies have also revealed that dysregulation of lncRNAs mediates the development of drug resistance through multiple molecules and pathways. In this review, we focus on the role and mechanism of lncRNAs in the progress of doxorubicin resistance in various cancers, which mainly include cellular drug transport, cell cycle disorder, anti-apoptosis, epithelial-mesenchymal transition, cancer stem cells, autophagy, tumor microenvironment, metabolic reprogramming and signaling pathways. This review is aimed to provide potential therapeutic targets for future cancer therapy, especially for the reversal of chemoresistance.

Increased Expression of Long Noncoding RNA LOC100506314 in T cells from Patients with Nonsegmental Vitiligo and Its Contribution to Vitiligo Pathogenesis.

This study aimed to identify the abnormal expression of long noncoding RNAs (lncRNAs) in T cells from patients with vitiligo and to investigate their functional roles in the immune system. Using microarray analysis, the expression levels of RNA transcripts in T cells from patients with vitiligo and controls were compared. We identified several genes and validated their expression levels in T cells from 41 vitiligo patients and 41 controls. The biological functions of the lncRNAs were studied in a transfection study using an RNA pull-down assay, followed by proteomic analysis and western blotting. The expression levels of 134 genes were significantly increased, and those of 142 genes were significantly decreased in T cells from vitiligo patients. After validation, six genes had increased expression, and three genes had decreased expression in T cells from patients with vitiligo. T-cell expression of LOC100506314 was increased in vitiligo, especially CD4+, but not CD8+ T cells. The expression levels of LOC100506314 in CD4+ T cells was positively and significantly associated with the severity of vitiligo. LOC100506314 was bound to the signal transducer and activator of transcription 3 (STAT3) and macrophage migration inhibitory factor (MIF). Enhanced expression of LOC100506314 inhibited the phosphorylation of STAT3, protein kinase B (AKT), and extracellular signal-regulated protein kinases (ERK), as well as the levels of nuclear protein of p65 and the expression of IL-6 and IL-17 in Jurkat cells and T cells from patients with vitiligo. In conclusion, this study showed that the expression of LOC100506314 was elevated in CD4+ T cells from patients with vitiligo and associated the severity of vitiligo. LOC100506314 interacted with STAT3 and MIF and inhibited IL-6 and IL-17 expression by suppressing the STAT3, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), AKT, and ERK pathways. Enhanced expression of LOC100506314 in T cells may be a potential treatment strategy for vitiligo.

The impact of Yiwei decoction on the LncRNA and CircRNA regulatory networks in premature ovarian insufficiency

Premature ovarian insufficiency（POI）is a female reproductive aging illness. Yiwei decoction（YWD） is a traditional treatment for Yangming nourishment. YWD can treat premature ovarian insufficiency, but the exact molecular mechanism is unknown. As a result, the differential expression of Long noncoding RNAs (LncRNAs) and Circular RNAs(CircRNAs) in the ovary of POI rats after YWD treatment was investigated in this paper, and the CeRNA regulatory network was built.The model was created using cyclophosphamide. The model group + YWD was in Group A, the model control group was in Group B, and the regular control group was in Group C. In this study, 177 differential expression Long noncoding RNAs(DELncRNAs) and 190 differential expression Circular RNAs (DECircRNAs) were discovered between A and B (P＜0.05,|LogFC|＞1). Following the analysis, 27 DELncRNAs and 96 DECircRNAs (P-adjusted＜0.05,|LogFC|＞1) were discovered. At the same time, we built the CeRNA network using differentially expressed mRNAs and microRNAs (miRNAs) expression between groups A and B. The DELncRNAs were used to construct a lncRNA-miRNA-mRNA ceRNA network with 27 LncRNAs, 4 miRNAs, and 19 mRNAs. The DECircRNAs were utilized to establish a CircRNA-miRNA-mRNA ceRNA network that was made up of 15 CircRNAs, 4 miRNAs, and 20 mRNA. The highly correlated regulatory networks were the LncMSTRG.22691.3/miR-3102/ANGPT4 and Circ10_34698898_34699378/miR-33-5p/TTC22. Circ20_12035276_12036793、Circ20_30693935_30696337、Circ4_157723097_157723378 and Circ4_157923266_157923904 occurred concurrently in AvsB, BvsC, and AvsC. MiRDB predicted eight target miRNAs for these CircRNAs. The miRanda(score = 140，energy = −1) binding energy calculation revealed that seven miRNAs were well combined with three CircRNA base complementary pairs. This implies that 3 DECircRNAs could serve as spongy bodies for these miRNAs. Network pharmacological analysis showed that ten active components in YWD may regulate the expression of LncRNAs and CircRNAs, such as Stigmasterol, Uridine, Ophiopogonanone A, Gamma-Aminobutyric Acid, and others.In conclusion, this study combined transcriptomics and network pharmacological analysis to identify differentially expressed lncRNAs as well as CircRNAs in ovaries of YWD-treated POI rats, thereby constructing ceRNA networks implicated in POI. This would contribute to clarifying the pathways by which Chinese herbal compounds regulate gene expression in POI.

Identification and functional prediction of long non-coding RNAs related to oxidative stress in the jejunum of piglets.

Oxidative stress (OS) is a pathological process arising from the excessive production of free radicals in the body. It has the potential to alter animal gene expression and cause damage to the jejunum. However, there have been few reports of changes in the expression of long noncoding RNAs (lncRNAs) in the jejunum in piglets under OS. The purpose of this research was to examine how lncRNAs in piglet jejunum change under OS. The abdominal cavities of piglets were injected with diquat (DQ) to produce OS. Raw reads were downloaded from the SRA database. RNA-seq was utilized to study the expression of lncRNAs in piglets under OS. Additionally, six randomly selected lncRNAs were verified using quantitative real-time polymerase chain reaction (qRT‒PCR) to examine the mechanism of oxidative damage. A total of 79 lncRNAs were differentially expressed (DE) in the treatment group compared to the negative control group. The target genes of DE lncRNAs were enriched in gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways. Chemical carcinogenesis-reactive oxygen species, the Foxo signaling pathway, colorectal cancer, and the AMPK signaling pathway were all linked to OS. Our results demonstrated that DQ-induced OS causes differential expression of lncRNAs, laying the groundwork for future research into the processes involved in the jejunum's response to OS.

The gut microbiota reprograms intestinal lipid metabolism through long noncoding RNA Snhg9.

The intestinal microbiota regulates mammalian lipid absorption, metabolism, and storage. We report that the microbiota reprograms intestinal lipid metabolism in mice by repressing the expression of long noncoding RNA (lncRNA) Snhg9 (small nucleolar RNA host gene 9) in small intestinal epithelial cells. Snhg9 suppressed the activity of peroxisome proliferator-activated receptor γ (PPARγ)-a central regulator of lipid metabolism-by dissociating the PPARγ inhibitor sirtuin 1 from cell cycle and apoptosis protein 2 (CCAR2). Forced expression of Snhg9 in the intestinal epithelium of conventional mice impaired lipid absorption, reduced body fat, and protected against diet-induced obesity. The microbiota repressed Snhg9 expression through an immune relay encompassing myeloid cells and group 3 innate lymphoid cells. Our findings thus identify an unanticipated role for a lncRNA in microbial control of host metabolism.

The expression and function of long noncoding RNAs in hepatocellular carcinoma.

With the deepening of the genome project study, attention on noncoding RNAs is increasing. Long noncoding RNAs (lncRNAs) have become a new research hotspot. A growing number of studies have revealed that lncRNAs are involved in tumorigenesis and tumor suppressor pathways. Aberrant expressions of lncRNAs have been found in a variety of human tumors including hepatocellular carcinoma (HCC). In this review, we provide a brief introduction to lncRNA and highlight recent research on the functions and clinical significance of lncRNAs in HCC.

The RNA helicases Dbp2 and Mtr4 regulate the expression of Xrn1-sensitive long non-coding RNAs in yeast.

The expression of yeast long non-coding (lnc)RNAs is restricted by RNA surveillance machineries, including the cytoplasmic 5'-3' exonuclease Xrn1 which targets a conserved family of lncRNAs defined as XUTs, and that are mainly antisense to protein-coding genes. However, the co-factors involved in the degradation of these transcripts and the underlying molecular mechanisms remain largely unknown. Here, we show that two RNA helicases, Dbp2 and Mtr4, act as global regulators of XUTs expression. Using RNA-Seq, we found that most of them accumulate upon Dbp2 inactivation or Mtr4 depletion. Mutants of the cytoplasmic RNA helicases Ecm32, Ski2, Slh1, Dbp1, and Dhh1 did not recapitulate this global stabilization of XUTs, suggesting that XUTs decay is specifically controlled by Dbp2 and Mtr4. Notably, Dbp2 and Mtr4 affect XUTs independently of their configuration relative to their paired-sense mRNAs. Finally, we show that the effect of Dbp2 on XUTs depends on a cytoplasmic localization. Overall, our data indicate that Dbp2 and Mtr4 are global regulators of lncRNAs expression and contribute to shape the non-coding transcriptome together with RNA decay machineries.

SDPS-31 EXTRACELLULAR VESICLES AND THEIR CARGO AS A PHARMACODYNAMIC REPORTER IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is a lethal brain tumor with limited treatment options, due in part to the low blood-brain-barrier (BBB) penetrance of many anti-cancer compounds. Development of a non-invasive test that can measure drug delivery and target engagement in GBM represents an opportunity to enhance knowledge during early phase clinical trials of a new compound’s pharmacological suitability and potential efficacy. We present here data supporting that the cargo within extracellular vesicles (EV) can potentially serve as a liquid biopsy that reports on the pharmacodynamics of GBM therapeutics. We treated short-term cell cultures established from four patient-derived xenografts with vehicle, temozolomide, MLN4924, or arsenic trioxide. EVs were isolated from culture supernatants using size exclusion chromatography and then analyzed by RNA sequencing and mass spectrometry (proteomics) to characterize and compare cargo profiles for each condition. We found that depending on treatment, there were shared alterations in EV cargo between models in the expression of long non-coding RNAs, genes that are overrepresented in biological processes such as cytoplasmic translation and cellular macromolecule biosynthesis, and circular RNAs. Mass spectrometry of protein cargo further demonstrated both upregulated and downregulated peptide abundances with treatment compared to vehicle. Thus, our preliminary results indicate that EV cargo post-treatment may be a novel pharmacodynamic reporter in GBM. To allow for the selective interrogation of patient GBM-derived EVs, we have simultaneously pursued the identification of biomarkers to allow for immunoprecipitation of EVs from patient biofluids. Potential biomarkers, including PTPRZ1, B7H3, and IL13RA2, were screened across isolated EVs from both pre-clinical and clinical patient samples. Future efforts are focused on evaluating immunoprecipitated GBM-EVs from longitudinally collected patient biofluids. Overall, we anticipate that the results of this study will lead to development of a clinical test that reflects BBB penetration and tumor response, which will likely aid in novel drug development efforts.

Abstract P1137: Novel Transcriptomic Interactions In Human Cardiomyopathic Heart Failure

Noncoding regions span ~98% of the human genome and are emerging as key players in cardiovascular pathologies. However, the role of human noncoding RNAs and their interactions with other RNAs and protein-coding genes in ischemic (ICM) and nonischemic cardiomyopathic (NICM) heart failure is not clear. Twenty-eight patients (53±11 years; mix of gender and race) were studied using Tukey’s analysis of variance and post-hoc analyses. Significantly increased left ventricular (LV) internal diameter (diastole and systole; p<0.01) and decreased ejection fraction (EF:p<1E-10) were observed in both ICM (EF:17.5%±3.5) and NICM (EF:20.6%±3.2) compared to organ donor controls (EF:56.6%±6). End-systolic volume was increased in ICM, and QTc and QRS duration were prolonged in NICM (p<0.05). Following blinded quality control validation of both LV RNA and data, blinded whole transcriptomics (n=4/group) revealed differential expression of numerous long noncoding (lnc)RNAs, micro(mi)RNAs, and mRNAs (p-corrected/adjusted; padj<0.05). We uncovered 251 novel lncRNAs, 137 novel mature miRNAs, and 286 novel mRNAs (padj<0.05). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome and Disease Ontology enrichment analyses revealed several significant (padj<0.05) pathways in cancer, neuro/macular degeneration, neuroembryological development, endocrine, inflammation, bone remodeling, vein disease, etc. Seven lncRNA-associated protein-protein interaction networks were identified in ICM (>700 confidence score). Co-expression analysis revealed highly significant lncRNA-mRNA interactions with Pearson Correlation Coefficient of <0.95 and >0.95 with p<0.00001. GO/KEGG also revealed miRNA-mRNA interactions related to bacterial/viral, parathyroid, cancer, mTOR, etc. (padj<0.05). Importantly, several genes and interactions in all the above RNA species and multiple enrichment pathways distinguished ICM versus NICM (padj<0.05). We also observed several lncRNA alternative splicing events (padj<0.0004; false discovery rate<0.05), numerous missense and silent mutations, and minimal nonsense mutations. Together, this study offers novel transcriptomic and interactomic understanding in human ICM and NICM heart failure.

Lnc-PLA2G4A-4 facilitates the progression of hepatocellular carcinoma by inducing versican expression via sponging miR-23b-3p

Aberrant expression of long non-coding RNAs (lncRNAs) is associated with progression of multiple human cancers including hepatocellular carcinoma (HCC). However, the role of lncRNAs in HCC is not been fully understood. Our study aimed to investigate the biological function and potential molecular mechanism of Lnc-PAL2G4A-4 in HCC. In the current study, we show that Lnc-PLA2G4A-4 was significantly up-regulated in HCC tissues and high Lnc-PLA2G4A-4 expression was remarkably associated with tumor size, microvascular invasion and poor prognosis of HCC patients. Functionally, Lnc-PLA2G4A-4 positively regulated cell proliferation, invasion and migration in vitro, and facilitated lung metastasis of HCC in vivo. Mechanistically, Lnc-PLA2G4A-4 functioned as a competing endogenous RNA (ceRNA) to bind to miR-23b-3p and subsequently facilitate miR-23b-3p's target gene versican (VCAN) expression in HCC cells. Over-expression of miR-23b-3p could reverse Lnc-PLA2G4A-4 induced cell phenotypes in HCC and suppress versican expression of by rescue analysis. Collectively, Lnc-PLA2G4A-4 promotes HCC progression by targeting the miR-23b-3p/versican axis, which may be a potential biomarker and therapeutic target for HCC.

Abnormal expression and related regulatory mechanism of long noncoding RNA in head and neck squamous cell carcinoma

The objective of this study was to investigate the effect and mechanism of long noncoding RNA (lncRNA) on head and neck squamous cell carcinoma (HNSCC). RNA was extracted from HNSCC tissue and adjacent tissues and sequenced. Differentially expressed lncRNA and microRNA (miRNA) were screened. Quantitative real-time polymerase chain reaction was used to compare the expression of target lncRNA between human oral keratinocyte (HOK) and cancer cell lines. LAMB1-210 in SCC-4 and CAL-27 cells was downregulated by transfecting short hairpin RNA (shRNA). Methyl thiazolyl tetrazolium, wound healing, and Transwell assay were used to evaluate viability, migration, and invasion ability in SCC-4 and CAL-27 cells. Eighty-one lncRNAs were differentially expressed, of which 40 were upregulated, 41 were downregulated, and 280 miRNAs were differentially expressed. Compared with HOK cells, the expression of LAMB1-210 was significantly upregulated in FADU, SCC-4, and CAL-27 cells (P < .001). LAMB1-210 was successfully downregulated by shRNA-LAMB1-210 (P < .001), and downregulated LAMB1-210 inhibited the cell viability, migration, and invasion ability of SCC-4 and CAL-27 cells (P < .001). The expression of lncRNA and miRNA in HNSCC tissues differs from that in normal tissues. LAMB1-210 is significantly overexpressed in tumor cells and is related to their survival, migration, and invasion.

Transcriptome-wide 5-methylcytosine modification profiling of long non-coding RNAs in A549 cells infected with H1N1 influenza A virus

BackgroundIn recent years, accumulating evidences have revealed that influenza A virus (IAV) infections induce significant differential expression of host long noncoding RNAs (lncRNAs), some of which play important roles in the regulation of virus-host interactions and determining the virus pathogenesis. However, whether these lncRNAs bear post-translational modifications and how their differential expression is regulated remain largely unknown. In this study, the transcriptome-wide 5-methylcytosine (m5C) modification of lncRNAs in A549 cells infected with an H1N1 influenza A virus was analyzed and compared with uninfected cells by Methylated RNA immunoprecipitation sequencing (MeRIP-Seq).ResultsOur data identified 1317 upregulated m5C peaks and 1667 downregulated peaks in the H1N1 infected group. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the differentially modified lncRNAs were associated with protein modification, organelle localization, nuclear export and other biological processes. Furthermore, conjoint analysis of the differentially modified (DM) and differentially expressed (DE) lncRNAs identified 143 ‘hyper-up’, 81 ‘hypo-up’, 6 ‘hypo-down’ and 4 ‘hyper-down’ lncRNAs. GO and KEGG analyses revealed that these DM and DE lncRNAs were predominantly associated with pathogen recognition and disease pathogenesis pathways, indicating that m5C modifications could play an important role in the regulation of host response to IAV replication by modulating the expression and/or stability of lncRNAs.ConclusionThis study presented the first m5C modification profile of lncRNAs in A549 cells infected with IAV and demonstrated a significant alteration of m5C modifications on host lncRNAs upon IAV infection. These data could give a reference to future researches on the roles of m5C methylation in virus infection.

PARP-1 inhibits DNMT1-mediated promoter methylation and promotes linc01132 expression in benzene-exposed workers and hydroquinone-induced malignant transformed cells

Hydroquinone (HQ), one of the main active metabolites of benzene, can induce the abnormal expression of long non-coding RNA (lncRNA). Studies have shown that lncRNA plays an important role in the occurrence of hematologic tumors induced by benzene or HQ. However, the molecular mechanism remains to be elucidated. Here, we investigated the molecular mechanism by which poly(ADP-ribose)polymerase 1 (PARP-1) interacts with DNA methyltransferase 1 (DNMT1) to regulate promoter methylation mediated linc01132 expression in HQ-induced TK6 malignant transformed cells (HQ-MT). The results revealed that the expression of linc01132 was increased in benzene-exposed workers and HQ-MT cells. The methylation of linc01132 promoter region was inhibited. Furthermore, in HQ-MT cells treated with 5-Aza-2′-deoxycytidine (5-AzaC) (DNA methyltransferase inhibitor) or trichostatin A (TSA) (histone deacetylation inhibitor), the expression of linc01132 was increased due to the regulation of DNA promoter methylation level by inhibiting DNMT1 expression. The methylation level of linc01132 promoter was correlated negatively with the expression of linc01132 in benzene-exposed workers, indicating that DNA methylation may contribute the expression of linc01132. Knockout of DNMT1, not DNMT3b, increased the expression of linc01132 as well as the demethylation of linc01132 promoter in HQ-MT cells. It was found that by knockdown PARP-1, the expression of DNMT1 in the nucleus was increased by immunofluorescence confocal microscopy, leading to the inhibition of hypermethylation in the promoter region of linc01132. Therefore, PARP-1 inhibits DNA methyltransferase (DNMT)-mediated promoter methylation and plays a role in linc01132 expression in benzene-exposed workers or HQ-MT cells, and is associated with benzene or HQ induced leukemia progression.

A monoclonal antibody raised against human EZH2 cross-reacts with the RNA-binding protein SAFB.

The Polycomb Repressive Complex 2 (PRC2) is a conserved enzyme that tri-methylates Lysine 27 on Histone 3 (H3K27me3) to promote gene silencing. PRC2 is remarkably responsive to the expression of certain long noncoding RNAs (lncRNAs). In the most notable example, PRC2 is recruited to the X-chromosome shortly after expression of the lncRNA Xist begins during X-chromosome inactivation. However, the mechanisms by which lncRNAs recruit PRC2 to chromatin are not yet clear. We report that a broadly used rabbit monoclonal antibody raised against human EZH2, a catalytic subunit of PRC2, cross-reacts with an RNA-binding protein called Scaffold Attachment Factor B (SAFB) in mouse embryonic stem cells (ESCs) under buffer conditions that are commonly used for chromatin immunoprecipitation (ChIP). Knockout of EZH2 in ESCs demonstrated that the antibody is specific for EZH2 by western blot (no cross-reactivity). Likewise, comparison to previously published datasets confirmed that the antibody recovers PRC2-bound sites by ChIP-Seq. However, RNA-IP from formaldehyde-crosslinked ESCs using ChIP wash conditions recovers distinct peaks of RNA association that co-localize with peaks of SAFB and whose enrichment disappears upon knockout of SAFB but not EZH2. IP and mass spectrometry-based proteomics in wild-type and EZH2 knockout ESCs confirm that the EZH2 antibody recovers SAFB in an EZH2-independent manner. Our data highlight the importance of orthogonal assays when studying interactions between chromatin-modifying enzymes and RNA.

Long Noncoding RNA ACTA2-AS1 Inhibits Cell Growth and Facilitates Apoptosis in Gastric Cancer by Binding with miR-6720-5p to Regulate ESRRB.

Gastric cancer (GC) is a common malignant tumor, posing a great threat to human's health and life. Previous studies have suggested aberrant expression of long non-coding RNAs (lncRNAs) in GC. This study elucidated the effects of lncRNA ACTA2-AS1 on the biological characteristics of GC. Gene expression in stomach adenocarcinoma (STAD) samples compared with normal tissues and the correlation between gene expression and prognosis of STAD patients were analyzed using bioinformatic tools. Gene expression at protein and mRNA levels in GC and normal cells was tested by western blotting and RT-qPCR. The subcellular localization of ACTA2-AS1 in AGS and HGC27 cells was identified by nuclear-cytoplasmic fractionation and FISH assay. EdU, CCK-8, flow cytometry analysis, TUNEL staining assays were conducted to evaluate the role of ACTA2-AS1 and ESRRB on GC cellular behaviors. The binding relationship among ACTA2-AS1, miR-6720-5p and ESRRB was verified by RNA pulldown, luciferase reporter assay and RIP assay. LncRNA ACTA2-AS1 was underexpressed in GC tissues and cell lines. ACTA2-AS1 elevation suppressed GC cell proliferation and induced apoptosis. Mechanistically, ACTA2-AS1 directly bound to miR-6720-5p and subsequently promoted the expression of target gene ESRRB in GC cells. Furthermore, ESRRB knockdown reversed the influence of ACTA2-AS1 overexpression on GC proliferation and apoptosis. ACTA2-AS1 plays an antioncogenic role in GC via binding with miR-6720-5p to regulate ESRRB expression.

Identification Invasion-Related Long Non-Coding RNAs in Lung Adenocarcinoma and Analysis of Competitive Endogenous RNA Regulatory Networks.

Cell invasion plays a vital role in cancer development and progression. Aberrant expression of long non-coding RNAs (lncRNAs) is also critical in carcinogenesis. However, the prognostic value of invasion-related lncRNAs in lung adenocarcinoma (LUAD) remains unknown. Differentially expressed mRNAs (DEmRNAs), lncRNAs (DElncRNAs), and microRNAs (DEmiRNAs) were between LUAD and control samples. Pearson correlation analyses were performed to screen for invasion-related DElncRNAs (DEIRLs). Univariate and multivariate Cox regression algorithms were applied to identify key genes and construct the risk score model, which was evaluated using receiver operating characteristic (ROC) curves. Gene set enrichment analysis (GSEA) was used to explore the underlying pathways of the risk model. Moreover, an invasion-related competitive endogenous RNA (ceRNA) regulatory network was constructed. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to detect the expression of prognostic lncRNAs in the LUAD and control samples. A total of 45 DElncRNAs were identified as DEIRLs. RP3-525N10.2, LINC00857, EP300-AS1, PDZRN3-AS1, and RP5-1102E8.3 were potential prognostic lncRNAs, the expression of which was verified by RT-qPCR in LUAD samples. Both the risk score model and nomogram used the prognostic lncRNAs. ROC curves showed the risk score model had moderate accuracy and the nomogram had high accuracy in predicting patient prognosis. GSEA results indicated that the risk score model was associated with many biological processes and pathways relevant to cell proliferation. A ceRNA regulatory network was constructed in which PDZRN3-miR-96-5p-CPEB1, EP300-AS1-miR-93-5p-CORO2B, and RP3-525N10.2-miR-130a-5p-GHR may be key invasion-related regulatory pathways in LUAD. Our study identified five novel invasion-related prognostic lncRNAs (RP3-525N10.2, LINC00857, EP300-AS1, PDZRN3-AS1, and RP5-1102E8.3) and established an accurate model for predicting the prognosis of patients with LUAD. These findings enrich our understanding of the relationships between cell invasion, lncRNAs, and LUAD and may provide novel treatment directions.

Topologically associating domain underlies tissue specific expression of long intergenic non-coding RNAs

Accumulating evidence indicates that long intergenic non-coding RNAs (lincRNAs) show more tissue-specific expression patterns than protein-coding genes (PCGs). However, although lincRNAs are subject to canonical transcriptional regulation like PCGs, the molecular basis for the specificity of their expression patterns remains unclear. Here, using expression data and coordinates of topologically associating domains (TADs) in human tissues, we show that lincRNA loci are significantly enriched in the more internal region of TADs compared to PCGs and that lincRNAs within TADs have higher tissue specificity than those outside TADs. Based on these, we propose an analytical framework to interpret transcriptional status using lincRNA as an indicator. We applied it to hypertrophic cardiomyopathy data and found disease-specific transcriptional regulation: ectopic expression of keratin at the TAD level and derepression of myocyte differentiation-related genes by E2F1 with down-regulation of LINC00881. Our results provide understanding of the function and regulation of lincRNAs according to genomic structure.