lncRNA MEG3 Research Articles

LncRNA MEG3: Targeting the Molecular Mechanisms and Pathogenic causes of Metabolic Diseases.

Non-coding RNA is a type of RNA that does not encode proteins, distributed among rRNA, tRNA, snRNA, snoRNA, microRNA and other RNAs with identified functions, where the Long non-coding RNA (lncRNA) displays a nucleotide length over 200. LncRNAs enable multiple biological processes in the human body, including cancer cell invasion and metastasis, apoptosis, cell autophagy, inflammation, etc. Recently, a growing body of studies has demonstrated the association of lncRNAs with obesity and obesity-induced insulin resistance and NAFLD, where MEG3 is related to glucose metabolism, such as insulin resistance. In addition, MEG3 has been demonstrated in the pathological processes of various cancers, such as mediating inflammation, cardiovascular disease, liver disease and other metabolic diseases. To explore the regulatory role of lncRNA MEG3 in metabolic diseases. It provides new ideas for clinical treatment or experimental research. In this paper, in order to obtain enough data, we integrate and analyze the data in the PubMed database. LncRNA MEG3 can regulate many metabolic diseases, such as insulin resistance, NAFLD, inflammation and so on. LncRNA MEG3 has a regulatory role in a variety of metabolic diseases, which are currently difficult to be completely cured, and MEG3 is a potential target for the treatment of these diseases. Here, we review the role of lncRNA MEG3 in mechanisms of action and biological functions in human metabolic diseases.

Determining the effect of long non-coding RNA maternally expressed Gene 3 (lncRNA MEG3) on the transcriptome profile in cervical cancer cell lines

This study investigates the role of the long non-coding RNA Maternally Expressed Gene3 (lncRNA MEG3) gene in cervical cancer, as evidenced by its downregulation in cancerous cell lines. The study demonstrates the effects of the overexpression of lncRNA MEG3 in cervical cancer cell lines, particularly in C33A and CaSki. Through comprehensive analyses, including Next-Generation Sequencing (NGS), alterations in global mRNA expression were analyzed. In C33A cells, 67 genes were upregulated, while 303 genes were downregulated. Similarly, in CaSki cells, 221 genes showed upregulation and 248 genes displayed downregulation. Gene ontology and KEGG pathway analyses were conducted to gain insight into potential mechanisms. Furthermore, the study delves into gene regulatory networks, uncovering intricate interactions among genes. The RNA sequencing data were confirmed for eight genes: PAX3, EGR2, ROR1, NRP1, OAS2, STRA6, CA9, and EDN2 by Real-time PCR. The findings illuminate the complex landscape of gene expression alterations and pathways impacted by the overexpression of lncRNA MEG3. The impact of MEG3 on the overall cervical cancer cells' mRNA profile is reported for the first time. New biomarkers for the prognosis of cervical cancer are also reported in this study. Moreover, identifying specific genes within the regulatory networks provides valuable insights into potential therapeutic targets for managing cervical cancer.

Up-regulation of HOXA-AS2 and MEG3 long non-coding RNAs acts as a potential peripheral biomarker for bipolar disorder.

Bipolar disorder (BD) is a psychiatric condition that is frequently misdiagnosed and linked to inadequate treatment. Long non-coding RNAs (lncRNAs) have lately gained recognition as crucial genetic elements and are now regarded as regulatory mechanisms in the neurological system. Our objective was to measure the quantities of HOXA-AS2 and MEG3 ncRNA transcripts. HOXA-AS2 and MEG3 ncRNA levels were checked in the peripheral blood of 50 type I BD and 50 control samples by real-time PCR. Furthermore, we conducted ROC curve analysis and correlation analysis to examine the association between gene expression and specific clinical characteristics in instances with BD. Additionally, a computational study was performed to investigate the binding sites of miRNAs on the HOXA-AS2 and MEG3 lncRNAs. BD subjects showed a significant increase in the expression of HOXA-AS2 and MEG3 compared to controls. The lncRNAs HOXA-AS2 and MEG3 have an area under the ROC curve (AUC) values of 0.70 and 0.71, respectively. There was a significant correlation between the expression levels of ncRNAs HOXA-AS2 and MEG3 in the peripheral blood of patients with BD and occupation scores. The data presented indicate a potential correlation between the expression of HOXA-AS2 and MEG3 lncRNAs with an elevated risk of BD. Furthermore, these lncRNAs may be linked to several molecular pathways. Our findings indicate that the amounts of lncRNAs HOXA-AS2 and MEG3 in transcripts might be a promising potential biomarker for patients with BD.

LncRNA MEG3 aggravates acute pulmonary embolism-induced pulmonary arterial hypertension by regulating miR-34a-3p/DUSP1 axis

LncRNA MEG3 aggravates acute pulmonary embolism-induced pulmonary arterial hypertension by regulating miR-34a-3p/DUSP1 axis

LncRNA MEG3 promotes aortic valve calcification by inducing osteoblast differentiation via Wnt catenin signaling pathway

Abstract Background Calcific aortic valve stenosis (CAVS) is the most common valvular heart disease in elderly population. This study aimed to investigate the role of long non-coding RNAs (lncRNAs) in the development of CAVS. Methods ldlr-/-mice were feeding with western diet for 6 months to induce CAVS. RNA-seq analysis was performed to identify differentially expressed molecular signaling between aortic valves of CAVS mice and control mice. The degree of aortic stenosis was detected by ultrasound. Calcium salt deposits were detected by Haematoxylin and eosin (H&E) staining, Von Kossa staining and alizarin red S staining. PCR and western blotting were employed to detect osteogenic differentiation markers after various interventions. Results A mouse CAVS model was successfully established, as evidenced by increased thickness and calcium deposition in the aortic valve leaflets and increased transvalvular peak jet velocity. RNA-seq analysis showed that lncRNA MEG3 was significantly enriched in aortic valves of CAVS mice as compared to that of control mice. MEG3 expression in aortic valve, assessed by RNA in situ hybridization and PCR, was also significantly upregulated during the osteoblast differentiation of AVICs in vitro. Gain- and loss-of-function experiments indicate that MEG3 could indeed promote osteoblast differentiation of primary AVICs as evidenced by increased calcified nodule formation and expressions of osteoblast differentiation markers (Runx2 and osterix). Consistent with these in vitro data, MEG3 knockdown in vivo through tail injection of antisense oligonucleotide chain MEG3 (ASO-MEG3) at 6 weeks interval for 12 weeks significantly attenuated aortic valve calcification in CAVS mice. Subsequent KEGG analysis demonstrated that the Wnt/β-catenin signaling pathway was significantly enriched during the CAVS progression. Overexpression of MEG3 activated the Wnt/β-catenin signaling pathway and upregulated osteogenic protein expressions, while this upregulation could be significantly ameliorated by Dickkopf 1, a Wnt/β-catenin pathway inhibitor. Conclusion Present study demonstrates that LncRNA MEG3 could promote aortic valve calcification by inducing osteoblast differentiation via activating Wnt/β-catenin signaling pathway. Our results provide experimental evidence of targeting MEG3 and Wnt/β-catenin signaling pathway as potential promising therapeutic option for prevention and treatment of aortic valve calcification.

LncRNA MEG3 suppresses hepatocellular carcinoma by stimulating macrophage M1 polarization and modulating immune system via inhibiting CSF-1 in vivo/vitro studies

Hepatocellular carcinoma (HCC) is characterized by a complex tumor microenvironment (TME), and long non-coding RNAs (lncRNAs) MEG3 emerged as regulators of macrophage polarization with a negative relationship with colony-stimulating factor 1 (CSF-1). Few studies are on the interplay among MEG3, CSF-1, T helper cells (Th), and the programmed cell death protein 1 and its ligands (PD-1/PD-Ls) in TME of HCC.MEG3 expression in THP-1 macrophages, monitored polarization, and PD-1/PD-Ls expression were through flow cytometry, WB, and RT-qPCR. In co-cultures, the interaction of MEG3, macrophage, and HCC was assayed by ELISA. The invasive and migratory of HCC were assessed through experiments such as CCK-8, clonogenic assay, wound healing, and Transwell. A xenograft mouse model of HCC was established, administered with MEG3 overexpression (OE) or knockdown (KD) constructs, and monitored tumor growth. In vitro, MEG3 OE induced a robust M1 macrophage phenotype, evidenced by elevated expression of M1 markers and a significant increase in Th1 cytokines, with a concomitant decrease in Th2 cytokines. This was paralleled by reduced CSF-1 and PD-1/PD-Ls expression. In contrast, MEG3 KD promoted an M2 phenotype with increased CSF-1 and PD-1/PD-Ls expression, and an upregulation of Th2 cytokines. MEG3 OE inhibited the growth, invasion, and migration of HCC, while the opposite was observed when MEG3 was downregulated. In vivo, MEG3 OE resulted in significantly reduced tumor growth, with decreased PD-1/PD-Ls expression on macrophages and enhanced Th1 response. Conversely, MEG3 KD promoted tumor growth with increased PD-1/PD-Ls and a Th2-skewed immune response. MEG3 modulates the TME by affecting TAMs through CSF-1, thereby influencing the balance of Th1/Th2 cells and altering the expression of PD-1/PD-L1s. This study demonstrates that targeting MEG3 is an effective therapeutic strategy for HCC.

Bulk and single-cell RNA-seq analyses reveal canonical RNA editing associated with microglia homeostasis and its role in sepsis-associated encephalopathy

Previous studies have demonstrated the roles of both microglia homeostasis and RNA editing in sepsis-associated encephalopathy (SAE), yet their relationship remains to be elucidated. In this study, we analyzed bulk and single-cell RNA-seq (scRNA) datasets containing 107 brain tissue and microglia samples from mice with microglial depletion and repopulation to explore canonical RNA editing associated with microglia homeostasis and evaluate its role in SAE. Analysis of mouse brain RNA-Seq revealed hallmarks of microglial repopulation, including peak expressions of Apobec1 and Apobec3 at Day 5 of repopulation and dramatically altered B2m RNA editing. Significant time-dependent changes in brain RNA editing during microglial depletion and repopulation were primarily observed in synapse-related genes, such as Tbc1d24 and Slc1a2. ScRNA-Seq revealed heterogeneous RNA editing among microglia subpopulations and their distinct changes associated with microglia homeostasis. Moreover, repopulated microglia from lipopolysaccharide (LPS)-induced sepsis mice exhibited intensified up-regulation of Apobec1 and Apobec3, with distinct RNA editing responses to LPS, mainly involved in immune-related pathways. The hippocampus from sepsis mice induced by peritoneal contamination and infection showed upregulated Apobec1 and Apobec3 expression, and altered RNA editing in immune-related genes, such as B2m and Mier1, and nervous-related lncRNA Meg3 and Snhg11, both of which were repressed by microglial depletion. Furthermore, the expression of complement-related genes, such as C4b and Cd47, was substantially correlated with RNA editing activity in microglia homeostasis and SAE. Our study demonstrates canonical RNA editing associated with microglia homeostasis and provides new insights into its potential role in SAE.

Functional role of lncRNA MEG3 on pyroptosis through interacting with EZH2 and YTHDC1 in postoperative cognitive dysfunction

BackgroundThe molecular biology mechanisms underlying postoperative cognitive dysfunction (POCD) remain unclear, resulting in a lack of specific therapeutic targets and limited clinical treatment options. The NLRP3 pyroptotic pathway, induced by neuroinflammation, is known to promote the development of POCD. Research has shown that lncRNA MEG3 exacerbates cell pyroptosis in various neurological injuries, though the precise mechanism remains to be investigated. MethodsIn vitro and in vivo models of POCD were established through treatment with sevoflurane. Gene and protein expression were investigated using qRT-PCR, Western blot analysis, ELISA, and histological staining. Additionally, cell viability and injury were assessed through CCK-8 and LDH assays. Hippocampal-dependent memory and cognitive abilities were evaluated using the Morris Water Maze (MWM) test. Furthermore, the interactions between MEG3 and EZH2/YTHDC1 were validated through RNA immunoprecipitation (RIP) and chromatin immunoprecipitation (ChIP). ResultsOur findings reveal that sevoflurane significantly reduced MEG3 and pyroptosis-related proteins in mice. The overexpression of MEG3 protected mice against sevoflurane-induced cognitive dysfunction and reversed sevoflurane-induced pyroptosis in hippocampal neurons. MEG3 induced the downregulation of NLRP3 expression and reduced mRNA stability through its interaction with EZH2/YTHDC1. ConclusionIn conclusion, our study elucidates that MEG3 inhibits the NLRP3 inflammasome and hippocampal neuron pyroptosis through the recruitment of EZH2/YTHDC1. These findings shed light on the underlying mechanism of MEG3 in the regulation of POCD and suggest that MEG3 could serve as a potential therapeutic target for the treatment of POCD.

Genetic variants of LncRNAs HOTTIP and MEG3 influence nasopharyngeal carcinoma susceptibility and clinicopathologic characteristics in the Southern Chinese population

ObjectiveRecent studies have indicated that HOTTIP and MEG3 are associated with the initiation and progression of various types of tumors, including nasopharyngeal carcinoma (NPC). This investigation aimed to elucidate the impact of HOTTIP and MEG3 polymorphisms on the susceptibility and clinicopathologic characteristics of NPC.MethodsThis research employed next-generation sequencing and multiplex PCR to assess the polymorphisms of HOTTIP rs1859168 and MEG3 rs7158663 in 200 NPC and 200 healthy individuals respectively. HOTTIP and MEG3 expression were assessed via qRT-PCR assessment. Furthermore, the genotypes and alleles frequency of rs1859168 and rs7158663 were compared between healthy and NPC individuals to elucidate their influence on NPC susceptibility and relation with clinicopathologic characteristics.ResultsIn comparison with the healthy cohort, the presence of HOTTIP rs1859168 CC genotype and the C allele were markedly linked with increased NPC incidence (p < 0.05). Furthermore, the MEG3 rs7158663 AA genotype and the A allele also indicated an increased risk of NPC (p < 0.05). The subgroup analysis of age, EBV infection, gender, nationality, smoking, and drinking status revealed no marked association between rs1859168 and rs7158663 genotypes and these potential confounding factors. Moreover, it was observed that rs1859168 CC and rs7158663 AA genotypes were related to local tumor invasion and lymph node metastasis. Additionally, HOTTIP indicated a marked elevation, while MEG3 substantially reduced in NPC samples than the normal nasopharyngeal biospecimens. Patients who carried CC or CA genotypes rather than the HOTTIP rs1859168 AA genotype, had substantially higher HOTTIP levels, while patients with rs7158663 AA or GA genotypes indicated notably lower expression of MEG3 than GG genotype carriers.ConclusionIndividuals with genetic variants of HOTTIP rs1859168 and MEG3 rs7158663 might have an increased risk of NPC susceptibility and related clinicopathologic characteristics, potentially by affecting the expression of HOTTIP and MEG3.

A possible role of lncRNA MEG3 and lncRNA MAFG-AS1 on miRNA 147-b in the pathogenesis of Behcet’s disease

Behcet’s disease (BD) is a multisystem disease with altered Toll-like receptors (TLRs) on macrophages. Long noncoding RNA Maternally expressed gene 3 (lncRNA MEG3) and lncRNA Musculoaponeurotic fibrosarcoma oncogene family, protein G antisense 1 (MAFG-AS1) are regulators of microRNA (miRNA) 147-b, which is induced upon TLR stimulation. We included fifty BD patients, and fifty age and sex-matched controls. Real-time polymerase chain reaction (PCR) was used to measure the expression levels of serum lncRNA MEG3, lncRNA MAFG-AS1, and miRNA 147-b. LncRNA MEG3 and lncRNA MAFG-AS1 were significantly downregulated while miRNA 147-b was significantly upregulated in the BD patients' serum compared to the controls with p-value <0.001. Receiver operation characteristics (ROC) curve analysis revealed that the three biomarkers can discriminate between BD and control subjects with 76%, 100%, and 70% sensitivity respectively, and 100% specificity for all of them. There was a lower expression level of lnc RNA MEG3 among patients who had new eye involvement in the last month in comparison to those without new eye involvement (p-value=0.017). So, LncRNA MEG3, lncRNA MAFG-AS1, and miRNA147-b are promising diagnostic markers and therapeutic targets for BD patients. LncRNA MEG3 can be used as a predictor for new BD ocular involvement.

MEG3-Mediated Oral Squamous-Cell-Carcinoma-Derived Exosomal miR-421 Activates Angiogenesis by Targeting HS2ST1 in Vascular Endothelial Cells.

Exosomal microRNAs (miRNAs) from cancer cells play a key role in mediating the oral squamous cell carcinoma (OSCC) microenvironment. The objective of this study was to investigate how the long non-coding RNA (lncRNA) MEG3 affects OSCC angiogenesis through exosomal miR-421. Global miRNA microarray analysis and quantitative real-time PCR (qRT-PCR) were performed to determine the level of miRNAs in OSCC cell-derived exosomes. Cell migration, invasion, tube formation, immunohistochemistry, and hemoglobin concentrations were used to study the effects of exosomal miR-421 in angiogenesis. Western blotting was used to determine the expression level of HS2ST1 and VEGFR2-related downstream proteins. MiRNA array and qRT-PCR identified the upregulation of miR-421 in OSCC cell-derived exosomes. Furthermore, exosomal miR-421 can be taken up by human umbilical vein endothelial cells (HUVECs) and then target HS2ST1 through VEGF-mediated ERK and AKT phosphorylation, thereby promoting HUVEC migration, invasion, and tube formation. Additionally, forced expression of the lncRNA MEG3 in OSCC cells reduced exosomal miR-421 levels and then increased HS2ST1 expression, thereby reducing the VEGF/VEGFR2 pathway in HUVECs. Our results demonstrate a novel mechanism by which lncRNA MEG3 can act as a tumor suppressor and regulate endothelial angiogenesis through the exosomal miR-421/HS2ST1 axis, which provides a potential therapeutic strategy for OSCC angiogenesis.

Adenovirus 36 seropositivity is related to the expression of anti-adipogenic lncRNAs GAS5 and MEG3 in adipose tissue obtained from subjects with obesity.

Human Adenovirus D-36 (HAdV-D36) promotes adipogenesis in cellular and animal models and may contribute to the development of human obesity. Induction of PPARγ by HAdV-D36 seems to have a central role in the maintenance of adipogenic status. There is limited information about epigenetic mechanisms contributing to this process in human adipose tissue. This study evaluated the expression of lncRNAs (ADINR, GAS5 and MEG3) and miRNAs (miR-18a and miR-140) involved in the adipogenic process in visceral adipose tissue (VAT) of subjects with obesity with previous HAdV-D36 infection (seropositive) and unexposed (seronegative) subjects with obesity. Individuals with obesity were grouped according to the presence of antibodies against HAdV-D36 (Seropositive: HAdV-D36[+], n = 29; and Seronegative: HAdV-D36[-], n = 28). Additionally, a group of individuals without obesity (n = 17) was selected as a control group. The HAdV-D36 serology was carried out by ELISA. Biopsies of VAT were obtained during an elective and clinically indicated surgery (bariatric or cholecystectomy). RNA extraction from VAT was performed and the expression of PPARG and non-coding RNAs was evaluated by qPCR. HAdV-D36[+] individuals had lower expression of anti-adipogenic lncRNAs GAS5 (p = 0.016) and MEG3 (p = 0.035) compared with HAdV-D36[-] subjects with obesity. HAdV-D36[+] subjects also presented increased expression of the adipogenic miRNA miR-18a (p = 0.042), which has been reported to be modulated by GAS5 through a RNA sponging mechanism during adipogenic differentiation. Additionally, an inverse correlation of GAS5 with PPARG expression was observed (r = -0.917, p = 0.01). Our results suggest that HAdV-D36 is related to non-coding RNAs implicated in adipogenesis, representing a potential mechanism by which previous HAdV-D36 infection could be associated with the long-term maintenance of adipogenic status, probably through the GAS5/miR-18a axis.

Long non-coding RNA MEG3 knockdown represses airway smooth muscle cells proliferation and migration via sponging miR-143-3p/FGF9 in asthma

BackgroundAsthma is a respiratory disease characterized by airway remodeling. We aimed to find out the role and mechanism of lncRNA MEG3 in asthma.MethodsWe established a cellular model of asthma by inducing human airway smooth muscle cells (HASMCs) with PDGF-BB, and detected levels of lncRNA MEG3, miR-143-3p and FGF9 in HASMCs through qRT-PCR. The functions of lncRNA MEG3 or miR-143-3p on HASMCs were explored by cell transfection. The binding sites of miR-143-3p and FGF9 were subsequently analyzed with bioinformatics software, and validated with dual-luciferase reporter assay. MTT, 5-Ethynyl-2’-deoxyuridine (EdU) assay, and Transwell were used to detect the effects of lncRNA MEG3 or miR-143-3p on proliferation and migration of HASMCs. QRT-PCR and western blot assay were used to evaluate the level of proliferation-related marker PCNA in HASMCs.ResultsThe study found that lncRNA MEG3 negatively correlated with miR-143-3p, and miR-143-3p could directly target with FGF9. Silence of lncRNA MEG3 can suppress migration and proliferation of PDGF-BB-induced HASMCs via increasing miR-143-3p. Further mechanistic studies revealed that miR-143-3p negatively regulated FGF9 expression in HASMCs. MiR-143-3p could inhibit PDGF-BB-induced HASMCs migration and proliferation through downregulating FGF9.ConclusionLncRNA MEG3 silencing could inhibit the migration and proliferation of HASMCs through regulating miR-143-3p/FGF9 signaling axis. These results imply that lncRNA MEG3 plays a protective role against asthma.

Study of lncRNAs expression profile in the response to biological drugs in Psoriatic Arthritis: MEG3 could be a potential genomic biomarker of therapy efficacy

We aimed to identify an expression profile of lncRNAs potentially related to treatment response in Psoriatic arthritis (PsA) patients, to be used as potential genomic biomarkers predictors of drug treatment effectiveness. In addition, we evaluated a possible association between lncRNAs genetic variants and the response to therapy using the clinical parameter of Disease Activity Index. For the expression study, we collected 48 treated PsA patients, monitoring the treatment response for 12 months. We initially used PCR Array and, then, we validated the results with qRT-PCR. We also retrospectively genotyped 163 treated PsA patients. Firstly, we observed a significant difference in the expression level between Responder and non-Responder patients, of 4 lncRNAs in the group of PsA patients treated with TNFi and of 3 lncRNAs in the group of patients treated with IL17i. Then, we confirmed a significant decrease of MEG3 expression in non-Responder patients compared to Responders, also considering separately the single groups of patients treated with TNFi and IL17i. In addition, our results seem to highlight a potential dose-dependent effect of rs941576 (MEG3) variant allele on Disease Activity Index. Our study suggests a possible role of the lncRNA MEG3 in the treatment response to biological drugs.

Retracted] Methylene blue relieves the development of osteoarthritis by upregulating lncRNA MEG3.

[This retracts the article DOI: 10.3892/etm.2018.5918.].

LncRNA MEG3 Restrains Hepatic Lipogenesis via the FOXO1 Signaling Pathway in HepG2 Cells

Nonalcoholic fatty liver disease (NAFLD) become a main public health concern, and is characterized by lipid accumulation in the hepatocytes. We found that overexpression of lncRNA MEG3 significantly reduced the expression of FOXO1, ACC1, and FAS, and subsequently decreased the lipid accumulation in HepG2 cells. Moreover, inhibition of lncRNA MEG3 could increase the lipid accumulation and the mRNA and protein levels of FOXO1, ACC1, and FAS. Further study showed that lncRNA MEG3 regulates the lipogenesis process by inhibiting the entry of FOXO1 into the nucleus translocation. Our study demonstrated that lncRNA MEG3 regulates de novo lipogenesis by decreasing the expression and nucleus translocation of FOXO1 in HepG2 cells, suggesting that lncRNA MEG3 could be a promising therapeutic target in lipid metabolic disorders.

MEG3 rs7158663 genetic polymorphism is associated with the risk of hepatocellular carcinoma

Recently, a meta-analysis has shown that a potentially functional genetic polymorphism (rs7158663 A > G) on the cancer-associated lncRNA MEG3 is associated with the risk of many types of cancer. Given the important role of MEG3 in the development of hepatocellular carcinoma (HCC), the current study evaluated the association of the rs7158663 genetic polymorphism with HCC risk. A total of 271 HCC patients and 267 healthy individuals were included in the current case-control study. Direct sequencing was used to detect the rs7158663 locus genotype of the included individuals. The case-control study showed that the MEG3 rs7158663 genetic polymorphism was associated with the increased risk of developing HCC [GA vs. GG: OR = 1.63, 95% CI = 1.14–2.34, p = 0.009; AA vs. GG: OR = 2.10, 95% CI = 1.10–4.08, p = 0.03; (GA + AA) vs. GG: OR = 1.70, 95% CI = 1.21–2.40, p = 0.003; A vs. G: OR = 1.53, 95% CI = 1.17–2.00, p = 0.002]. In addition, the genotype-tissue expression showed that the rs7158663 AA or GA genotype was associated with reduced MEG3 expression. Bioinformatic analysis showed that the rs7158663 genetic polymorphism not only affects the binding of transcription factors but also interacts with multiple genes through chromatin loops. In summary, the current findings suggest that the rs7158663 genetic polymorphism affecting MEG3 expression is associated with HCC risk and may serve as a marker of genetic susceptibility to HCC. However, the specific molecular mechanisms of the rs7158663 genetic polymorphism in the development of HCC need to be further revealed.

Biophysical Investigation of RNA ⋅ DNA : DNA Triple Helix and RNA : DNA Heteroduplex Formation by the lncRNAs MEG3 and Fendrr.

Long non-coding RNAs (lncRNAs) are important regulators of gene expression and can associate with DNA as RNA : DNA heteroduplexes or RNA ⋅ DNA : DNA triple helix structures. Here, we review in vitro biochemical and biophysical experiments including electromobility shift assays (EMSA), circular dichroism (CD) spectroscopy, thermal melting analysis, microscale thermophoresis (MST), single-molecule Förster resonance energy transfer (smFRET) and nuclear magnetic resonance (NMR) spectroscopy to investigate RNA ⋅ DNA : DNA triple helix and RNA : DNA heteroduplex formation. We present the investigations of the antiparallel triplex-forming lncRNA MEG3 targeting the gene TGFB2 and the parallel triplex-forming lncRNA Fendrr with its target gene Emp2. The thermodynamic properties of these oligonucleotides lead to concentration-dependent heterogeneous mixtures, where a DNA duplex, an RNA : DNA heteroduplex and an RNA ⋅ DNA : DNA triplex coexist and their relative populations are modulated in a temperature-dependent manner. The in vitro data provide a reliable readout of triplex structures, as RNA ⋅ DNA : DNA triplexes show distinct features compared to DNA duplexes and RNA : DNA heteroduplexes. Our experimental results can be used to validate computationally predicted triple helix formation between novel disease-relevant lncRNAs and their DNA target genes.

Transcriptomics analysis of long non-coding RNAs in smooth muscle cells from patients with peripheral artery disease and diabetes mellitus

Diabetes mellitus (DM) is a significant risk factor for peripheral arterial disease (PAD), and PAD is an independent predictor of cardiovascular disorders (CVDs). Growing evidence suggests that long non-coding RNAs (lncRNAs) significantly contribute to disease development and underlying complications, particularly affecting smooth muscle cells (SMCs). So far, no study has focused on transcriptome analysis of lncRNAs in PAD patients with and without DM. Tissue samples were obtained from our Vascular Biobank. Due to the sample’s heterogeneity, expression analysis of lncRNAs in whole tissue detected only ACTA2-AS1 with a 4.9-fold increase in PAD patients with DM. In contrast, transcriptomics of SMCs revealed 28 lncRNAs significantly differentially expressed between PAD with and without DM (FDR < 0.1). Sixteen lncRNAs were of unknown function, six were described in cancer, one connected with macrophages polarisation, and four were associated with CVDs, mainly with SMC function and phenotypic switch (NEAT1, MIR100HG, HIF1A-AS3, and MRI29B2CHG). The enrichment analysis detected additional lncRNAs H19, CARMN, FTX, and MEG3 linked with DM. Our study revealed several lncRNAs in diabetic PAD patients associated with the physiological function of SMCs. These lncRNAs might serve as potential therapeutic targets to improve the function of SMCs within the diseased tissue and, thus, the clinical outcome.

The long non-coding RNA Meg3 mediates imprinted gene expression during stem cell differentiation.

The imprinted Dlk1-Dio3 domain comprises the developmental genes Dlk1 and Rtl1, which are silenced on the maternal chromosome in different cell types. On this parental chromosome, the domain's imprinting control region activates a polycistron that produces the lncRNA Meg3 and many miRNAs (Mirg) and C/D-box snoRNAs (Rian). Although Meg3 lncRNA is nuclear and associates with the maternal chromosome, it is unknown whether it controls gene repression in cis. We created mouse embryonic stem cells (mESCs) that carry an ectopic poly(A) signal, reducing RNA levels along the polycistron, and generated Rian-/- mESCs as well. Upon ESC differentiation, we found that Meg3 lncRNA (but not Rian) is required for Dlk1 repression on the maternal chromosome. Biallelic Meg3 expression acquired through CRISPR-mediated demethylation of the paternal Meg3 promoter led to biallelic Dlk1 repression, and to loss of Rtl1 expression. lncRNA expression also correlated with DNA hypomethylation and CTCF binding at the 5'-side of Meg3. Using Capture Hi-C, we found that this creates a Topologically Associating Domain (TAD) organization that brings Meg3 close to Dlk1 on the maternal chromosome. The requirement of Meg3 for gene repression and TAD structure may explain how aberrant MEG3 expression at the human DLK1-DIO3 locus associates with imprinting disorders.