MEG3 Silencing Research Articles

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.

Multifaceted roles of Meg3 in cellular senescence and atherosclerosis

Background and aimsLong noncoding RNAs are involved in the pathogenesis of atherosclerosis. As long noncoding RNAs maternally expressed gene 3 (Meg3) prevents cellular senescence of hepatic vascular endothelium and obesity-induced insulin resistance, we decided to examine its role in cellular senescence and atherosclerosis. Methods and ResultsBy analyzing our data and human and mouse data from the Gene Expression Omnibus database, we found that Meg3 expression was reduced in humans and mice with cardiovascular disease, indicating its potential role in atherosclerosis. In Ldlr−/− mice fed a Western diet for 12 weeks, Meg3 silencing by chemically modified antisense oligonucleotides attenuated the formation of atherosclerotic lesions by 34.9% and 20.1% in male and female mice, respectively, revealed by en-face Oil Red O staining, which did not correlate with changes in plasma lipid profiles. Real-time quantitative PCR analysis of cellular senescence markers p21 and p16 revealed that Meg3 deficiency aggravates hepatic cellular senescence but not cellular senescence at aortic roots. Human Meg3 transgenic mice were generated to examine the role of Meg3 gain-of-function in the development of atherosclerosis induced by PCSK9 overexpression. Meg3 overexpression promotes atherosclerotic lesion formation by 29.2% in Meg3 knock-in mice independent of its effects on lipid profiles. Meg3 overexpression inhibits hepatic cellular senescence, while it promotes aortic cellular senescence likely by impairing mitochondrial function and delaying cell cycle progression. ConclusionsOur data demonstrate that Meg3 promotes the formation of atherosclerotic lesions independent of its effects on plasma lipid profiles. In addition, Meg3 regulates cellular senescence in a tissue-specific manner during atherosclerosis. Thus, we demonstrated that Meg3 has multifaceted roles in cellular senescence and atherosclerosis.

LncRNA MEG3 suppresses erastin-induced ferroptosis of chondrocytes via regulating miR-885-5p/SLC7A11 axis.

Ferroptosis is involved in osteoarthritis development; however, the roles of long noncoding RNAs (lncRNAs), including lncRNA MEG3, in the regulation of ferroptosis in osteoarthritis are still unclear. In this study, qRT‒PCR and Western blotting assays were used to detect the expression of lncRNA MEG3, miR-885-5p, SLC7A11 and GPX4; MDA and CCK-8 assays were applied to analyse cellular MDA levels and cell viability, respectively. Erastin elevated cellular MDA levels and decreased the viability of chondrocytes and the erastin-induced decline in cell viability was reversed by a ferroptosis inhibitor (ferrostatin-1). Erastin downregulated lncRNA MEG3, SLC7A11 and GPX4 and upregulated miR-885-5p. Silencing of lncRNA MEG3 increased miR-885-5p and downregulated SLC7A11 and GPX4 and further sensitized chondrocytes to erastin-induced ferroptosis. In contrast, overexpression of lncRNA MEG3 had opposite effects. Dual luciferase assays confirmed binding between lncRNA MEG3 and miR-885-5p and between miR-885-5p and the 3'UTR of SLC7A11. In the synovial fluids from patients with osteoarthritis compared with synovial fluids from normal controls, the RNA levels of lncRNA MEG3 and SLC7A11 were decreased and the miR-885-5p expression level was increased. Our findings indicated that lncRNA MEG3 overexpression alleviated ferroptosis in chondrocytes by affecting the miR-885-5p/SLC7A11 signalling pathway.

Silencing of the MEG3 gene promoted anti-cancer activity and drug sensitivity in glioma.

Aberrant expression of MEG3 has been shown in various cancers. The purpose of this study is to evaluate the effect of MEG3 on glioma cells and the use of potential chemotherapeutics in glioma by modulating MEG3 expression. Cell viability, migration and chemosensitivity were assayed. Cell death was evaluated in MEG3 overexpressing and MEG3 suppressed cells. MEG3 expression was compared in patient-derived glioma cells concerning IDH1 mutation and WHO grades. Silencing of MEG3 inhibited cell proliferation and reduced cell migration while overexpression of MEG3 promoted proliferation in glioma cells. MEG3 inhibition improved the chemosensitivity of glioma cells to 5-fluorouracil (5FU) but not to navitoclax. On the other hand, there is no significant effect of MEG3 expression on temozolamide (TMZ) treatment which is a standard chemotherapeutic agent in glioma. Suppression of the MEG3 gene in patient-derived oligodendroglioma cells also showed the same effect whereas glioblastoma cell proliferation and chemosensitivity were not affected by MEG3 inhibition. Further, as a possible cell death mechanism of action apoptosis was investigated. Although MEG3 is a widely known tumour suppressor gene and its loss is associated with several cancer types, here we reported that MEG3 inhibition can be used for improving the efficiency of known chemotherapeutic drug sensitivity. We propose that the level of MEG3 should be evaluated in the treatment of different glioma subtypes that are resistant to effective drugs to increase the potential effective drug applications.

Acupuncture regulates the apoptosis of ovarian granulosa cells in polycystic ovarian syndrome-related abnormal follicular development through LncMEG3-mediated inhibition of miR-21-3p

BackgroundThe main features of polycystic ovary syndrome (PCOS) are abnormal follicular development and ovulatory dysfunction, which are caused by excessive apoptosis of ovarian granulosa cells. Acupuncture has been shown to improve follicular development abnormalities in patients with PCOS, but its mechanism is unknown. This study hypothesized that the mechanism of acupuncture on follicular development abnormalities in PCOS patients is the inhibition of granulosa cell apoptosis through LncMEG3-mediated regulation of miR-21-3p.MethodsA PCOS-like rat model was established using subcutaneous injection of dehydroepiandrosterone (DHEA). Acupuncture was performed on rats for 15 d (CV-4, RN-3, CV-6, SP-6 and EX-CA 1). Ovarian morphology was observed by HE staining, and sex hormone and AMH levels were detected by ELISA. Primary granulosa cells were isolated from each group of rats to assess the association of acupuncture treatment, LncMEG3, miR-21-3p, and granulosa cell apoptosis in rats with PCOS.ResultsLncMEG3 and miR-21-3p were highly expressed in the ovarian granulosa cells of rats with PCOS, and LncMEG3-mediated regulation of miR-21-3p was involved in the development of PCOS in rats. Silencing of MEG3 attenuated sex hormone dysregulation and ovarian histopathological changes in PCOS rats and promoted follicle cell development and maturation. In addition, silencing MEG3 increased the viability and number of granulosa cells. In addition, silencing MEG3 further inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats. Acupuncture improved polycystic ovarian morphology and sex hormone levels in PCOS rats. Acupuncture intervention increased the viability and number of granulosa cells. Acupuncture intervention inhibited early and late apoptosis of ovarian granulosa cells in PCOS rats by targeting miR-21-3p via LncMEG3.ConclusionThese results suggest that acupuncture can downregulate LncMEG3, thereby targeting and regulating miR-21-3p to suppress early and late granulosa cell apoptosis and normalize their proliferation. These factors ultimately compensate for abnormal follicular development. These findings shed light on the clinical potential of acupuncture as a safe treatment for follicular developmental abnormalities in PCOS.

The long noncoding RNA Meg3 mediates TLR4-induced inflammation in experimental obstructive nephropathy.

Kidney inflammation contributes to the progression of chronic kidney disease (CKD). Modulation of Toll-like receptor 4 (TLR4) signaling is a potential therapeutic strategy for this pathology, but the regulatory mechanisms of TLR4 signaling in kidney tubular inflammation remains unclear. Here, we demonstrated that tubule-specific deletion of TLR4 in mice conferred protection against obstruction-induced kidney injury, with reduction in inflammatory cytokine production, macrophage infiltration and kidney fibrosis. Transcriptome analysis revealed a marked down-regulation of long noncoding RNA (lncRNA) Meg3 in the obstructed kidney from tubule-specific TLR4 knockout mice compared with wild-type control. Meg3 was also induced by lipopolysaccharide in tubular epithelial cells via a p53-dependent signaling pathway. Silencing of Meg3 suppressed LPS-induced cytokine production of CCL-2 and CXCL-2 and the activation of p38 MAPK pathway in vitro and ameliorated kidney fibrosis in mice with obstructive nephropathy. Together, these findings identify a proinflammatory role of lncRNA Meg3 in CKD and suggest a novel regulatory pathway in TLR4-driven inflammatory responses in tubular epithelial cells.

Retraction.

Journal of Cellular and Molecular MedicineVolume 27, Issue 2 p. 309-309 RETRACTIONOpen Access Retraction This article retracts the following: Retracted: Long non-coding RNA MEG3 silencing and microRNA-214 restoration elevate osteoprotegerin expression to ameliorate osteoporosis by limiting TXNIP ChangSheng Yang, ZhengTao Gu, Rui Ding, CaiQiang Huang, QingChu Li, DengHui Xie, RongKai Zhang, YiYan Qiu, Volume 25Issue 4Journal of Cellular and Molecular Medicine pages: 2025-2039 First Published online: January 3, 2021 First published: 02 November 2022 https://doi.org/10.1111/jcmm.17601AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Retraction: Yang, C, Gu, Z, Ding, R, et al. Long non-coding RNA MEG3 silencing and microRNA-214 restoration elevate osteoprotegerin expression to ameliorate osteoporosis by limiting TXNIP. J Cell Mol Med. 2021; 25: 2025–2039. doi:10.1111/jcmm.16096. The above article published online on 03 January 2021 in Wiley Online Library (wileyonlinelibrary.com) has been retracted by agreement between the authors, the journal Editor in Chief, and John Wiley and Sons Ltd. The retraction has been agreed following an investigation based on concerns raised by the authors and allegations raised by a third party. Several flaws and inconsistencies between results presented and experimental methods described were found, the editors consider the conclusions of this article to be invalid. REFERENCES 1Yang C, Gu Z, Ding R, et al. Long non-coding RNA MEG3 silencing and microRNA-214 restoration elevate osteoprotegerin expression to ameliorate osteoporosis by limiting TXNIP. J Cell Mol Med. 2021; 25: 2025– 2039. https://doi.org/10.1111/jcmm.16096. Volume27, Issue2January 2023Pages 309-309 ReferencesRelatedInformation

LncRNA Meg3 promotes oxygen and glucose deprivation injury by decreasing angiogenesis in hBMECs by targeting the miR‑122‑5p/NDRG3 axis.

Oxygen-glucose deprivation (OGD) is widely used as an in vitro model for stroke. The present study aimed to explore the mechanisms of action of long non-coding RNA (lncRNA) maternally expressed gene 3 (Meg3) in angiogenesis following OGD. The human brain microvascular endothelial cell line, hCMEC/D3, was used to establish the OGD model. lncRNA Meg3 was highly expressed in hCMEC/D3 cells subjected to OGD. Furthermore, it was found that the overexpression of lncRNA Meg3 decreased the proliferation, migration and angiogenesis of hCMEC/D3 cells subjected to OGD, and increased cell apoptosis. Meg3 silencing exerted the opposite effects. Subsequently, lncRNA Meg3 increased the expression of NDRG family member 3 (NDRG3) by directly binding to miR-122-5p. The overexpression of miR-122-5p and the knockdown of NDRG3 reversed the inhibitory effects of Meg3 overexpression on the proliferation, migration and angiogenesis of hCMEC/D3 cells subjected to OGD, as well as the promoting effects of Meg3 overexpression on cell apoptosis. The present study demonstrated that lncRNA Meg3 functions as a competing endogenous RNA by targeting the miR-122-5p/NDRG3 axis in regulating OGD injury.

Downregulation of MEG3 and upregulation of EZH2 cooperatively promote neuroblastoma progression.

Neuroblastoma (NB), an embryonic tumour originating from sympathetic crest cells, is the most common extracranial solid tumour type in children with poor overall prognosis. Accumulating evidence has demonstrated the involvement of long non‐coding RNA (lncRNA) in numerous biological processes and their associations with embryonic development and multiple diseases. Ectopic lncRNA expression is linked to malignant tumours. Previous studies by our team indicate that MEG3 attenuates NB autophagy through inhibition of FOXO1 and epithelial‐mesenchymal transition via the mTOR pathway in vitro. Moreover, MEG3 and EZH2 negatively regulate each other. In present study, we first collected 60 NB tissues and 20 adjacent tissues for Quantitative real‐time polymerase chain reaction (Q‐PCR) experiments and performed clinical correlation analysis of the results. At the same time, nude mice were used for subcutaneous tumour formation to detect the effect of MEG3 in vivo. Two NB cell lines, SK‐N‐AS and SK‐N‐BE(2)C, were overexpressed MEG3 and rescued with EZH2 and then were subjected to proliferation, migration, invasion, apoptosis and autophagy experiments. RNA‐binding protein immunoprecipitation (RIP) and Co‐Immunoprecipitation (Co‐IP) experiments were performed to explore the molecular mechanism of MEG3 and EZH2 interaction. Q‐PCR revealed that MEG3 expression was negatively correlated with INSS stage and risk grade of NB. Moreover, MEG3 overexpression was associated with inhibition of NB growth in vivo. MEG3 exerted an anti‐cancer effect via stimulatory effects on EZH2 ubiquitination leading to its degradation. Conversely, EZH2 interacted with DNMT1 and HDAC1 to induce silencing of MEG3. The EZH2 inhibitor, DZNep, and HDAC inhibitor, SAHA, displayed synergistic activity against NB. Combined treatment with DZNep and SAHA inhibited proliferation, migration and invasion of NB through suppression of the PI3K/AKT/mTOR/FOXO1 pathway. In conclusion, downregulation of MEG3 and upregulation of EZH2 forms a feedback loop that concertedly promotes the development of NB. Combined blockage of EZH2 and HDAC1 with the appropriate inhibitors may therefore present an effective treatment strategy for NB cases with low MEG3 and high EZH2 expression.

Se alleviates homocysteine-induced fibrosis in cardiac fibroblasts via downregulation of lncRNA MEG3.

Selenium (Se) is considered to have antioxidant properties, which are beneficial for heart condition. Hyperhomocysteinemia (HHCY) has been suggested to potentially lead to heart failure and is characterized by cardiac fibrosis; however, investigation on the role of Se and HHCY in cardiac fibrosis is rare. Since previous studies demonstrated the important role of the long non-coding RNA maternally expressed 3 (MEG3) in some heart diseases, the present study aimed to determine how Se and MEG3 might exert regulatory effects on HCY-induced fibrosis in cardiac fibroblasts (CFs). Mouse CFs were isolated and treated with HCY and Se. The expression of α-smooth muscle actin (α-SMA), collagen I and III was detected by western blotting to reflect CF fibrosis. Reverse transcription-quantitative PCR was performed to determine the expression levels of MEG3. Inflammation and oxidative stress responses were analyzed by measuring TNF-α, IL-1β (ELISA) and reactive oxygen species levels (using a commercial kit), respectively. Cell Counting Kit-8 was used to evaluate CF proliferation. Total and phosphorylated (p) expression of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) was evaluated by western blotting. CFs were transfected with adenovirus expressing MEG3 short-hairpin RNA to knock down MEG3 expression. Se treatment downregulated the expression level of MEG3 in HCY-stimulated CFs, whilst inhibiting the inflammatory and oxidative stress response. Furthermore, Se inhibited the increased proliferation of CFs following HCY treatment. In addition, MEG3-knockdown in CFs could improve fibrosis caused by HCY. Furthermore, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 were decreased following treatment with Se or MEG3 silencing. Taken together, the findings from the present study suggested that Se may alleviate cardiac fibrosis by downregulating the expression of MEG3 and reducing the inflammatory and oxidative stress response in CFs. This suggests that Se may be a potential therapeutic option for treating cardiac fibrosis in the future.

Long non-coding RNA MEG3 silencing and microRNA-214 restoration elevate osteoprotegerin expression to ameliorate osteoporosis by limiting TXNIP.

Studies have shown that long non‐coding RNA (lncRNA) MEG3 plays a key role in osteoporosis (OP), but its regulatory mechanism is somewhat incompletely clear. Here, we intend to probe into the mechanism of MEG3 on OP development by modulating microRNA‐214 (miR‐214) and thioredoxin‐interacting protein (TXNIP). Rat models of OP were established. MEG3, miR‐214 and TXNIP mRNA expression in rat femoral tissues were detected, along with TXNIP, OPG and RANKL protein expression. BMD, BV/TV, Tb.N and Tb.Th in tissue samples were measured. Ca, P and ALP contents in rat serum were also determined. Primary osteoblasts were isolated and cultured. Viability, COL‐I, COL‐II and COL‐Χ mRNA expression, PCNA, cyclin D1, OCN, RUNX2 and osteolix protein expresion, ALP content and activity, and mineralized nodule area of rat osteoblasts were further detected. Dual‐luciferase reporter gene and RNA‐pull down assays verified the targeting relationship between MEG3, miR‐214 and TXNIP. MEG3 and TXNIP were up‐regulated while miR‐214 was down‐regulated in femoral tissues of OP rats. MEG3 silencing and miR‐214 overexpression increased BMD, BV/TV, Tb.N, Tb.Th, trabecular bone area, collagen area and OPG expression, and down‐regulated RANKL of femoral tissues in OP rats. MEG3 silencing and miR‐214 overexpression elevated Ca and P and reduced ALP in OP rat serum, elevated osteoblast viability, differentiation ability, COL‐I and COL‐Χ expression and ALP activity, and reduced COL‐II expression of osteoblasts. MEG3 specifically bound to miR‐214 to regulate TXNIP. MEG3 silencing and miR‐214 overexpression promote proliferation and differentiation of osteoblasts in OP by down‐regulating TXNIP, which further improves OP.

Long non-coding RNA MEG3 inhibits M2 macrophage polarization by activating TRAF6 via microRNA-223 down-regulation in viral myocarditis.

Viral myocarditis (VMC) commonly triggers heart failure, for which no specific treatments are available. This study aims to explore the specific role of long non‐coding RNA (lncRNA) maternally expressed 3 (MEG3) in VMC. A VMC mouse model was induced by Coxsackievirus B3 (CVB3). Then, MEG3 and TNF receptor‐associated factor 6 (TRAF6) were silenced and microRNA‐223 (miR‐223) was over‐expressed in the VMC mice, followed by determination of ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS). Dual‐luciferase reporter assay was introduced to test the interaction among MEG3, TRAF6 and miR‐223. Macrophages were isolated from cardiac tissues and bone marrow, and polarization of M1 or M2 macrophages was induced. Then, the expressions of components of NLRP3 inflammatory body (NLRP3, ASC, Caspase‐1), M1 markers (CD86, iNOS and TNF‐α) and M2 markers (CD206, Arginase‐1 and Fizz‐1) were measured following MEG3 silencing. In the VMC mouse model, MEG3 and TRAF6 levels were obviously increased, while miR‐223 expression was significantly reduced. Down‐regulation of MEG3 resulted in the inhibition of TRAF6 by promoting miR‐223. TRAF6 was negatively correlated with miR‐223, but positively correlated with MEG3 expression. Down‐regulations of MEG3 or TRAF6 or up‐regulation of miR‐223 was observed to increase mouse weight, survival rate, LVEF and LVFS, while inhibiting myocarditis and inflammation via the NF‐κB pathway inactivation in VMC mice. Down‐regulation of MEG3 decreased M1 macrophage polarization and elevated M2 macrophage polarization by up‐regulating miR‐223. Collectively, down‐regulation of MEG3 leads to the inhibition of inflammation and induces M2 macrophage polarization via miR‐223/TRAF6/NF‐κB axis, thus alleviating VMC.

Competing endogenous network analysis identifies lncRNA Meg3 activates inflammatory damage in UVB induced murine skin lesion by sponging miR-93-5p/epiregulin axis.

In this study, we obtained the RNA expression data of murine skin tissues of control, and UVB irradiated groups. After the re-annotation of lncRNAs, a gene expression similarity analysis was done by WGCNA. The target mRNA prediction of lncRNAs, miRNAs, and ceRNA regulatory networks were constructed by five lncRNAs, 14 miRNAs and 54 mRNAs, respectively. Based on the ceRNA network of UVB-induced skin lesions, it was evident that the dysregulation of Meg3 has critical effects on the UVB-induced inflammatory lesion of murine skin tissues. The overexpression of Meg3 after UVB irradiation was observed in primary murine skin fibroblasts, and the up-regulated Meg3 expression was related to the activation of the inflammatory cytokines. These functional experiments demonstrated that the RNA silencing of Meg3 in murine skin fibroblasts could suppress the expression of the cytokines (in vitro) and UVB-induced skin lesions (in vivo). Moreover, the Meg3 functioned as a competing endogenous RNA (ceRNA) that acted as a sponge for miR-93-5p and thereby modulated the expression of Epiregulin (Ereg). Our results proved that Meg3 was involved in UVB-induced skin inflammation and that the ceRNA networks, which includes miR-93-5p and Ereg, could prove to be a potential therapeutic target for UVB-induced skin damage.

High lncRNA MEG3 expression is associated with high mortality rates in patients with sepsis and increased lipopolysaccharide-induced renal epithelial cell and cardiomyocyte apoptosis.

Long non-coding RNA (lncRNA) maternally expressed 3 (MEG3) has been reported to be a key component in cancer biology. Preliminary microarray data from a previous study indicated that lncRNA MEG3 expression was altered in plasma of patients with sepsis. The present study found that the plasma levels of lncRNA MEG3 were significantly higher in sepsis patients compared with healthy controls. Patients were subsequently divided into high and low lncRNA MEG3 expression groups according to the Youden's index. Patients from the high lncRNA MEG3 expression group exhibited a significantly higher mortality rate compared with those from the low lncRNA MEG3 expression group. According to treatment outcomes, patients were divided into survival and mortality groups. It was observed that the pre-therapy expression levels of lncRNA MEG3 were significantly higher in mortality group compared with the survival group. Cell experiments in vitro revealed that lncRNA MEG3 overexpression and silencing using siRNA promoted and inhibited renal epithelial cell and cardiomyocyte apoptosis induced by lipopolysaccharide, respectively. In conclusion, these data suggest that lncRNA MEG3 overexpression may be involved in sepsis, and the downregulation of lncRNA MEG3 may serve as a potential therapeutic target for sepsis.

Silencing long non-coding RNA MEG3 accelerates tibia fraction healing by regulating the Wnt/β-catenin signalling pathway.

As fracture healing is related to gene expression, fracture healing is prospected to be implicated in long non‐coding RNAs (lncRNAs). This study focuses on the effects of epigenetic silencing of long non‐coding RNA maternally expressed gene 3 (lncRNA MEG3) on fracture healing by regulating the Wnt/β‐catenin signalling pathway. Genes expressed in fracture were screened using bioinformatics and the subcellular location of MEG3 was determined using FISH. Next, we successfully established tibia fracture (TF) models of C57BL/6J and Col2a1‐ICAT mice and the effect of silencing lncRNA MEG3 on fracture healing was detected after TF mice were treated with phosphate buffer saline (PBS), MEG3 siRNA and scramble siRNA. X‐ray imaging, Safranin‐O/fast green and haematoxylin‐eosin (HE) staining and histomorphometrical and biomechanical analysis were adopted to observe and to detect the fracture healing conditions. Additionally, the positive expression of collagen II and osteocalcin was examined using immunohistochemistry. At last, in the in vitro experiment, the relationship of MEG3 and the Wnt/β‐catenin signalling pathway in fraction healing was investigated. MEG3 was located in the cell nucleus. In addition, it was found that MEG3 and the Wnt/β‐catenin signalling pathway were associated with fraction healing. Moreover, silencing MEG3 was proved to elevate callus area and maximum bending load and to furthermore enhance the recanalization of bone marrow cavity. Finally, MEG3 knockdown elevated levels of Col10a1, Runx2, Osterix, Osteocalcin, Wnt10b and β‐catenin/β‐catenin whereas it reduced p‐GSK‐3β/GSK‐3β levels. Taken together, our data supported that epigenetic silencing of lncRNA MEG3 could promote the tibia fracture healing by activating the Wnt/β‐catenin signalling pathway.

Silencing of MEG3 inhibited ox-LDL-induced inflammation and apoptosis in macrophages via modulation of the MEG3/miR-204/CDKN2A regulatory axis.

Atherosclerosis (AS) is one of the most common chronic inflammatory diseases and a major cause of morbidity and mortality. However, the underlying molecular mechanisms of the progression of AS are still largely unknown. Increasing evidence has demonstrated that long noncoding RNAs (lncRNAs) play important roles in a variety of biological processes and the physiological and pathological progression of human diseases. In this study, we aimed to explore the role and underlying molecular mechanism of lncRNA MEG3 in Raw264.7 cells treated with oxidized low-density lipoprotein (ox-LDL). First, we found that ox-LDL inhibited the cell viability and proliferation, increased TNFα and IL1β secretion and induced the apoptosis of Raw264.7 cells. Second, we demonstrated that ox-LDL upregulated MEG3 expression and that knockdown of MEG3 inhibited the action of ox-LDL in Raw264.7 cells. Third, we showed that MEG3 sponged miR-204 in Raw264.7 cells treated with ox-LDL. Fourth, we demonstrated that miR-204 regulated the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A) in Raw264.7 cells treated with ox-LDL. Finally, we revealed that MEG3 exerted its function via the regulation of the MEG3/miR-204/CDKN2A regulatory axis in Raw264.7 cells treated with ox-LDL. In summary, our study identified the role of the MEG3/miR-204/CDKN2A pathway in Raw264.7 cells treated with ox-LDL, revealed a novel regulatory pathway in AS and indicated potential novel characteristic biomarkers and therapeutic targets for AS.

Allele-specific RNA-seq expression profiling of imprinted genes in mouse isogenic pluripotent states

BackgroundGenomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA).ResultsAs homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin.ConclusionsAltogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential.

Silencing of long noncoding RNA MEG3 enhances cerebral protection of dexmedetomidine against hypoxic-ischemic brain damage in neonatal mice by binding to miR-129-5p.

Hypoxic-ischemic brain damage (HIBD) is a leading cause of neonatal acute mortality and chronic nervous system injury. Recently, it has been found that long noncoding RNAs (lncRNAs) play a significant role in the neurodevelopment and etiopathogenesis of HIBD. Here, the researchers aimed to determine the role of lncRNA maternally expressed gene (MEG3) in the therapeutic effect of dexmedetomidine (DEX) in neonatal mice with HIBD through the regulation of microRNA-129-5p (miR-129-5p). HIBD models were established in C57/BL6 neonatal mice. Subsequently, the target relationship between MEG3 and miR-129-5p was predicted and verified. The neonatal mice were injected with DEX, ad-shMEG3, and mimics and inhibitors of miR-129-5p to identify roles of MEG3 and miR-129-5p in therapeutic effects of DEX on neuronal apoptosis and injury, cerebral atrophy, and learning and memory ability of neonatal mice with HIBD. MEG3 directly targeted and inhibited the expression of miR-129-5p. Silencing of MEG3 or upregulation of miR-129-5p effectively promoted the therapeutic effect of DEX on neonatal mice with HIBD. Silencing of MEG3 or upregulation of miR-129-5p reduced the neuronal apoptosis rate and degree of cerebral atrophy, and also enhanced the learning and memory ability of HIBD neonatal mice. Collectively, the key findings obtained from the present study support the notion that MEG3 silencing enhances the therapeutic effect of DEX on neonatal mice with HIBD by binding to miR-129-5p.

LncRNA Meg3 protects endothelial function by regulating the DNA damage response.

The role of long non-coding RNAs (lncRNAs) in regulating endothelial function through the DNA damage response (DDR) remains poorly understood. In this study, we demonstrate that lncRNA maternally expressed gene 3 (Meg3) interacts with the RNA binding protein polypyrimidine tract binding protein 3 (PTBP3) to regulate gene expression and endothelial function through p53 signaling ─ a major coordinator of apoptosis and cell proliferation triggered by the DDR. Meg3 expression is induced in endothelial cells (ECs) upon p53 activation. Meg3 silencing induces DNA damage, activates p53 signaling, increases the expression of p53 target genes, promotes EC apoptosis, and inhibits EC proliferation. Mechanistically, Meg3 silencing reduces the interaction of p53 with Mdm2, induces p53 expression, and promotes the association of p53 with the promoters of a subset of p53 target genes. PTBP3 silencing recapitulates the effects of Meg3 deficiency on the expression of p53 target genes, EC apoptosis and proliferation. The Meg3-dependent association of PTBP3 with the promoters of p53 target genes suggests that Meg3 and PTBP3 restrain p53 activation. Our studies reveal a novel role of Meg3 and PTBP3 in regulating p53 signaling and endothelial function, which may serve as novel targets for therapies to restore endothelial homeostasis.

Long non-coding RNA MEG3 silencing protects against light-induced retinal degeneration

Excessive light exposure leads to retinal degeneration and accelerates the progression and severity of several ocular diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa. Long non-coding RNAs (LncRNAs) have emerged as important regulators of photoreceptor development and ocular diseases. In this study, we investigated the role of lncRNA-MEG3 in light-induced retinal degeneration. MEG3 expression was significantly up-regulated after light insult in vivo and in vitro. MEG3 silencing protected against light-induced retinal degeneration in vivo and light-induced photoreceptor cell apoptosis in vitro. Mechanistically, MEG3 regulated retinal photoreceptor cell function by acting as p53 decoy. MEG3 silencing decreased caspase 3/7 activity, up-regulated anti-apoptotic protein (Bcl-2) expression, and down-regulated pro-apoptotic protein (Bax) expression. Taken together, this study provides a promising method of MEG3 silencing for treating light-induced retinal degeneration.