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.

LncRNA MEG3 regulates ferroptosis of lens epithelial cells via PTBP1/GPX4 axis to participate in age-related cataract.

Age-related cataract (ARC) is regarded as the principal cause of vision impairment among the aged. The regulatory role of long noncoding RNAs (LncRNAs) in ARC remains unclear. The lncRNA maternally expressed gene 3 (MEG3) has been reported to promote ARC progression, and the underlying mechanism was further investigated in this study. Lens epithelium samples were collected to verify the expression of MEG3. Lens epithelial cells (LECs) were treated with H2O2 to mimic microenvironment of ARC in vitro. Cell viability, reactive oxygen species, and ferroptosis were evaluated during the in viro experiments. In the present work, lncRNA MEG3 was highly expressed in ARC group, compared with normal group. MEG3 was induced, cell viability and glutathione peroxidase 4 (GPX4) level were inhibited, and ferroptosis was promoted in H2O2 treated LECs. LncRNA MEG3 silence reversed the effects of H2O2 on viability and ferroptosis in LECs. Thereafter, lncRNA MEG3 was found to bind to PTBP1 for GPX4 degradation. Silencing of GPX4 reversed the regulation of lncRNA MEG3 inhibition in H2O2-treated LECs. To sum up, lncRNA MEG3 exhibited high expression in ARC. In H2O2-induced LECs, inhibition of lncRNA MEG3 accelerated cell viability and repressed ferroptosis by interaction with PTBP1 for GPX4 messenger RNA decay. Targeting lncRNA MEG3 may be a novel treatment of ARC.

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

BACKGROUND: Asthma is a respiratory disease characterized by airway remodeling. We aimed to find out the role and mechanism of lncRNA MEG3 in asthma. METHODS: We 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. RESULTS: The 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. CONCLUSION: LncRNA 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.

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.

Cigarette Smoke Extract-Treated Mouse Airway Epithelial Cells-Derived Exosomal LncRNA MEG3 Promotes M1 Macrophage Polarization and Pyroptosis in Chronic Obstructive Pulmonary Disease by Upregulating TREM-1 via m6A Methylation.

Cigarette smoke extract (CSE)-treated mouse airway epithelial cells (MAECs)-derived exosomes accelerate the progression of chronic obstructive pulmonary disease (COPD) by upregulating triggering receptor expressed on myeloid cells 1 (TREM-1); however, the specific mechanism remains unclear. We aimed to explore the potential mechanisms of CSE-treated MAECs-derived exosomes on M1 macrophage polarization and pyroptosis in COPD. In vitro, exosomes were extracted from CSE-treated MAECs, followed by co-culture with macrophages. In vivo, mice exposed to cigarette smoke (CS) to induce COPD, followed by injection or/and intranasal instillation with oe-TREM-1 lentivirus. Lung function and pathological changes were evaluated. CD68+ cell number and the levels of iNOS, TNF-α, IL-1ß (M1 macrophage marker), and pyroptosis-related proteins (NOD-like receptor family pyrin domain containing 3, apoptosis-associated speck-like protein containing a caspase-1 recruitment domain, caspase-1, cleaved-caspase-1, gasdermin D [GSDMD], and GSDMD-N) were examined. The expression of maternally expressed gene 3 (MEG3), spleen focus forming virus proviral integration oncogene (SPI1), methyltransferase 3 (METTL3), and TREM-1 was detected and the binding relationships among them were verified. MEG3 increased N6-methyladenosine methylation of TREM-1 by recruiting SPI1 to activate METTL3. Overexpression of TREM-1 or METTL3 negated the alleviative effects of MEG3 inhibition on M1 polarization and pyroptosis. In mice exposed to CS, EXO-CSE further aggravated lung injury, M1 polarization, and pyroptosis, which were reversed by MEG3 inhibition. TREM-1 overexpression negated the palliative effects of MEG3 inhibition on COPD mouse lung injury. Collectively, CSE-treated MAECs-derived exosomal long non-coding RNA MEG3 may expedite M1 macrophage polarization and pyroptosis in COPD via the SPI1/METTL3/TREM-1 axis.

RhoGDIß inhibition via miR-200c/AUF1/SOX2/miR-137 axis contributed to lncRNA MEG3 downregulation-mediated malignant transformation of human bronchial epithelial cells.

Nickel pollution is a recognized factor contributing to lung cancer. Understanding the molecular mechanisms of its carcinogenic effects is crucial for lung cancer prevention and treatment. Our previous research identified the downregulation of a long noncoding RNA, maternally expressed gene 3 (MEG3), as a key factor in transforming human bronchial epithelial cells (HBECs) into malignant cells following nickel exposure. In our study, we found that deletion of MEG3 also reduced the expression of RhoGDIß. Notably, artificially increasing RhoGDIß levels counteracted the malignant transformation caused by MEG3 deletion in HBECs. This indicates that the reduction in RhoGDIß contributes to the transformation of HBECs due to MEG3 deletion. Further exploration revealed that MEG3 downregulation led to enhanced c-Jun activity, which in turn promoted miR-200c transcription. High levels of miR-200c subsequently increased the translation of AUF1 protein, stabilizing SOX2 messenger RNA (mRNA). This stabilization affected the regulation of miR-137, SP-1 protein translation, and the suppression of RhoGDIß mRNA transcription and protein expression, leading to cell transformation. Our study underscores the co-regulation of RhoGDIß expression by long noncoding RNA MEG3, multiple microRNAs (miR-200c and miR-137), and RNA-regulated transcription factors (c-Jun, SOX2, and SP1). This intricate network of molecular events sheds light on the nature of lung tumorigenesis. These novel findings pave the way for developing targeted strategies for the prevention and treatment of human lung cancer based on the MEG3/RhoGDIß pathway.

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

BACKGROUND: 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. METHODS: 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. RESULT: 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. CONCLUSION: Our findings indicated that lncRNA MEG3 overexpression alleviated ferroptosis in chondrocytes by affecting the miR-885-5p/SLC7A11 signalling pathway.

[Overexpression of LncRNA MEG3 promotes ferroptosis and enhances chemotherapy sensitivity of hepatocellular carcinoma cells to cisplatin].

OBJECTIVE: To investigate the effect of overexpression of LncRNA MEG3 on proliferation, migration and cisplatin sensitivity of hepatoma cells HepG2 and LM3 and explore the underlying and mechanism. METHODS: The expression of MEG3 in healthy individuals and patients with hepatocellular carcinoma (HCC) was analyzed by online bioinformatics analysis, and Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect MEG3 expression in different HCC cell lines. A MEG3-overexpresing plasmid was transfected in HepG2 and LM3 cells, and the changes in cell proliferation and migration were examined using CCK8 assay and scratch assay. CCK8 assay was used to determine the inhibitory rate of cisplatin on the transfected cells. A reactive oxygen species (ROS) fluorescence probe (DCFH-DA) and malondialdehyde (MDA) kit were used to assess the changes in ROS production and MDA level in the cells. Western blotting was performed to detect the expression levels of ferroptosis-related proteins glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1). RESULTS: The expression of MEG3 was significantly lower in HCC cells than in LO2 cells, which was consistent with the results of bioinformatic analysis (P < 0.05). Overexpression of MEG3 in the HCC cell lines significantly suppressed cell proliferation and migration (P < 0.05), increased the cell inhibition rate of cisplatin (P < 0.05), enhanced cellular ROS production and increased MDA levels in the cells (P < 0.05). MEG3 overexpression significantly decreased the expressions of GPX4 and FTH1 in the HCC cell lines. CONCLUSION: The expression of MEG3 is decreased in HCC cells, and its overexpression inhibits proliferation and migration and enhances cisplatin sensitivity of HCC cells by promoting ferroptosis of the cells.

Altered expression of long noncoding RNA MEG3 in the offspring of gestational diabetes mellitus induces impaired glucose tolerance in adulthood.

AIM: Gestational diabetes mellitus (GDM) affects a significant number of women worldwide and has been associated with lifelong health consequences for their offspring, including increased susceptibility to obesity, insulin resistance, and type II diabetes. Recent studies have suggested that aberrant expression of the long non-coding RNA Meg3 in the liver may contribute to impaired glucose metabolism in individuals. In this study, we aimed to investigate whether intrauterine exposure to hyperglycemia affects glucose intolerance in puberty by mediating the overexpression of LncMeg3 in the liver. METHODS: To test our hypothesis, we established an animal model of intrauterine hyperglycemia to mimic GDM. The progeny was observed for phenotypic changes, and intraperitoneal glucose tolerance tests, insulin tolerance tests, and pyruvate tolerance tests were conducted to assess glucose and insulin tolerance. We also measured LncMeg3 expression in the liver using real-time quantitative PCR and examined differential methylation areas (DMRs) in the Meg3 gene using pyrophosphoric sequencing. To investigate the role of LncMeg3 in glucose tolerance, we conducted Meg3 intervention by vein tail and analyzed the changes in the phenotype and transcriptome of the progeny using bioinformatics analysis. RESULTS: We found that intrauterine exposure to hyperglycemia led to impaired glucose and insulin tolerance in the progeny, with a tendency toward increased fasting blood glucose in fat offspring at 16 weeks (P = 0.0004). LncMeg3 expression was significantly upregulated (P = 0.0061), DNMT3B expression downregulated (P = 0.0226), and DNMT3A (P = 0.0026), TET2 (P = 0.0180) expression upregulated in the liver. Pyrophosphoric sequencing showed hypomethylation in Meg3-DMRs (P = 0.0005). Meg3 intervention by vein tail led to a decrease in the percentage of obese and emaciated offspring (emaciation: 44% vs. 23%; obesity: 25% vs. 15%) and attenuated glucose intolerance. Bioinformatics analysis revealed significant differences in the transcriptome of the progeny, particularly in circadian rhythm and PPAR signaling pathways. CONCLUSION: In conclusion, our study suggests that hypomethylation of Meg3-DMRs increases the expression of the imprinted gene Meg3 in the liver of males, which is associated with impaired glucose tolerance in GDM-F1. MEG3 interference may attenuate glucose intolerance, which may be related to transcriptional changes. Our findings provide new insights into the mechanisms underlying the long-term effects of intrauterine hyperglycemia on progeny health and highlight the potential of Meg3 as an intervention target for glucose intolerance.

Niraparib restrains prostate cancer cell proliferation and metastasis and tumor growth in mice by regulating the lncRNA MEG3/miR-181-5p/GATA6 pathway.

Background: Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi), have gained approval for treating patients with castration-resistant prostate cancer (CRPC). Maternally expressed gene 3 (MEG3), a long non-coding RNA (lncRNA), plays a role in inhibiting tumorigenesis through regulating DNA repair genes. This study aimed to investigate the association between the anti-prostate cancer (PCa) effect of niraparib, a representative PARPi, and MEG3 expression, as well as explore the downstream pathway involved. Methods: The levels of MEG3, miR-181-5p, GATA binding protein 6 (GATA6) in clinical samples from PCa patients were accessed by RT-qPCR. PC3 cells were treated with niraparib, and the expression of MEG3, miR-181-5p, GATA6 expression was tested. PC3 cell proliferation, migration, and invasion were tested by CCK-8, wound healing, and Transwell assays, respectively. The bindings between miR-181-5p and MEG3/GATA6 were determined by dual-luciferase reporter gene assay. Furthermore, rescue experiments were conducted to investigate the underlying mechanism of MEG3/miR-181-5p/GATA6 axis in PCa progression. Additionally, mice were injected with PC3 cells transfected with sh-MEG3 and treated with niraparib, and the xenograft tumor growth was observed. Results: MEG3 and GATA6 were upregulated and miR-181-5p was downregulated in PCa patients. Niraparib treatment substantially upregulated MEG3 and GATA6, and downregulated miR-181-5p expression in PCa cells. Niraparib effectively restrained PC3 cell proliferation, migration, and invasion. MiR-181-5p targeted to MEG3, and the inhibitory effects of MEG3 overexpression on PC3 cell proliferation and metastasis were abrogated by miR-181-5p overexpression. Moreover, GATA6 was identified as a target of miR-181-5p, and GATA6 silencing abolished the inhibitory effects of miR-181-5p inhibition on PC3 cell proliferation and metastasis. Besides, MEG3 silencing could abrogate niraparib-mediated tumor growth inhibition in mice. Conclusions: Niraparib restrains prostate cancer cell proliferation and metastasis and tumor growth in mice by regulating the lncRNA MEG3/miR-181-5p/GATA6 pathway.

Epigallocatechin gallate prevents cardiomyocytes from pyroptosis through lncRNA MEG3/TAF15/AIM2 axis in myocardial infarction.

BACKGROUND: ( -)-Epigallocatechin-3-gallate (EGCG), a bioactive polyphenol isolated from green tea, has recently garnered attention for its potential protective role against acute myocardial infarction (MI) via inhibiting inflammation. Herein, we tested whether EGCG participates in modulating cardiac ischemia reperfusion-induced injury and elucidate its potential mechanisms. METHODS: To induce MI in mice, we employed coronary artery ligation, while cell models utilized oxygen glucose deprivation/re-oxygenation (OGD/R)-treated HL-1 cells. TTC, HE and Massion staining evaluated the pathological changes of heart tissues. Besides, RNA-pull down and RIP assays analyzed the interactions of MEG3/TAF15 and AIM2 mRNA/TAF15. FISH associated with immunofiuorescence (IF) double staining was conducted to measure the co-localization of MEG3 and TAF15. RESULTS: In vitro and in vivo evidence supported that EGCG treatment improved cardiomyocytes viability while inhibiting the expressions of AIM2, C-caspase-1, ASC, GSDMD-N, IL-18 and IL-1ß. Knockdown of MEG3 intensified EGCG's therapeutic effects both in vitro and in vivo. LncRNA MEG3 and AIM2 mRNA interacted with TAF15, and MEG3, in turn, promoted the stability of AIM2 mRNA through regulating TAF15. Overexpression of TAF15 reversed the promoting effect of EGCG and MEG3 knockdown on cell viability, and the inhibiting effect on cell pyroptosis. CONCLUSION: EGCG protected cardiomyocytes from pyroptosis by the MEG3/TAF15/AIM2 axis, indicating EGCG as a potential novel therapeutic strategy for managing MI.

MiR-23a promotes autophagy of yak cumulus cells to alleviate apoptosis via the apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase pathway.

The ultimate fate of Graafian follicles is ovulation or atresia which relies on the highly coordinated processes of apoptosis and autophagy in ovarian cells. Long non-coding RNA maternally expressed gene 3 (LncRNA MEG3), miR-23a, and apoptosis signal-regulating kinase 1 (ASK1) are factors associated with autophagy. However, whether these factors can regulate autophagy in cumulus cells (CCs) of yak is unclear. Here, miR-23a overexpression upregulated the LC3-II/LC3-I ratio and Beclin1 abundance while reducing p62 accumulation (p < 0.05). The monodansylcadaverine assay exhibited a marked increase in punctate green fluorescence, and the GFP-LC3B displayed increased yellow fluorescence (p < 0.05). The opposite effect was observed for miR-23a inhibitors. Furthermore, miR-23a overexpression downregulated the abundance of ASK1 mRNA and total ASK1 protein (t-ASK1), whereas miR-23a inhibitors up-regulated them (p < 0.05). The effects of miR-23a overexpression on ASK1 phosphorylated protein at serine 845 (P-845), total JNK (c-Jun N-terminal kinase) (t-JNK) and the JNK phosphorylated protein (p-JNK) were similar to those of t-ASK1 but elicited the opposite effect on ASK1 phosphorylated protein at serine 967 (P-967) (p < 0.05). We further demonstrated that ASK1 expression can be silenced by small-interfering RNA (siRNA), which had no significant effect on t-JNK abundance (p > 0.05) but significantly suppressed the p-JNK expression (p < 0.05). Silencing ASK1 significantly improved Beclin1 abundance and the LC3-II/LC3-I ratio, but decreased p62 abundance (p < 0.05). An increase in yellow GFP-LC3B puncta and green MDC staining puncta were observed (p < 0.05). Overexpression of LncRNA MEG3 significantly increased the expression of t-ASK1, P-845, and JNK and decreased the abundance of P-967 and miR-23a (p < 0.05). In addition, miR-23a upregulation reduced the number of the TUNEL-positive cells, and the addition of 8 mM 3-methyladenine (3-MA) reversed this downregulation (p < 0.05). Similar trends were observed for the Bax/Bcl2 ratio and cleaved-caspase3 abundance. In summary, miR-23a promotes autophagy by inhibiting ASK1 abundance, which reduces apoptosis of yak CCs. This effect can be inhibited by LncRNA MEG3, which has implications for decreasing abnormal Graafian follicular atresia and maintaining development.

Overexpression of lncRNA-MEG3 inhibits endometrial cell proliferation and invasion via miR-21-5p/DNMT3B/Twist.

Recent studies have found that lncRNA-MEG3(MEG3) plays an important role in the development of EMs (Endometriosis), but the specific mechanism needs to be further explored. This study aimed to investigate the effect of MEG3 on the proliferation, invasion of EMs cells. The authors used RT-qPCR to detect the expression of MEG3 and miR-21-5p in EMs tissues and hESCs cells, MTT and Transwell to detect cell proliferation and invasion, western blotting assay to detect the expression of DNMT3B and Twist, MSP to detect the methylation of Twist. The present study's detection results showed that MEG3 was lowly expressed in EMs tissues and hESCs cells, and overexpression of MEG3 could down-regulate miR-21-5p and inhibit endometrial cell proliferation and invasion. In addition, overexpression of MEG3 upregulated the expression of DNMT3B and promoted the methylation of TWIST. In conclusion, the present findings suggest that MEG3 is downregulated in EMs tissues, and overexpression of MEG3 can promote the activity of DNA methyltransferase DNMT3B by downregulating miR-21-5p, thereby promoting the methylation of Twist, downregulating Twist level to inhibits hESCs cells proliferation and invasion.

lncRNA MEG3 Promotes Osteogenic Differentiation of Tendon Stem Cells Via the miR-129-5p/TCF4/ß-Catenin Axis and thus Contributes to Trauma-Induced Heterotopic Ossification.

BACKGROUND: Heterotopic ossification (HO) is one of the most intractable conditions following injury to the musculoskeletal system. In recent years, much attention has been paid to the role of lncRNA in musculoskeletal disorders, but its role in HO was still unclear. Therefore, this study attempted to determine the role of lncRNA MEG3 in the formation of post-traumatic HO and further explore the underlying mechanisms. RESULTS: On the basis of high-throughput sequencing and qPCR validation, elevated expression of the lncRNA MEG3 was shown during traumatic HO formation. Accordingly, in vitro experiments demonstrated that lncRNA MEG3 promoted aberrant osteogenic differentiation of tendon-derived stem cells (TDSCs). Mechanical exploration through RNA pulldown, luciferase reporter gene assay and RNA immunoprecipitation assay identified the direct binding relationship between miR-129-5p and MEG3, or miR-129-5p and TCF4. Further rescue experiments confirmed the miR-129-5p/TCF4/ß-catenin axis to be downstream molecular cascade responsible for the osteogenic-motivating effects of MEG3 on the TDSCs. Finally, experiments in a mouse burn/tenotomy model corroborated the promoting effects of MEG3 on the formation of HO through the miR-129-5p/TCF4/ß-catenin axis. CONCLUSIONS: Our study demonstrated that the lncRNA MEG3 promoted osteogenic differentiation of TDSCs and thus the formation of heterotopic ossification, which could be a potential therapeutic target.

HNRNPA2B1-mediated m6A modification of lncRNA MEG3 facilitates tumorigenesis and metastasis of non-small cell lung cancer by regulating miR-21-5p/PTEN axis.

BACKGROUND: Accumulating data indicate that N6-methyladenosine (m6A) RNA methylation and lncRNA deregulation act crucial roles in cancer progression. Heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) as an m6A "reader" has been reported to be an oncogene in multiple malignancies. We herein aimed to elucidate the role and underlying mechanism by which HNRNPA2B1-mediated m6A modification of lncRNAs contributes to non-small cell lung cancer (NSCLC). METHODS: The expression levels of HNRNPA2B1 and their association with the clinicopathological characteristics and prognosis in NSCLC were determined by RT-qPCR, Western blot, immunohistochemistry and TCGA dataset. Then, the role of HNRNPA2B1 in NSCLC cells was assessed by in vitro functional experiments and in vivo tumorigenesis and lung metastasis models. HNRNPA2B1-mediated m6A modification of lncRNAs was screened by m6A-lncRNA epi-transcriptomic microarray and verified by methylated RNA immunoprecipitation (Me-RIP). The lncRNA MEG3-specific binding with miR-21-5p was evaluated by luciferase gene report and RIP assays. The effects of HNRNPA2B1 and (or) lncRNA MEG3 on miR-21-5p/PTEN/PI3K/AKT signaling were examined by RT-qPCR and Western blot analyses. RESULTS: We found that upregulation of HNRNPA2B1 was associated with distant metastasis and poor survival, representing an independent prognostic factor in patients with NSCLC. Knockdown of HNRNPA2B1 impaired cell proliferation and metastasis in vitro and in vivo, whereas ectopic expression of HNRNPA2B1 possessed the opposite effects. Mechanical investigations revealed that lncRNA MEG3 was an m6A target of HNRNPA2B1 and inhibition of HNRNPA2B1 decreased MEG3 m6A levels but increased its mRNA levels. Furthermore, lncRNA MEG3 could act as a sponge of miR-21-5p to upregulate PTEN and inactivate PI3K/AKT signaling, leading to the suppression of cell proliferation and invasion. Low expression of lncRNA MEG3 or elevated expression of miR-21-5p indicated poor survival in patients with NSCLC. CONCLUSIONS: Our findings uncover that HNRNPA2B1-mediated m6A modification of lncRNA MEG3 promotes tumorigenesis and metastasis of NSCLC cells by regulating miR-21-5p/PTEN axis and may provide a therapeutic target for NSCLC.

LncRNA MEG3 aggravates adipocyte inflammation and insulin resistance by targeting IGF2BP2 to activate TLR4/NF-κB signaling pathway.

Recently, emerging evidence has shown that LncRNA MEG3 is involved in adipocyte inflammation and insulin resistance progression, however, the specific mechanism of action remains unclear. In this study, we found that LncRNA MEG3 expression was increased in TNF-α stimulated 3T3-L1 mature adipocytes, and inflammatory factors IL-6 and MCP-1 secretion levels were increased, cell apoptosis and caspase3 activity was enhanced, ROS content was increased, and iNOS protein expression was increased. Moreover, TNF-α treatment attenuated glucose uptake, promoted triglyceride accumulation, inhibited GLUT4 protein expression at the plasma membrane, and reduced the phosphorylation levels of AMPK and ACC in the cells. Interestingly, we found that transfection of si-MEG3 reversed TNF-α caused inflammatory injury and insulin resistance of 3T3-L1 mature adipocytes. Next, we found that IGF2BP2 is an RNA binding protein of LncRNA MGE3 and transfection of si-IGF2BP2 reversed TNF-α caused inflammatory injury and insulin resistance in 3T3-L1 mature adipocytes, the same effects as transfection of si-MEG3. Mechanistically, LncRNA MGE3 was able to aggravate adipocyte inflammatory injury and dysregulation of insulin sensitivity by activating TLR4 pathway through upregulating the protein expression of IGF2BP2. In vivo findings showed that HFD mice with knockdown of MEG3 had reduced body weight, lower glucose concentrations and insulin levels in plasma, decreased inflammatory factors secretion, and reduced MEG3 and IGF2BP2 expression in epididymal adipose tissues and reduced fat accumulation in mice compared with HFD mice. Our results indicate that LncRNA MEG3 can aggravate chronic inflammation and insulin resistance in adipocytes by activating TLR4/NF-κB signaling pathway via targeting IGF2BP2.

Knockdown of lncRNA Meg3 delays the onset of puberty in female rats.

This study investigated how lncRNA Meg3 affects the onset of puberty in female rats. We determined Meg3 expression in the hypothalamus-pituitary-ovary axis of female rats at the infancy, prepubertal, pubertal, and adult life stages, using quantitative reverse transcription polymerase chain reaction (qRT-PCR). We also assessed the effects of Meg3 knockdown on the expression levels of puberty-related genes and Wnt/ß-catenin proteins in the hypothalamus, time of puberty onset, levels of reproductive genes and hormones, and ovarian morphology in female rats. Meg3 expression in the ovary varied significantly between prepuberty and puberty (P < 0.01). Meg3 knockdown decreased the expression of Gnrh, and Kiss1 mRNA (P < 0.05) and increased the expression of Wnt (P < 0.01) and ß-catenin proteins (P < 0.05) in the hypothalamic cells. Onset of puberty in Meg3 knockdown rats was delayed compared to the control group (P < 0.05). Meg3 knockdown decreased Gnrh mRNA levels (P < 0.05) and increased Rfrp-3 mRNA levels (P < 0.05) in the hypothalamus. The serum concentrations of progesterone (P4) and estradiol (E2) of Meg3 knockdown rats were lower than those in the control animals (P < 0.05). Higher longitudinal diameter and ovary weight were found in Meg3 knockdown rats (P < 0.05). These findings suggest that Meg3 regulates the expression of Gnrh, Kiss-1 mRNA and Wnt/ß-catenin proteins in the hypothalamic cells, and Gnrh, Rfrp-3 mRNA of the hypothalamus and the serum concentration of P4 and E2, and its knockdown delays the onset of puberty in female rats.

LncRNA MEG3 regulates ASK1/JNK axis-mediated apoptosis and autophagy via sponging miR-23a in granulosa cells of yak tertiary follicles.

Apoptosis and autophagy in granulosa cells (GCs) are highly related to follicular development and atresia. It has also been reported that they are related to LncRNA MEG3, miR-23a and apoptosis signal-regulating kinase 1 (ASK-1). However, their relationship to follicular development and the extent to which follicle stimulating hormone (FSH) or luteinizing hormone (LH) can regulate this process remain unknown. Here, we found that ASK1 and JNK were expressed in the GCs of gonadotropin-dependent follicles, and those levels were significantly higher (p < 0.05) in yak Tertiary follicles compared to that of Secondary follicles and Graafian follicles. Then, the effect of LncRNA MEG3 / miR-23a on apoptosis and autophagy via ASK1/JNK (c-Jun N-terminal kinase) in yak GCs was studied. Overexpressing LncRNA MEG3 reduced miR-23a levels and p-967 protein expression, but enhanced ASK1 and JNK mRNA levels as well as t-ASK1, p-845, t-JNK, and p-JNK proteins levels. And Up-regulation of LncRNA MEG3 promoted apoptosis while attenuating autophagy. The targeting relationship between miR-23a and the binding sites of LncRNA MEG3 and ASK1 was also confirmed with the dual luciferase reporter assay. And, the relationship between LncRNA MEG3 and miR-23a was observed as a negative feedback regulation, and changes in LncRNA MEG3 and miR-23a levels can alter the expression of ASK1/JNK axis in yaks GCs. In addition, FSH (10 µg/mL) or LH (100 µg/mL) ability to reverse the effects of LncRNA MEG3 on miR-23a levels and ASK1/JNK axis-mediated apoptosis and autophagy was verified in yak GCs. This is significantly beneficial for decreasing abnormal follicular atresia for yaks tertiary follicles.

IL-27 induces autophagy through regulation of the DNMT1/lncRNA MEG3/ERK/p38 axis to reduce pulmonary fibrosis.

PURPOSE: Previous studies have shown that interleukin-27 (IL-27) can reduce bleomycin (BLM)-induced pulmonary fibrosis (PF). However, the underlying mechanism by which IL-27 attenuates PF is not fully clear. METHODS: In this research, we used BLM to construct a PF mouse model, and MRC-5 cells stimulated by transforming growth factor-ß1 (TGF-ß1) were used to construct a PF model in vitro. The lung tissue status was observed by Masson and hematoxylin and eosin (HE) staining. To detect gene expression, RT‒qPCR was used. The protein levels were detected by western blotting and immunofluorescence staining. EdU and ELISA were used to detect cell proliferation viability and hydroxyproline (HYP) content, respectively. RESULTS: Aberrant IL-27 expression was observed in BLM-induced mouse lung tissues, and the use of IL-27 attenuated mouse lung tissue fibrosis. TGF-ß1 induced autophagy inhibition in MRC-5 cells, and IL-27 alleviated MRC-5 cell fibrosis by activating autophagy. The mechanism is inhibition of DNA methyltransferase 1 (DNMT1)-mediated lncRNA MEG3 methylation and ERK/p38 signaling pathway activation. Overexpression of DNMT1, knockdown of lncRNA MEG3, autophagy inhibitor or ERK/p38 signaling pathway inhibitors reversed the positive effect of IL-27 in a lung fibrosis model in vitro. CONCLUSION: In conclusion, our study shows that IL-27 upregulates MEG3 expression through inhibition of DNMT1-mediated lncRNA MEG3 promoter methylation, which in turn inhibits ERK/p38 signaling pathway-induced autophagy and attenuates BLM-induced PF, providing a contribution to the elucidation of the potential mechanisms by which IL-27 attenuates PF.

Marsdenia tenacissima extract prevents the malignant progression of glioma through upregulating lncRNA MEG3 and SFRP1-dependent inhibition of Wnt/ß-catenin pathway.

BACKGROUND/AIM: Recent studies have highlighted the tumor-suppressive effect of Marsdenia tenacissima extract (MTE) on human cancers. This research unveils the potential impact of MTE on glioma and ascertains the relevant molecular mechanisms. METHODS: Glioma cells were treated with MTE, with normal human astrocytes (NHAs) as controls. A battery of function experiments, including the CCK-8 viability test, colony formation assay, scratch migration assay, and Transwell invasion assay, was executed to address the responses of glioma cells to MTE treatment and gain or loss of function of lncMEG3, miR-542-3p, and SFRP1. FISH, RIP, and dual-luciferase reporter assays were adopted for assessing gene interactions. U251-GFP-Luc cells were delivered into nude mice through intracranial injection to develop an orthotopic glioma model for in vivo validation. RESULTS: 200 mg/mL MTE could suppress the proliferating, migrating, and invading properties of glioma cells but not affect those of NHAs. MTE treatment enhanced the expression of lncMEG3, which competes with SFRP1 for binding miR-542-3p. SFRP1 could inactivate the Wnt/ß-catenin pathway. Animal experimentation substantiated the antitumor activity and mechanism of MTE in nude mice. CONCLUSIONS: MTE suppresses glioma via the lncMEG3/miR-542-3p/SFRP1/Wnt/ß-catenin axis. These findings contribute to a theoretical basis for the use of MTE for glioma patients.