METTL3-mediated m6A modification of ATG7 regulates autophagy-GATA4 axis to promote cellular senescence and osteoarthritis progression

ObjectiveThe aim of the study was to investigate the role and regulatory mechanisms of fibroblast-like synoviocytes (FLSs) and their senescence in the progression of osteoarthritis (OA).MethodsSynovial tissues from normal patients and patients with OA were collected. Synovium FLS senescence was analysed by immunofluorescence and western blotting. The role of methyltransferase-like 3 (METTL3) in autophagy regulation was explored using N6-methyladenosine (m6A)-methylated RNA and RNA immunoprecipitation assays. Mice subjected to destabilisation of the medial meniscus (DMM) surgery were intra-articularly injected with or without pAAV9 loaded with small interfering RNA (siRNA) targeting METTL3. Histological analysis was performed to determine cartilage damage.ResultsSenescent FLSs were markedly increased with the progression of OA in patients and mouse models. We determined that impaired autophagy occurred in OA-FLS, resulting in the upregulation of senescence-associated secretory phenotype (SASP). Re-establishment of autophagy reversed the senescent phenotype by suppressing GATA4. Further, we observed for the first time that excessive m6A modification negatively regulated autophagy in OA-FLS. Mechanistically, METTL3-mediated m6A modification decreased the expression of autophagy-related 7, an E-1 enzyme crucial for the formation of autophagosomes, by attenuating its RNA stability. Silencing METTL3 enhanced autophagic flux and inhibited SASP expression in OA-FLS. Intra-articular injection of synovium-targeted METTL3 siRNA suppressed cellular senescence propagation in joints and ameliorated DMM-induced cartilage destruction.ConclusionsOur study revealed the important role of FLS senescence in OA progression. Targeted METTL3 inhibition could alleviate the senescence of FLS and limit OA development in experimental animal models, providing a potential strategy for OA therapy.

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circRNA N6-methyladenosine methylation in preeclampsia and the potential role of N6-methyladenosine-modified circPAPPA2 in trophoblast invasion

Scientific Reports ◽

10.1038/s41598-021-03662-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Yonggang Zhang ◽

Hongling Yang ◽

Yan Long ◽

Yipeng Zhang ◽

Ronggui Chen ◽

...

Keyword(s):

Rna Stability ◽

Start Codon ◽

Trophoblast Invasion ◽

Reverse Transcription Pcr ◽

Mrna Binding Protein ◽

Rna Immunoprecipitation ◽

New Vision ◽

Mrna Binding

AbstractHere, we performed N6-methyladenosine (m6A) RNA sequencing to determine the circRNA m6A methylation changes in the placentas during the pathogenesis of preeclampsia (PE). We verified the expression of the circRNA circPAPPA2 using quantitative reverse transcription-PCR. An invasion assay was carried out to identify the role of circPAPPA2 in the development of PE. Mechanistically, we investigated the cause of the altered m6A modification of circPAPPA2 through overexpression and knockdown cell experiments, RNA immunoprecipitation, fluorescence in situ hybridization and RNA stability experiments. We found that increases in m6A-modified circRNAs are prevalent in PE placentas and that the main changes in methylation occur in the 3’UTR and near the start codon, implicating the involvement of these changes in PE development. We also found that the levels of circPAPPA2 are decreased but that m6A modification is augmented. Furthermore, we discovered that methyltransferase‑like 14 (METTL14) increases the level of circPAPPA2 m6A methylation and that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) maintains circPAPPA2 stability. Decreases in IGF2BP3 levels lead to declines in circPAPPA2 levels. In summary, we provide a new vision and strategy for the study of PE pathology and report that placental circRNA m6A modification appears to be an important regulatory mechanism.

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METTL3 Promotes Lung Adenocarcinoma Tumorigenesis and Inhibits Ferroptosis by Stabilizing SLC7A11 m6A Modification

10.21203/rs.3.rs-1085921/v1 ◽

2021 ◽

Author(s):

Yiming Xu ◽

Dandan Lv ◽

Chao Yan ◽

Hua Su ◽

Xue Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Lung Adenocarcinoma ◽

Rna Stability ◽

Underlying Mechanism ◽

Rna Immunoprecipitation ◽

Direct Target ◽

Novel Target

Abstract Background: N6-methyladenosine (m 6 A) has emerged as a significant regulator of the progress of various cancers. However, its role in lung adenocarcinoma (LUAD) remains unclear. Here, we explored the biological function and underlying mechanism of methyltransferase-like 3 (METTL3), the main catalyst of m 6 A, in LUAD progression. Methods: The expression of m 6 A, METTL3, YTHDF1 and SLC7A11 were detected by immunochemistry or/and online datasets in LUAD patients. The effects of METTL3 on LUAD cell proliferation, apoptosis and ferroptosis were assessed through in vitro loss-and gain-of-function experiments. The in vivo effect on tumorigenesis of METTL3 was evaluated using the LUAD cell xenograft mouse model. MeRIP-seq, RNA immunoprecipitation and RNA stability assay were conducted to explore the molecular mechanism of METTL3 in LUAD. Results: The results showed that the m 6 A level, as well as the methylase METTL3 were both significantly elevated in LUAD patients and lung cancer cells. Functionally, we found that METTL3 could promote proliferation and inhibit ferroptosis in different LUAD cell models, while METTL3 knockdown suppressed LUAD growth in cell-derived xenografts. Mechanistically, solute carrier 7A11 (SLC7A11), the subunit of system Xc - , was identified as the direct target of METTL3 by mRNA-seq and MeRIP-seq. METTL3-mediated m 6 A modification could stabilize SLC7A11 mRNA and promote its translation, thus promoting LUAD cell proliferation and inhibiting cell ferroptosis, a novel form of programmed cell death. Additionally, we demonstrated that YTHDF1, a m 6 A reader, was recruited by METTL3 to enhance SLC7A11 m 6 A modification. Moreover, the expression of YTHDF1 and SLC7A11 were positively correlated with METTL3 and m 6 A in LUAD tissues.Conclusions: These findings reinforced the oncogenic role of METTL3 in LUAD progression and revealed its underlying correlation with cancer cell ferroptosis; these findings also indicate that METTL3 is a promising novel target in LUAD diagnosis and therapy.

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CCL2/CCR2, but not CCL5/CCR5, mediates monocyte recruitment, inflammation and cartilage destruction in osteoarthritis

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2016-210426 ◽

2016 ◽

Vol 76 (5) ◽

pp. 914-922 ◽

Cited By ~ 82

Author(s):

Harini Raghu ◽

Christin M Lepus ◽

Qian Wang ◽

Heidi H Wong ◽

Nithya Lingampalli ◽

...

Keyword(s):

Chemokine Receptors ◽

Cartilage Damage ◽

Cartilage Destruction ◽

Synthesis Inhibitor ◽

Promising Therapeutic Approach ◽

Concomitant Reduction ◽

Synovial Tissues ◽

And Control ◽

Microarray Analyses ◽

And Cartilage

ObjectivesWhile various monocyte chemokine systems are increased in expression in osteoarthritis (OA), the hierarchy of chemokines and chemokine receptors in mediating monocyte/macrophage recruitment to the OA joint remains poorly defined. Here, we investigated the relative contributions of the CCL2/CCR2 versus CCL5/CCR5 chemokine axes in OA pathogenesis.MethodsCcl2-, Ccr2-, Ccl5- and Ccr5-deficient and control mice were subjected to destabilisation of medial meniscus surgery to induce OA. The pharmacological utility of blocking CCL2/CCR2 signalling in mouse OA was investigated using bindarit, a CCL2 synthesis inhibitor, and RS-504393, a CCR2 antagonist. Levels of monocyte chemoattractants in synovial tissues and fluids from patients with joint injuries without OA and those with established OA were investigated using a combination of microarray analyses, multiplexed cytokine assays and immunostains.ResultsMice lacking CCL2 or CCR2, but not CCL5 or CCR5, were protected against OA with a concomitant reduction in local monocyte/macrophage numbers in their joints. In synovial fluids from patients with OA, levels of CCR2 ligands (CCL2, CCL7 and CCL8) but not CCR5 ligands (CCL3, CCL4 and CCL5) were elevated. We found that CCR2+ cells are abundant in human OA synovium and that CCR2+ macrophages line, invade and are associated with the erosion of OA cartilage. Further, blockade of CCL2/CCR2 signalling markedly attenuated macrophage accumulation, synovitis and cartilage damage in mouse OA.ConclusionsOur findings demonstrate that monocytes recruited via CCL2/CCR2, rather than by CCL5/CCR5, propagate inflammation and tissue damage in OA. Selective targeting of the CCL2/CCR2 system represents a promising therapeutic approach for OA.

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Synovial macrophage M1 polarisation exacerbates experimental osteoarthritis partially through R-spondin-2

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2018-213450 ◽

2018 ◽

Vol 77 (10) ◽

pp. 1524-1534 ◽

Cited By ~ 35

Author(s):

Haiyan Zhang ◽

Chuangxin Lin ◽

Chun Zeng ◽

Zhenyu Wang ◽

Hua Wang ◽

...

Keyword(s):

Cartilage Damage ◽

Research Society ◽

M1 Macrophages ◽

Myeloid Lineage ◽

Synovial Macrophage ◽

Osteoarthritis Research Society ◽

Synovial Tissues ◽

Experimental Osteoarthritis ◽

And Control

ObjectivesTo investigate the roles and regulatory mechanisms of synovial macrophages and their polarisation in the development of osteoarthritis (OA).MethodsSynovial tissues from normal patients and patients with OA were collected. M1 or M2-polarised macrophages in synovial tissues of patients with OA and OA mice were analysed by immunofluorescence and immunohistochemical staining. Mice with tuberous sclerosis complex 1 (TSC1) or Rheb deletion specifically in the myeloid lineage were generated and subjected to intra-articular injection of collagenase (collagenase-induced osteoarthritis, CIOA) and destabilisation of the medial meniscus (DMM) surgery to induce OA. Cartilage damage and osteophyte size were measured by Osteoarthritis Research Society International score and micro-CT, respectively. mRNA sequencing was performed in M1 and control macrophages. Mice and ATDC5 cells were treated with R-spondin-2 (Rspo2) or anti-Rspo2 to investigate the role of Rspo2 in OA.ResultsM1 but not M2-polarised macrophages accumulated in human and mouse OA synovial tissue. TSC1 deletion in the myeloid lineage constitutively activated mechanistic target of rapamycin complex 1 (mTORC1), increased M1 polarisation in synovial macrophages and exacerbated experimental OA in both CIOA and DMM models, while Rheb deletion inhibited mTORC1, enhanced M2 polarisation and alleviated CIOA in mice. The results show that promoting the macrophage M1 polarisation leads to exacerbation of experimental OA partially through secretion of Rspo2 and activation of β-catenin signalling in chondrocytes.ConclusionsSynovial macrophage M1 polarisation exacerbates experimental CIOA partially through Rspo2. M1 macrophages and Rspo2 are potential therapeutic targets for OA treatment.

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METTL3 promotes lung adenocarcinoma tumor growth and inhibits ferroptosis by stabilizing SLC7A11 m6A modification

Cancer Cell International ◽

10.1186/s12935-021-02433-6 ◽

2022 ◽

Vol 22 (1) ◽

Author(s):

Yiming Xu ◽

Dandan Lv ◽

Chao Yan ◽

Hua Su ◽

Xue Zhang ◽

...

Keyword(s):

Cell Proliferation ◽

Lung Adenocarcinoma ◽

Rna Stability ◽

Underlying Mechanism ◽

Rna Immunoprecipitation ◽

Direct Target ◽

Novel Target

Abstract Background N6-methyladenosine (m6A) has emerged as a significant regulator of the progress of various cancers. However, its role in lung adenocarcinoma (LUAD) remains unclear. Here, we explored the biological function and underlying mechanism of methyltransferase-like 3 (METTL3), the main catalyst of m6A, in LUAD progression. Methods The expression of m6A, METTL3, YTHDF1 and SLC7A11 were detected by immunochemistry or/and online datasets in LUAD patients. The effects of METTL3 on LUAD cell proliferation, apoptosis and ferroptosis were assessed through in vitro loss-and gain-of-function experiments. The in vivo effect on tumorigenesis of METTL3 was evaluated using the LUAD cell xenograft mouse model. MeRIP-seq, RNA immunoprecipitation and RNA stability assay were conducted to explore the molecular mechanism of METTL3 in LUAD. Results The results showed that the m6A level, as well as the methylase METTL3 were both significantly elevated in LUAD patients and lung cancer cells. Functionally, we found that METTL3 could promote proliferation and inhibit ferroptosis in different LUAD cell models, while METTL3 knockdown suppressed LUAD growth in cell-derived xenografts. Mechanistically, solute carrier 7A11 (SLC7A11), the subunit of system Xc−, was identified as the direct target of METTL3 by mRNA-seq and MeRIP-seq. METTL3-mediated m6A modification could stabilize SLC7A11 mRNA and promote its translation, thus promoting LUAD cell proliferation and inhibiting cell ferroptosis, a novel form of programmed cell death. Additionally, we demonstrated that YTHDF1, a m6A reader, was recruited by METTL3 to enhance SLC7A11 m6A modification. Moreover, the expression of YTHDF1 and SLC7A11 were positively correlated with METTL3 and m6A in LUAD tissues. Conclusions These findings reinforced the oncogenic role of METTL3 in LUAD progression and revealed its underlying correlation with cancer cell ferroptosis; these findings also indicate that METTL3 is a promising novel target in LUAD diagnosis and therapy.

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OP0243 SERPINA3N LIMITS CARTILAGE DESTRUCTION IN OSTEOARTHRITIS BY INHIBITING MACROPHAGE-DERIVED LEUKOCYTE ELASTASE

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.4135 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 153-154

Author(s):

A. Latourte ◽

A. Combier ◽

S. Jaulerry ◽

C. Cherifi ◽

Y. Jouan ◽

...

Keyword(s):

Serine Protease ◽

Serine Proteases ◽

Cartilage Damage ◽

Cartilage Destruction ◽

Cartilage Explants ◽

Wild Type ◽

French Society ◽

Leukocyte Elastase ◽

Ct Analysis

Background:Interleukin-6 (IL-6) plays an important role in osteoarthritis (OA). Transcriptomic analyses (RNAseq) revealed that SerpinA3N, a serine protease inhibitor, is a key target of IL-6 in chondrocyte.Objectives:This study aimed to examine the role of SerpinA3N and Leukocyte Elastase (Elane), a serine protease targeted by SerpinA3N, in cartilage destruction during OA.Methods:The role of SerpinA3N was investigated in the destabilization of medial meniscus (DMM) model of murine OA with 1) mice with conditional inducible knockdown ofSerpina3nin cartilage (Col2CreER;Serpina3nfl/flmice [ΔSerpina3nCol2]) and 2) C57BL/6 wild type (WT) mice treated with intra-articular injection of SerpinA3N (1,5 or 15 nM/week). OA joint lesions were assessed by histology (OARSI and synovitis scores) and micro-CT analysis (osteophyte volume, subchondral bone remodeling).Because serine proteases targeted by SerpinA3N are not produced by murine chondrocytes,Elaneexpression (qRT-PCR) was determined in murine macrophages (Raw) stimulated or not by IL-6 (100 ng/ml). Recombinant SerpinA3N (30 nM) and a specific Elane inhibitor, Sivelestat (100 µg/ml) were used on cartilage explants treated by conditioned medium of macrophages pre-treated or not by IL-6 (CM–IL-6). Cartilage catabolism was determined by histology and matrix metalloproteinase MMP-3 production was evaluated by Western Blot and immunohistochemistry (IHC). Weekly intra-articular injections of Sivelestat (1mM) were performed in the DMM to determine the role of Elane in OA.Results:ΔSerpina3nCol2mice had more severe OA lesions than control littermates 6 weeks after DMM, with greater cartilage damage (mean±SD OARSI score: 5.6±0.4 vs 3.4±0.5, p=0.01), increased synovitis scores (3.0±0.3 vs 1.9±0.3, p=0.03) and bigger osteophytes (7.2±0.8x107 vs 3.8±0.8x107 µ3, p=0.048). Conversely, WT mice treated with intra-articular injections of SerpinA3N 15nM exhibited less severe cartilage loss than mice treated with PBS after DMM (OARSI score: 2.1±0.4 vs 3.9±0.5, p=0.02). Elane mRNA expression was increased in macrophages upon IL-6 stimulation. In cartilage explants, CM–IL-6 activated cartilage catabolism and MMP-3 production, and effect that was blunted by SerpinA3N and Sivelestat. Finally, mice treated with intra-articular injections of Sivelestat had less severe cartilage damage than those treated with PBS after DMM (OARSI score: 3.3±0.47 vs 5.8±0.53, p=0.0046).Conclusion:SerpinA3N protects against experimental OA via the inhibition of Elane, a pro-catabolic serine protease produced by macrophages. This results highlight the crosstalk between cartilage and surrounding macrophages and open up new therapeutic perspectives.Acknowledgments:This work has been supported by French Society of Rheumatology and ART Viggo association.Disclosure of Interests:None declared

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Role of small nuclear RNAs in eukaryotic gene expression

Essays in Biochemistry ◽

10.1042/bse0540079 ◽

2013 ◽

Vol 54 ◽

pp. 79-90 ◽

Cited By ~ 34

Author(s):

Saba Valadkhan ◽

Lalith S. Gunawardane

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Rna Stability ◽

Splice Sites ◽

Branch Site ◽

Small Nuclear Rnas ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

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MiRNA-145 and Its Direct Downstream Targets in Digestive System Cancers: A Promising Therapeutic Target

Current Pharmaceutical Design ◽

10.2174/1381612826666201029095702 ◽

2020 ◽

Vol 26 ◽

Author(s):

Yini Ma ◽

Xiu Cao ◽

Guojuan Shi ◽

Tianlu Shi

Keyword(s):

Digestive System ◽

Target Genes ◽

Malignant Neoplasm ◽

Vital Role ◽

Diagnostic Biomarkers ◽

Cellular Processes ◽

Promising Therapeutic Target ◽

Direct Target ◽

Potential Strategy

: MicroRNAs (miRNAs) play a vital role in the onset and development of many diseases, including cancers. Emerging evidence shows that numerous miRNAs have the potential to be used as diagnostic biomarkers for cancers, and miRNA-based therapy may be a promising therapy for the treatment of malignant neoplasm. MicroRNA-145 (miR-145) has been considered to play certain roles in various cellular processes, such as proliferation, differentiation and apoptosis, via modulating expression of direct target genes. Recent reports show that miR-145 participates in the progression of digestive system cancers, and plays crucial and novel roles for cancer treatment. In this review, we summarize the recent knowledge concerning the function of miR-145 and its direct targets in digestive system cancers. We discuss the potential role of miR-145 as valuable biomarkers for digestive system cancers and how miR-145 regulates these digestive system cancers via different targets to explore the potential strategy of targeting miR-145.

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Group III phospholipase A2 downregulation attenuated survival and metastasis in ovarian cancer and promotes chemo-sensitization

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-021-01985-9 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Upasana Ray ◽

Debarshi Roy ◽

Ling Jin ◽

Prabhu Thirusangu ◽

Julie Staub ◽

...

Keyword(s):

Ovarian Cancer ◽

Cell Viability ◽

Phospholipase A2 ◽

Mtt Assay ◽

Metabolic Inhibitor ◽

Autophagic Flux ◽

Ciliated Cells ◽

Group Iii

Abstract Background Aberrant lipogenicity and deregulated autophagy are common in most advanced human cancer and therapeutic strategies to exploit these pathways are currently under consideration. Group III Phospholipase A2 (sPLA2-III/PLA2G3), an atypical secretory PLA2, is recognized as a regulator of lipid metabolism associated with oncogenesis. Though recent studies reveal that high PLA2G3 expression significantly correlates with poor prognosis in several cancers, however, role of PLA2G3 in ovarian cancer (OC) pathogenesis is still undetermined. Methods CRISPR-Cas9 and shRNA mediated knockout and knockdown of PLA2G3 in OC cells were used to evaluate lipid droplet (LD) biogenesis by confocal and Transmission electron microscopy analysis, and the cell viability and sensitization of the cells to platinum-mediated cytotoxicity by MTT assay. Regulation of primary ciliation by PLA2G3 downregulation both genetically and by metabolic inhibitor PFK-158 induced autophagy was assessed by immunofluorescence-based confocal analysis and immunoblot. Transient transfection with GFP-RFP-LC3B and confocal analysis was used to assess the autophagic flux in OC cells. PLA2G3 knockout OVCAR5 xenograft in combination with carboplatin on tumor growth and metastasis was assessed in vivo. Efficacy of PFK158 alone and with platinum drugs was determined in patient-derived primary ascites cultures expressing PLA2G3 by MTT assay and immunoblot analysis. Results Downregulation of PLA2G3 in OVCAR8 and 5 cells inhibited LD biogenesis, decreased growth and sensitized cells to platinum drug mediated cytotoxicity in vitro and in in vivo OVCAR5 xenograft. PLA2G3 knockdown in HeyA8MDR-resistant cells showed sensitivity to carboplatin treatment. We found that both PFK158 inhibitor-mediated and genetic downregulation of PLA2G3 resulted in increased number of percent ciliated cells and inhibited cancer progression. Mechanistically, we found that PFK158-induced autophagy targeted PLA2G3 to restore primary cilia in OC cells. Of clinical relevance, PFK158 also induces percent ciliated cells in human-derived primary ascites cells and reduces cell viability with sensitization to chemotherapy. Conclusions Taken together, our study for the first time emphasizes the role of PLA2G3 in regulating the OC metastasis. This study further suggests the therapeutic potential of targeting phospholipases and/or restoration of PC for future OC treatment and the critical role of PLA2G3 in regulating ciliary function by coordinating interface between lipogenesis and metastasis.

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