N6 ‐methyladenosine‐modified circRNA RERE modulates osteoarthritis by regulating β‐catenin ubiquitination and degradation

ObjectivesN⁶‐methyladenosine (m6A) is one of the most abundant internal RNA modifications. We investigated the role of m6A‐modified circRERE in osteoarthritis (OA) and its mechanism.Materials and MethodsCircRERE and IRF2BPL were screened by microarrays. The role of m6A‐modification in circRERE was examined by methylated RNA precipitation and morpholino oligo (MOs) treatment. The axis of circRERE/miR‐195‐5p/IRF2BPL/β‐catenin was determined using flow cytometry, western blotting and immunofluorescence in human chondrocytes (HCs) and corroborated using a mouse model of destabilization of medial meniscus (DMM) with intra‐articular (IA) injection of adeno‐associated viruses (AAV).ResultsCircRERE was decreased in OA cartilage and chondrocytes compared with control. CircRERE downregulation was likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2‐HRSP12‐RNase P/MRP in OA chondrocytes. MOs transfection targeting HRSP12 binding motifs in circRERE partially reversed decreased circRERE expression and increased apoptosis in HCs treated with IL‐1β for 6 h. CircRERE exerted chondroprotective effects by targeting miR‐195‐5p/IRF2BPL, thus regulating the ubiquitination and degradation of β‐catenin. CircRere (mouse homologue) overexpression by IA‐injection of AAV‐circRere into mice attenuated the severity of DMM‐induced OA, whereas AAV‐miR‐195a‐5p or AAV‐sh‐Irf2bpl reduced the protective effects. The detrimental effects of AAV‐sh‐Irf2bpl on DMM‐induced OA were substantially counteracted by ICG‐001, an inhibitor of β‐catenin.ConclusionsOur study is a proof‐of‐concept demonstration for targeting m6A‐modified circRERE and its target miR‐195‐5p/IRF2BPL/β‐catenin as potential therapeutic strategies for OA treatment.

Liu et al. demonstrate that circRERE expression is downregulated in human osteoarthritis (OA) cartilage and chondrocytes, and circRERE downregulation is likely attributed to its increased m6A modification prone to endoribonucleolytic cleavage by YTHDF2‐HRSP12‐RNase P/MRP. Mechanistically, circRERE downregulation leads to aberrant β‐catenin ubiquitin and degradation by targeting miR‐195‐5p/IRF2BPL during OA pathogenesis.     

Novel insights into the interaction between N6‐methyladenosine methylation and noncoding RNAs in musculoskeletal disorders

BackgroundMusculoskeletal disorder (MSD) are a class of inflammatory and degener‐ative diseases, but the precise molecular mechanisms are still poorly understood. Noncoding RNA (ncRNA) N6‐methyladenosine (m6A) modification plays an essential role in the pathophysiological process of MSD. This review summarized the interaction between m6A RNA methylation and ncRNAs in the molecular regulatory mechanism of MSD. It provides a new perspective for the pathophysiological mechanism and ncRNA m6A targeted therapy of MSD.MethodsA comprehensive search of databases was conducted with musculoskeletal disorders, noncoding RNA, N6‐methyladenosine, intervertebral disc degeneration, osteoporosis, osteosarcoma, osteoarthritis, skeletal muscle, bone, and cartilage as the key‐words. Then, summarized all the relevant articles.ResultsIntervertebral disc degeneration (IDD), osteoporosis (OP), osteosarcoma (OS), and osteoarthritis (OA) are common MSDs that affect muscle, bone, cartilage, and joint, leading to limited movement, pain, and disability. However, the precise pathogenesis remains unclear, and no effective treatment and drug is available at present. Numerous studies confirmed that the mutual regulation between m6A and ncRNAs (i.e., microRNAs, long ncRNAs, and circular RNAs) was found in MSD, m6A modification can regulate ncRNAs, and ncRNAs can also target m6A regulators. ncRNA m6A modification plays an essential role in the pathophysiological process of MSDs by regulating the homeostasis of skeletal muscle, bone, and cartilage.Conclusionm6A interacts with ncRNAs to regulate multiple biological processes and plays important roles in IDD, OP, OS, and OA. These studies provide new insights into the pathophysiological mechanism of MSD and targeting m6A‐modified ncRNAs may be a promising therapy approach.

m6A regulates the expression and function of ncRNAs (i.e., miRNAs, lncRNAs and circRNAs), ncRNAs can also affect m6A‐related protein. m6A‐related proteins in ncRNA are abnormally expressed and closely associated with bone osteogenic, osteoclastogenic processes and myogenesis, participating in modulating the homoeostasis of skeletal muscle, bone and cartilage. ncRNA m6A modification regulates the pathological and physiological processes of musculoskeletal disorders (i.e., IDD, OP, OS and OA). In IDD, ncRNA m6A modification regulated NP cells glucose metabolism, senescence and pyroptosis. In OP, ncRNA m6A modification regulated BMSCs osteogenic differentiation, osteoblast proliferation, differentiation and bone formation. In OS, ncRNA m6A modification regulated OS cells growth, migration, proliferation and apoptosis. In OA, ncRNA m6A modification regulated chondrocyte apoptosis and ECM degradation.     

circ‐AKT3 aggravates renal ischaemia‐reperfusion injury via regulating miR‐144‐5p /Wnt/β‐catenin pathway and oxidative stress

Renal ischaemia‐reperfusion (RI/R) injury is one major pathological state of acute kidney injury (AKI) with a mortality rate ranking 50% to 80%. MiR‐144‐5p acts as a molecular trigger in various diseases. We presumed that miR‐144‐5p might be involved RI/R injury progression. We found that RI/R injury decreased miR‐144‐5p expression in rat models. MiR‐144‐5p downregulation promoted cell apoptosis rate and activated Wnt/β‐catenin signal in RI/R injury rats. By performing bioinformatic analysis, RIP, RNA pull‐down, luciferase reporter experiments, we found that circ‐AKT3 sponged to miR‐144‐5p and decreased its expression in RI/R injury rats. Moreover, we found that circ‐AKT3 promoted cell apoptosis rate and activated Wnt/β‐catenin signal, and miR‐144‐5p mimic reversed the promotive effect of circ‐AKT3 in rat models. We also found that circ‐AKT3 increased the oxidative stress level in rat models. In conclusion, our study suggests that the circAKT3 is involved RI/R injury progression through regulating miR‐144‐5p/Wnt/β‐catenin pathway and oxidative stress.     

Circular RNA circ‐RCCD promotes cardiomyocyte differentiation in mouse embryo development via recruiting YY1 to the promoter of MyD88

Congenital heart disease (CHD) is the most common birth defect, affecting approximately 1% of live births. Genetic and environmental factors are leading factors to CHD, but the mechanism of CHD pathogenesis remains unclear. Circular RNAs (circRNAs) are kinds of endogenous non‐coding RNAs (ncRNAs) involved in a variety of physiological and pathological processes, especially in heart diseases. In this study, three significant differently expressed circRNA between maternal embryonic day (E) E13 and E17 was found by microarray assay. Among them, the content of circ‐RCCD increases with the development of heart and was enriched in primary cardiomyocytes of different species, which arouses our attention. Functional experiments revealed that inhibition of circ‐RCCD dramatically suppressed the formation of beating cell clusters, the fluorescence intensity of cardiac differentiation marker MF20, and the expression of the myocardial‐specific markers CTnT, Mef2c, and GATA4. Next, we found that circ‐RCCD was involved in cardiomyocyte differentiation through negative regulation of MyD88 expression. Further experiments proved that circ‐RCCD inhibited MyD88 levels by recruiting YY1 to the promoter of MyD88; circ‐RCCD inhibited nuclear translocation of YY1. These results reported that circ‐RCCD promoted cardiomyocyte differentiation by recruiting YY1 to the promoter of MyD88. And, this study provided a potential role and molecular mechanism of circ‐RCCD as a target for the treatment of CHD.     

G6PD overexpression protects from oxidative stress and age‐related hearing loss

Aging of the auditory system is associated with the incremental production of reactive oxygen species (ROS) and the accumulation of oxidative damage in macromolecules, which contributes to cellular malfunction, compromises cell viability, and, ultimately, leads to functional decline. Cellular detoxification relies in part on the production of NADPH, which is an important cofactor for major cellular antioxidant systems. NADPH is produced principally by the housekeeping enzyme glucose‐6‐phosphate dehydrogenase (G6PD), which catalyzes the rate‐limiting step in the pentose phosphate pathway. We show here that G6PD transgenic mice (G6PD‐Tg), which show enhanced constitutive G6PD activity and NADPH production along life, have lower auditory thresholds than wild‐type mice during aging, together with preserved inner hair cell (IHC) and outer hair cell (OHC), OHC innervation, and a conserved number of synapses per IHC. Gene expression of antioxidant enzymes was higher in 3‐month‐old G6PD‐Tg mice than in wild‐type counterparts, whereas the levels of pro‐apoptotic proteins were lower. Consequently, nitration of proteins, mitochondrial damage, and TUNEL⁺ apoptotic cells were all lower in 9‐month‐old G6PD‐Tg than in wild‐type counterparts. Unexpectedly, G6PD overexpression triggered low‐grade inflammation that was effectively resolved in young mice, as shown by the absence of cochlear cellular damage and macrophage infiltration. Our results lead us to propose that NADPH overproduction from an early stage is an efficient mechanism to maintain the balance between the production of ROS and cellular detoxification power along aging and thus prevents hearing loss progression.     

IDH2 stabilizes HIF‐1α‐induced metabolic reprogramming and promotes chemoresistance in urothelial cancer

Drug resistance contributes to poor therapeutic response in urothelial carcinoma (UC). Metabolomic analysis suggested metabolic reprogramming in gemcitabine‐resistant urothelial carcinoma cells, whereby increased aerobic glycolysis and metabolic stimulation of the pentose phosphate pathway (PPP) promoted pyrimidine biosynthesis to increase the production of the gemcitabine competitor deoxycytidine triphosphate (dCTP) that diminishes its therapeutic effect. Furthermore, we observed that gain‐of‐function of isocitrate dehydrogenase 2 (IDH2) induced reductive glutamine metabolism to stabilize Hif‐1α expression and consequently stimulate aerobic glycolysis and PPP bypass in gemcitabine‐resistant UC cells. Interestingly, IDH2‐mediated metabolic reprogramming also caused cross resistance to CDDP, by elevating the antioxidant defense via increased NADPH and glutathione production. Downregulation or pharmacological suppression of IDH2 restored chemosensitivity. Since the expression of key metabolic enzymes, such as TIGAR, TKT, and CTPS1, were affected by IDH2‐mediated metabolic reprogramming and related to poor prognosis in patients, IDH2 might become a new therapeutic target for restoring chemosensitivity in chemo‐resistant urothelial carcinoma.     

Exosomal hsa_circ_0125310 promotes cell proliferation and fibrosis in diabetic nephropathy via sponging miR‐422a and targeting the IGF1R/p38 axis

Diabetic nephropathy (DN) is still on the rise worldwide, and millions of patients have to be treated through dialysis or transplant because of kidney failure caused by DN. Recent reports have highlighted circRNAs in the treatment of DN. Herein, we aimed to investigate the mechanism by which high glucose‐induced exo‐circ_0125310 promotes diabetic nephropathy progression. circ_0125310 is highly expressed in diabetic nephropathy and exosomes isolated from high glucose‐induced mesangial cells (MCs). High glucose‐induced exosomes promote the proliferation and fibrosis of MCs. However, results showed that the effects of exosomes on MCs can be reversed by the knockdown of circ_0125310. miR‐422a, which targets IGF1R, was the direct target of circ_0125310. circ_0125310 regulated IGF1R/p38 axis by sponging miR‐422a. Exo‐circ_0125310 increased the luciferase activity of the WT‐IGF1R reporter in the dual‐luciferase reporter gene assays and upregulated the expression level of IGF1R and p38. Finally, in vivo research indicated that the overexpression of circ_0125310 promoted the diabetic nephropathy progression. Above results demonstrated that the high glucose‐induced exo‐circ_0125310 promoted cell proliferation and fibrosis in diabetic nephropathy via sponging miR‐422a and targeting the IGF1R/p38 axis.     

Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.     

m6A modification of HSATIII lncRNAs regulates temperature‐dependent splicing

Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress‐inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature‐dependent Ser/Arg‐rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA‐binding proteins revealed HSATIII association with proteins related to N⁶‐methyladenosine (m⁶A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m⁶A‐modified. nSBs sequester the m⁶A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m⁶A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m⁶A modification of pre‐mRNAs, leading to repression of m⁶A‐dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature‐dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m⁶A‐modified architectural lncRNAs.     

Effect of MiR‐100‐5p on proliferation and apoptosis of goat endometrial stromal cell in vitro and embryo implantation in vivo

The growth of endometrial stromal cells (ESCs) at implantation sites may be a potential factor affecting the success rate of embryo implantation. Incremental proofs demonstrated that ncRNAs (e.g. miRNAs, lncRNAs and circRNAs) were involved in various biological procedures, including proliferation and apoptosis. In this study, the role of miR‐100‐5p on proliferation and apoptosis of goat ESCs in vitro and embryo implantation in vivo was determined. The mRNA expression of miR‐100‐5p was significantly inhibited in the receptive phase (RE) rather than in the pre‐receptive phase (PE). Overexpression of miR‐100‐5p suppressed ESCs proliferation and induced apoptosis. The molecular target of MiR‐100‐5p, HOXA1, was confirmed by 3′‐UTR assays. Meanwhile, the product of HOXA1 mRNA RT‐PCR increased in the RE more than that in the PE. The HOXA1‐siRNA exerted significant negative effects on growth arrest. Instead, incubation of ESCs with miR‐100‐5p inhibitor or overexpressed HOXA1 promoted the cell proliferation. In addition, Circ‐9110 which acted as a sponge for miR‐100‐5p reversed the relevant biological effects of miR‐100‐5p. The intrinsic apoptosis pathway was suppressed in ESCs, revealing a crosstalk between Circ‐9110/miR‐100‐5p/HOXA1 axis, PI3K/AKT/mTOR, and ERK1/2 pathways. To further evaluate the progress in study on embryo implantation regulating mechanism of miR‐100‐5p in vivo, the pinopodes of two phases were observed and analysed, suggesting that, as similar as in situ, miR‐100‐5p was involved in significantly regulating embryo implantation in vivo. Mechanistically, miR‐100‐5p performed its embryo implantation function through regulation of PI3K/AKT/mTOR and ERK1/2 pathways by targeting Circ‐9110/miR‐100‐5p/HOXA1 axis in vivo.