Circ‐CTNNB1 drives aerobic glycolysis and osteosarcoma progression via m6A modification through interacting with RBM15

ObjectivesCircular RNAs (circRNAs) are a subclass of noncoding RNAs, playing essential roles in tumorigenesis and aggressiveness. Recent studies have revealed the pivotal functions of circ‐CTNNB1 (a circular RNA derived from CTNNB1) in cancer progression. However, little is known about the role of circ‐CTNNB1 in osteosarcoma (OS), a highly malignant bone tumour in children and adolescents.MethodsCirc‐CTNNB1 was analysed by qRT‐PCR, and the results were confirmed by Sanger sequencing. The interaction and effects between circ‐CTNNB1 and RNA binding motif protein 15 (RBM15) were analysed through biotin‐labelled RNA pull‐down and mass spectrometry, in vitro binding, and RNA electrophoretic mobility shift assays. In vitro and in vivo experiments were performed to evaluate the biological functions and underlying mechanisms of circ‐CTNNB1 and RBM15 in OS cells.ResultsCirc‐CTNNB1 was highly expressed in OS tissues and predominantly detected in the nucleus of OS cells. Ectopic expression of circ‐CTNNB1 promoted the growth, invasion, and metastasis of OS cells in vitro and in vivo. Mechanistically, circ‐CTNNB1 interacted with RBM15 and subsequently promoted the expression of hexokinase 2 (HK2), glucose‐6‐phosphate isomerase (GPI) and phosphoglycerate kinase 1 (PGK1) through N6‐methyladenosine (m6A) modification to facilitate the glycolysis process and activate OS progression.ConclusionsCirc‐CTNNB1 drives aerobic glycolysis and OS progression by facilitating RBM15‐mediated m6A modification.

Circ‐CTNNB1 was highly expressed in osteosarcoma (OS) tissues and predominantly detected in the nucleus of OS cells. Ectopic expression of circ‐CTNNB1 promoted the growth, invasion, and metastasis of OS cells in vitro and in vivo. Mechanistically, circ‐CTNNB1 interacted with RBM15 and subsequently promoted the expression of hexokinase 2 (HK2), glucose‐6‐phosphate isomerase (GPI), and phosphoglycerate kinase 1 (PGK1) through N6‐methyladenosine (m6A) modification to facilitate the glycolysis process and activate OS progression. Circ‐CTNNB1 drives aerobic glycolysis and OS progression by facilitating RBM15‐mediated m6A modification.     

Effect of TLR4/MyD88/NF‐kB axis in paraventricular nucleus on ventricular arrhythmias induced by sympathetic hyperexcitation in post‐myocardial infarction rats

Sympathetic activation after myocardial infarction (MI) leads to ventricular arrhythmias (VAs), which can result in sudden cardiac death (SCD). The toll‐like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/nuclear factor‐kappa B (NF‐kB) axis within the hypothalamic paraventricular nucleus (PVN), a cardiac‐neural sympathetic nerve centre, plays an important role in causing VAs. An MI rat model and a PVN‐TLR4 knockdown model were constructed. The levels of protein were detected by Western blotting and immunofluorescence, and localizations were visualized by multiple immunofluorescence staining. Central and peripheral sympathetic activation was visualized by immunohistochemistry for c‐fos protein, renal sympathetic nerve activity (RSNA) measurement, heart rate variability (HRV) analysis and norepinephrine (NE) level detection in serum and myocardial tissue measured by ELISA. The arrhythmia scores were measured by programmed electrical stimulation (PES), and cardiac function was detected by the pressure–volume loop (P‐V loop). The levels of TLR4 and MyD88 and the nuclear translocation of NF‐kB within the PVN were increased after MI, while sympathetic activation and arrhythmia scores were increased and cardiac function was decreased. However, inhibition of TLR4 significantly reversed these conditions. PVN‐mediated sympathetic activation via the TLR4/MyD88/NF‐kB axis ultimately leads to the development of VAs after MI.     

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 hsa_circ_0008003 facilitates tumorigenesis and development of non‐small cell lung carcinoma via modulating miR‐488/ZNF281 axis

As one of the most aggressive malignancies, non‐small cell lung carcinoma (NSCLC) has high risks of death. It has been demonstrated that circRNAs accelerate NSCLC progression, but the underlying molecular mechanisms of circRNAs in NSCLC were still obscure. In the first place, the circRNA microarray of NSCLC was investigated in this study, and hsa_circ_0008003 (circ‐0008003) was chosen as the research object. Then, it was unveiled that the expression of circ‐0008003 examined via qRT‐PCR was elevated in tumour tissues relative to the non‐tumour tissues, which was associated with TNM stage and lymphatic metastasis in NSCLC. Additionally, the prognosis of NSCLC patients with high circ‐0008003 level was poor. Besides, circ‐0008003 silencing dampened the invasion and proliferation of NSCLC cells. Next, according to the mechanistic studies, circ‐0008003 functioned as a ceRNA of ZNF281 in NSCLC by acting as the endogenous sponge for miR‐488, which was proved to be a tumour suppressor in NSCLC. Additionally, ZNF281 overexpression and miR‐488 suppression recovered the influences of repressed circ‐0008003 on NSCLC cellular processes. It was validated in this research that circ‐0008003 triggered tumour formation in NSCLC, which was adjusted via miR‐488/ZNF281 axis, casting a novel light on the resultful target for treating NSCLC and predicting the prognosis.     

Dental pulp stem cell‐derived exosomes alleviate cerebral ischaemia‐reperfusion injury through suppressing inflammatory response

ObjectivesThe study aimed to determine whether dental pulp stem cell‐derived exosomes (DPSC‐Exos) exert protective effects against cerebral ischaemia‐reperfusion (I/R) injury and explore its underlying mechanism.Materials and MethodsExosomes were isolated from the culture medium of human DPSC. Adult male C57BL/6 mice were subjected to 2 hours transient middle cerebral artery occlusion (tMCAO) injury followed by 2 hours reperfusion, after which singular injection of DPSC‐Exos via tail vein was administrated. Brain oedema, cerebral infarction and neurological impairment were measured on day 7 after exosomes injection. Then, oxygen‐glucose deprivation–reperfusion (OGD/R) induced BV2 cells were studied to analyse the therapeutic effects of DPSC‐Exos on I/R injury in vitro. Protein levels of TLR4, MyD88, NF‐κB p65, HMGB1, IL‐6, IL‐1β and TNF‐α were determined by western blot or enzyme‐linked immunosorbent assay. The cytoplasmic translocation of HMGB1 was detected by immunofluorescence staining.ResultsDPSC‐Exos alleviated brain oedema, cerebral infarction and neurological impairment in I/R mice. DPSC‐Exos inhibited the I/R‐mediated expression of TLR4, MyD88 and NF‐κB significantly. DPSC‐Exos also reduced the protein expression of IL‐6, IL‐1β and TNF‐α compared with those of the control both in vitro and in vivo. Meanwhile, DPSC‐Exos markedly decreased the HMGB1 cytoplasmic translocation induced by I/R damage.ConclusionsDPSC‐Exos can ameliorate I/R‐induced cerebral injury in mice. Its anti‐inflammatory mechanism might be related with the inhibition of the HMGB1/TLR4/MyD88/NF‐κB pathway.     

Sparstolonin B suppresses free fatty acid palmitate‐induced chondrocyte inflammation and mitigates post‐traumatic arthritis in obese mice

Abnormal lipid metabolism, such as systemic increased free fatty acid, results in overproduction of pro‐inflammatory enzymes and cytokines, which is crucial in the development of obesity‐related osteoarthritis (OA). However, there are only a few drugs that target the lipotoxicity of OA. Recent researches have documented that the traditional Chinese medicine, Sparstolonin B (Ssn B), exerted anti‐inflammatory effects in various diseases, but not yet in OA. On the basis of this evidence, our works purposed to evaluate the effect of Ssn B on free fatty acid (FFA) palmitate (PA)‐stimulated human osteoarthritic chondrocytes and obesity‐associated mouse OA model. We found that Ssn B suppressed PA‐triggered inflammatory response and extracellular matrix catabolism in a concentration‐dependent approach. In vivo, Ssn B treatment inhibited cartilage degeneration and subchondral bone calcification caused by joint mechanical imbalance and alleviated metabolic inflammation in obesity. Mechanistically, co‐immunoprecipitine and molecular docking analysis showed that the formation of toll­like receptor 4 (TLR4)/myeloid differentiation protein‐2 (MD‐2) complex caused by PA was blocked by Ssn B. Subsequently, it leads to inactivation of PA‐caused myeloid differentiation factor 88 (MyD88)‐dependent nuclear factor‐kappaB (NF‐κB) cascade. Together, these findings demonstrated that Ssn B is a potential treatment agent for joint degenerative diseases in obese individuals.     

Tacrolimus alleviates LPS‐induced AKI by inhibiting TLR4/MyD88/NF‐κB signalling in mice

Lipopolysaccharide (LPS)‐induced sepsis‐associated acute kidney injury (SA‐AKI) is a model of clinical serious care syndrome, with high morbidity and mortality. Tacrolimus (TAC), a novel immunosuppressant that inhibits inflammatory response, plays a pivotal role in kidney diseases. In this study, LPS treated mice and cultured podocytes were used as the models of SA‐AKI in vivo and in vitro, respectively. Medium‐ and high‐dose TAC administration significantly attenuated renal function and renal pathological manifestations at 12, 24 and 48 h after LPS treatment in mice. Moreover, the Toll‐like receptor 4 (TLR4)/myeloid differential protein‐88 (MyD88)/nuclear factor‐kappa (NF‐κB) signalling pathway was also dramatically inhibited by medium‐ and high‐dose TAC administration at 12, 24 and 48 h of LPS treatment mice. In addition, TAC reversed LPS‐induced podocyte cytoskeletal injury and podocyte migratory capability. Our findings indicate that TAC has protective effects against LPS‐induced AKI by inhibiting TLR4/MyD88/NF‐κB signalling pathway and podocyte dysfunction, providing another potential therapeutic effects for the LPS‐induced SA‐AKI.     

Circular RNA circEIF4G2 aggravates renal fibrosis in diabetic nephropathy by sponging miR‐218

Circular RNAs play essential roles in the development of various human diseases. However, how circRNAs are involved in diabetic nephropathy (DN) are not fully understood. Our study aimed to investigate the effects of circRNA circEIF4G2 on DN. Experiments were performed in the db/db mouse model of type 2 diabetes and NRK‐52E cells. We found that circEIF4G2 was significantly up‐regulated in the kidneys of db/db mice and NRK‐52E cells stimulated by high glucose. circEIF4G2 knockdown inhibited the expressions of TGF‐β1, Collagen I and Fibronectin in high glucose‐stimulated NRK‐52E cells, which could be rescued by miR‐218 inhibitor. Knockdown of SERBP1 reduced the expression of TGF‐β1, Collagen I and Fibronectin in HG‐stimulated NRK‐52E cells. In summary, our findings suggested that circEIF4G2 promotes renal tubular epithelial cell fibrosis via the miR‐218/SERBP1 pathway, presenting a novel insight for DN treatment.     

LncRNA SNHG1 promotes sepsis‐induced myocardial injury by inhibiting Bcl‐2 expression via DNMT1

Myocardial injury is a frequently occurring complication of sepsis. This study aims to investigate the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1)‐mediated DNA methyltransferase 1/B‐cell lymphoma‐2 (DNMT1/Bcl‐2) axis in sepsis‐induced myocardial injury. Mice and HL‐1 cells were treated with lipopolysaccharide (LPS) to establish animal and cellular models simulating sepsis and inflammation. LncRNA SNHG1 was screened out as a differentially expressed lncRNA in sepsis samples through microarray profiling, and the upregulated expression of lncRNA SNHG1 was confirmed in myocardial tissues of LPS‐induced septic mice and HL‐1 cells. Further experiments suggested that silencing of lncRNA SNHG1 reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. LncRNA SNHG1 inhibited Bcl‐2 expression by recruiting DNMT1 to Bcl‐2 promoter region to cause methylation. Inhibition of Bcl‐2 promoter methylation reduced the inflammation and apoptotic rate of LPS‐induced HL‐1 cells. In vivo experiments substantiated that lncRNA SNHG1 silencing alleviated sepsis‐induced myocardial injury in mice. Taken together, lncRNA SNHG1 promotes LPS‐induced myocardial injury in septic mice by downregulating Bcl‐2 through DNMT1‐mediated Bcl‐2 methylation.     

Nicorandil inhibits TLR4/MyD88/NF-κB/NLRP3 signaling pathway to reduce pyroptosis in rats with myocardial infarction

Pyroptosis is an inflammatory cell death that regulates cardiomyocyte loss after myocardial infarction. Reports indicate that nicorandil has a strong anti-inflammatory effect and protects the myocardium from myocardial infarction. However, its relationship with pyroptosis is largely unreported. Here, we investigated to influence and mechanism of action of nicorandil on cardiomyocyte pyroptosis. Forty Sprague Dawley rats were randomly assigned to sham, MI, MI + nicorandil, and MI + nicorandil + TAK242 groups (10 per group). Myocardial infarction modeling was performed through ligation of the anterior descending branch of the left coronary artery. The function of cardiac was evaluated through echocardiography, detection of myocardial adenine nucleotides, cTnI, LDH, TTC, and HE staining. Moreover, we used qRT-PCR, immunohistochemistry, and Western blotting to examine the expression of pyroptosis-related molecules and the inflammasome pathway of TLR4/MyD88/NF-κB/NLRP3. Myocardial infarction caused the activation of GSDMD, aggravated myocardial injury, and triggered cardiac dysfunction. Myocardial infarction induced pyroptotic cell death, manifested as upregulation in mRNA and protein levels associated with pyroptosis, including caspase-1 cleavage and increased expression of IL-1β and IL-18. These changes were mitigated by nicorandil. The achieved data implicate that myocardial infarction induces pyroptosis via the TLR4/MyD88/NF-κB/NLRP3 pathway, which can be inhibited by nicorandil pretreatment. Therefore, nicorandil exerts cardioprotective effects by activating K_ATP channels, and at least in part through inhibition of the TLR4/MyD88/NF-κB/NLRP3 pathway to reduce myocardial infarction-induced pyroptosis. As such, it is a potential therapy for ischemic heart disease.     

LncRNA FAF attenuates hypoxia/ischaemia‐induced pyroptosis via the miR‐185‐5p/PAK2 axis in cardiomyocytes

Pyroptosis is associated with various cardiovascular diseases. Increasing evidence suggests that long noncoding RNAs (lncRNAs) have been implicated in gene regulation, but how lncRNAs participate in the regulation of pyroptosis in the heart remains largely unknown. In this study, we aimed to explore the antipyroptotic effects of lncRNA FGF9‐associated factor (FAF) in acute myocardial infarction (AMI). The expression patterns of lncRNA FAF, miR‐185‐5p and P21 activated kinase 2 (PAK2) were detected in hypoxia/ischaemia‐induced cardiomyocytes. Hoechst 33342/PI staining, lactate dehydrogenase (LDH) release assay, immunofluorescence and Western blotting were conducted to assay cell pyroptosis. The interaction between lncRNA FAF, miR‐185‐5p and PAK2 was verified by bioinformatics analysis, small RNA sequencing luciferase reporter assay and qRT‐PCR. The expression of LncRNA FAF was downregulated in hypoxic cardiomyocytes and myocardial tissues. Overexpression of lncRNA FAF could attenuate cardiomyocyte pyroptosis, improve cell viability and reduce infarct size during the procession of AMI. Moreover, lncRNA FAF was confirmed as a sponge of miR‐185‐5p and promoted PAK2 expression in cardiomyocytes. Collectively, our findings reveal a novel lncRNA FAF/miR‐185‐5p/PAK2 axis as a crucial regulator in cardiomyocyte pyroptosis, which might be a potential therapeutic target of AMI.     

The role of E2A in ATPR‐induced cell differentiation and cycle arrest in acute myeloid leukaemia cells

Acute myeloid leukaemia (AML) is a biologically heterogeneous disease with an overall poor prognosis; thus, novel therapeutic approaches are needed. Our previous studies showed that 4‐amino‐2‐trifluoromethyl‐phenyl retinate (ATPR), a new derivative of all‐trans retinoic acid (ATRA), could induce AML cell differentiation and cycle arrest. The current study aimed to determine the potential pharmacological mechanisms of ATPR therapies against AML. Our findings showed that E2A was overexpressed in AML specimens and cell lines, and mediate AML development by inactivating the P53 pathway. The findings indicated that E2A expression and activity decreased with ATPR treatment. Furthermore, we determined that E2A inhibition could enhance the effect of ATPR‐induced AML cell differentiation and cycle arrest, whereas E2A overexpression could reverse this effect, suggesting that the E2A gene plays a crucial role in AML. We identified P53 and c‐Myc were downstream pathways and targets for silencing E2A cells using RNA sequencing, which are involved in the progression of AML. Taken together, these results confirmed that ATPR inhibited the expression of E2A/c‐Myc, which led to the activation of the P53 pathway, and induced cell differentiation and cycle arrest in AML.     

Circular RNA CircSLC8A1 contributes to osteogenic differentiation in hBMSCs via CircSLC8A1/miR‐144‐3p/RUNX1 in periprosthetic osteolysis

Circular RNAs (circRNAs) are often found in eukaryocyte and have a role in the pathogenesis of a variety of human disorders. Our related research has shown the differential expression of circRNAs in periprosthetic osteolysis (PPOL). However, the involvement of circRNAs in the exact process is yet unknown. CircSLC8A1 expression was evaluated in clinical samples and human bone marrow mesenchymal stem cells (hBMSCs) in this investigation using quantitative real‐time PCR. In vitro and in vivo studies were conducted to explicate its functional role and pathway. We demonstrated CircSLC8A1 is involved in PPOL using gain‐ and loss‐of‐function methods. The association of CircSLC8A1 and miR‐144‐3p, along with miR‐144‐3p and RUNX1, was predicted using bioinformatics. RNA pull‐down and luciferase assays confirmed it. The impact of CircSLC8A1 in the PPOL‐mouse model was also investigated using adeno‐associated virus. CircSLC8A1 was found to be downregulated in PPOL patients'' periprosthetic tissues. Overexpression of CircSLC8A1 promoted osteogenic differentiation (OD) and inhibited apoptosis of hBMSCs in vitro. The osteogenic markers of RUNX1, osteopontin (OPN) and osteocalcin (OCN) were significantly upregulated in hBMSCs after miR‐144‐3p inhibitor was transferred. Mechanistic analysis demonstrated that CircSLC8A1 directly bound to miR‐144‐3p and participated in PPOL through the miR‐144‐3p/RUNX1 pathway in hBMSCs. Micro‐CT and quantitative analysis showed that CircSLC8A1 markedly inhibited PPOL, and osteogenic markers (RUNX1, OPN and OCN) were significantly increased (P<0.05) in the mice model. Our findings prove that CircSLC8A1 exerted a regulatory role in promoting osteogenic differentiation in hBMSCs, and CircSLC8A1/miR‐144‐3p/RUNX1 pathway may provide a potential target for prevention of PPOL.     

Cinnamic Aldehyde,the main monomer component of Cinnamon,exhibits anti‐inflammatory property in OA synovial fibroblasts via TLR4/MyD88 pathway

Cinnamon is a wildly used traditional Chinese herbal medicine for osteoarthritis (OA) treatment, but the underlying mechanism remains ambiguous. The purpose of this study is to explore the mechanism of cinnamic aldehyde (CA), a bioactive substance extracted from Cinnamon, on synovial inflammation in OA. A total of 144 CA‐OA co‐targeted genes were identified by detect databases (PubChem, HIT, TCMSP, TTD, DrugBank and GeneCards). The results of GO enrichment analysis indicated that these co‐targeted genes have participated in many biological processes including ‘inflammatory response’, ‘cellular response to lipopolysaccharide’, ‘response to drug’, ‘immune response’, ‘lipopolysaccharide‐mediated signalling pathway’, etc. KEGG pathway analysis showed these co‐targeted genes were mainly enriched in ‘Toll‐like receptor signalling pathway’, ‘TNF signalling pathway’, ‘NF‐kappa B signalling pathway’, etc. Molecular docking demonstrated that CA could successfully bind to TLR2 and TLR4. The results of in vitro experiments showed no potential toxicity of 10, 20 and 50 μM/L CA on human OA FLS, and CA can significantly inhibit the inflammation in LPS‐induced human FLS. Further experimental mechanism evidence confirmed CA can inhibited the inflammation in LPS‐induced human OA FLS via blocking the TLR4/MyD88 signalling pathway. Our results demonstrated that CA exhibited strong anti‐inflammation effect in OA FLS through blocking the activation of TLR4/MyD88 signalling pathway, suggesting its potential as a hopeful candidate for the development of novel agents for the treatment of OA.     

Overexpression of FOXD2‐AS1 enhances proliferation and impairs differentiation of glioma stem cells by activating the NOTCH pathway via TAF‐1

Emerging data have highlighted the importance of long noncoding RNAs (lncRNAs) in exerting critical biological functions and roles in different forms of brain cancer, including gliomas. In this study, we sought to investigate the role of lncRNA FOXD2 adjacent opposite strand RNA 1 (FOXD2‐AS1) in glioma cells. First, we used sphere formation assay and flow cytometry to select U251 glioma stem cells (GSCs). Then, we quantified the expression of lncRNA FOXD2‐AS1, TATA‐box binding protein associated factor 1 (TAF‐1) and NOTCH1 in glioma tissues and GSCs, as well as the expression of GSC stem markers, OCT4, SOX2, Nanog, Nestin and CD133 in GSCs. Colony formation assay, sphere formation assay, and flow cytometry were used to evaluate GSC stemness. Next, the correlations among lncRNA FOXD2‐AS1, TAF‐1 and NOTCH1 were investigated. LncRNA FOXD2‐AS1, TAF‐1 and NOTCH1 were found to be elevated in glioma tissues and GSCs, and silencing lncRNA FOXD2‐AS1 inhibited stemness and proliferation, while promoting apoptosis and differentiation of GSCs. LncRNA FOXD2‐AS1 overexpression also led to increased NOTCH1 by recruiting TAF‐1 to the NOTCH1 promoter region, thereby promoting stemness and proliferation, while impairing cell apoptosis and differentiation. Mechanistically, lncRNA FOXD2‐AS1 elevation promoted glioma in vivo by activating the NOTCH signalling pathway via TAF‐1 upregulation. Taken together, the key findings of our investigation support the proposition that downregulation of lncRNA FOXD2‐AS1 presents a viable and novel molecular candidate for improving glioma treatment.