LncRNA DLX6-AS1 regulates osteosarcoma progression via the miR-200a-3p/GPM6P axis

LncRNA DLX6-AS1 takes part in the progression of various cancers. However, it is not elaborated clearly in osteosarcoma (OS) development. Therefore, we aimed to explore the impacts and specific mechanisms of DLX6-AS1 on the progression of OS. We estimated the pattern of DLX6-AS1 expression in Ost tissues and cells via quantitative reverse transcription polymerase chain reaction. A number of biochemical assays were carried out to assess the effects of DLX6-AS1. Target genes were predicted by bioinformatics methods. Then we used the transfection of si-RNA, miRNA inhibitor, and miRNA mimics to explore the underlying mechanisms and built tumor xenograft models for the in vivo experiments. A higher expression of DLX6-AS1 was found in OS tissues and cell lines, while knockdown of DXL6-AS1 suppressed OS cell metastasis and proliferation in vitro and in vivo. Mechanistically, it was revealed that DXL6-AS1 sponged miR-200a-3p, thus positively regulating the downstream GPM6B. In summary, DLX6-AS1 knockdown would inhibit OS cell migration, cell invasion, and cell proliferation, in which the DXL6-AS1/ miR-200a-3p/ GPM6B axis played a critical role.

Enpp2 Expression by Dendritic Cells Is a Key Regulator in Migration

Enpp2 is an enzyme that catalyzes the conversion of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), which exhibits a wide variety of biological functions. Here, we examined the biological effects of Enpp2 on dendritic cells (DCs), which are specialized antigen-presenting cells (APCs) characterized by their ability to migrate into secondary lymphoid organs and activate naïve T-cells. DCs were generated from bone marrow progenitors obtained from C57BL/6 mice. Enpp2 levels in DCs were regulated using small interfering (si)RNA or recombinant Enpp2. Expression of Enpp2 in LPS-stimulated mature (m)DCs was high, however, knocking down Enpp2 inhibited mDC function. In addition, the migratory capacity of mDCs increased after treatment with rmEnpp2; this phenomenon was mediated via the RhoA-mediated signaling pathway. Enpp2-treated mDCs showed a markedly increased capacity to migrate to lymph nodes in vivo. These findings strongly suggest that Enpp2 is necessary for mDC migration capacity, thereby increasing our understanding of DC biology. We postulate that regulating Enpp2 improves DC migration to lymph nodes, thus improving the effectiveness of cancer vaccines based on DC.

circRNA-Mediated Inhibin–Activin Balance Regulation in Ovarian Granulosa Cell Apoptosis and Follicular Atresia

Ovarian granulosa cells (GC) play an essential role in the development and atresia of follicles. Emerging studies suggest that non-coding RNAs are involved in the regulation of GC apoptosis. Here, we aimed to analyze the function of ssc-circINHA-001, coded by the first exon of the inhibin subunit α gene (INHA), in resisting GC apoptosis and follicular atresia by enhancing the expression of the inhibin subunit β A (INHBA) through a cluster of miRNAs. A higher expression of ssc-circINHA-001 in healthy follicles compared to early atretic follicles was detected by qRT-PCR. Its circular structure was confirmed by RNase R treatment and reversed PCR. The function of ssc-circINHA-001 in GC resistance to apoptosis was detected by in vitro transfection of its si-RNA. Furthermore, the dual-luciferase reporter assay suggested that ssc-circINHA-001 adsorbed three miRNAs, termed miR-214-5p, miR-7144-3p, and miR-9830-5p, which share the common target INHBA. A low expression of ssc-circINHA-001 increased the levels of the free miRNAs, inhibited INHBA expression, and thus raised GCs apoptosis through a shift from the secretion of activin to that of inhibin. Our study demonstrated the existence of a circRNA–microRNAs–INHBA regulatory axis in follicular GC apoptosis and provides insight into the relationship between circRNA function and its coding gene in inhibin/activin balance and ovarian physiological functions.

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina

Extracellular vesicles (EVs) have great potential as novel drug carriers for the treatment of various diseases. These lipid bilayer vesicles are naturally abundant in mammalian tissues and circulation, can be loaded with therapeutic small molecule drugs, (si)RNA, proteins and CRISPR/Cas9, and may be engineered for retention by specific tissues. However, many questions remain on the optimal dosing, administration route, and pharmacokinetics of EVs. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available from larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had markedly longer circulation times in plasma than previously reported in mice, and were detectable in CSF after 30-60 minutes. Already after one minute following IV administration, we observed EV uptake by PBMCs, most notably B-cells. EVs were detected in liver and spleen within one hour of IV administration. None of the IN doses resulted in readily detectable EV levels in plasma, CSF, or organs, suggesting that IN delivery of EVs in large animals including humans may require reconsideration. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

Glutamine deficiency shifts the asthmatic state toward neutrophilic airway inflammation

Background: The administration of L-glutamine (Gln) suppresses allergic airway inflammation via the rapid upregulation of MAPK phosphatase (MKP)-1, which functions as a negative regulator of inflammation by deactivating p38 and JNK mitogen-activated protein kinases (MAPKs). However, the role of endogenous Gln remains to be elucidated. Therefore, we investigated the mechanism by which endogenous Gln regulates MKP-1 induction and allergic airway inflammation in an ovalbumin-based murine asthma model. Methods: We depleted endogenous Gln levels using l-γ-glutamyl- p-nitroanilide (GPNA), an inhibitor of the Gln transporter ASCT2, and glutamine synthetase small interfering (si)RNA. Lentivirus expressing MKP-1 was injected to achieve overexpression of MKP-1. Asthmatic phenotypes were assessed using our previously developed ovalbumin-based murine model, which is suitable for examining sequential asthmatic events, including neutrophil infiltration. Gln levels were analyzed using a Gln assay kit. Results: GPNA or glutamine synthetase siRNA successfully depleted endogenous Gln levels. Importantly, homeostatic MKP-1 induction did not occur at all, which resulted in prolonged p38 MAPK and cytosolic phospholipase A (cPLA ) phosphorylation in Gln-deficient mice. Gln deficiency augmented all examined asthmatic reactions, but it exhibited a strong bias toward increasing the neutrophil count, which was not observed in MKP-1-overexpressing lungs. This neutrophilia was inhibited by a cPLA inhibitor and a leukotriene B4 inhibitor, but not by dexamethasone. Conclusion: Gln deficiency leads to the impairment of MKP-1 induction and activation of p38 MAPK and cPLA , resulting in the augmentation of neutrophilic, more so than eosinophilic, airway inflammation.

Hypoxia-induced preadipocyte factor 1 expression in human lung fibroblasts through ERK/PEA3/c-Jun pathway

Background Several studies have reported that hypoxia plays a pathological role in severe asthma and tissue fibrosis. Our previous study showed that hypoxia induces A disintegrin and metalloproteinase 17 (ADAM17) expression in human lung fibroblasts. Moreover, preadipocyte factor 1 (Pref-1) is cleaved by ADAM17, which participates in adipocyte differentiation. Furthermore, Pref­1 overexpression is involved in tissue fibrosis including liver and heart. Extracellular signal-regulated kinase (ERK) could active downstram gene expression through polyoma enhancer activator 3 (PEA3) phosphorylation. Studies have demonstrated that PEA3 and activator protein 1 (AP-1) play crucial roles in lung fibrosis, and the Pref-1 promoter region contains PEA3 and AP-1 binding sites as predicted. However, the roles of ERK, PEA3, and AP-1 in hypoxia-stimulated Pref-1 expression in human lung fibroblasts remain unknown. Methods The protein expression in ovalbumin (OVA)-induced asthmatic mice was performed by immunohistochemistry and immunofluorescence. The protein expression or the mRNA level in human lung fibroblasts (WI-38) was detected by western blot or quantitative PCR. Small interfering (si) RNA was used to knockdown gene expression. The collaboration with PEA3 and c-Jun were determined by coimmunoprecipitation. Translocation of PEA3 from the cytosol to the nucleus was observed by immunocytochemistry. The binding ability of PEA3 and AP-1 to Pref-1 promoter was assessed by chromatin immunoprecipitation. Results Pref-1 and hypoxia-inducible factor 1α (HIF-1α) were expressed in the lung sections of OVA-treated mice. Colocalization of PEA3 and Fibronectin was detected in lung sections from OVA-treated mice. Futhermore, Hypoxia induced Pref­1 protein upregulation and mRNA expression in human lung fibroblasts (WI­38 cells). In 60 confluent WI-38 cells, hypoxia up-regulated HIF-1α and Pref-1 protein expression. Moreover, PEA3 small interfering (si) RNA decreased the expression of hypoxia-induced Pref­1 in WI­38 cells. Hypoxia induced PEA3 phosphorylation, translocation of PEA3 from the cytosol to the nucleus, PEA3 recruitment and AP-1 binding to the Pref­1 promoter region, and PEA3-luciferase activity. Additionally, hypoxia induced c-Jun-PEA3 complex formation. U0126 (an ERK inhibitor), curcumin (an AP­1 inhibitor) or c-Jun siRNA downregulated hypoxia-induced Pref-1 expression. Conclusions These results implied that ERK, PEA3, and AP­1 participate in hypoxia­induced Pref­1 expression in human lung fibroblasts.

An Aedes Aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections

Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

The Rho kinase signaling pathway participates in tubular mitochondrial oxidative injury and apoptosis in uric acid nephropathy

Introduction Oxidative stress is a pathologic feature of hyperuricemia that is highly prevalent and that contributes to kidney tubular interstitial fibrosis. Rho-kinase is closely related to mitochondrial-induced oxidative stress. Herein, we designed a study to explore the expression and role of Rho-kinase in hyperuricemia nephropathy. The secondary objective was to investigate whether the Rho-kinase signaling pathway regulates hyperuricemic tubular oxidative injury and apoptosis via the mitochondrial pathway in addition to the mechanisms that are involved. Materials and methods HK-2 cells were divided into the following five groups: normal; uric acid (UA); UA+Fasudil; UA+ROCK1 si-RNA; and UA+sc-siRNA. Rho-kinase activity, mitochondrial oxidative injury, and apoptosis-related protein levels were measured in each group. A t-test was used to analyze the difference between groups. Results Myosin phosphatase target subunit 1 (MYPT1) overexpression was shown in HK-2 cells, which was caused by UA. High concentrations of UA also up-regulated Rho-kinase expression and mitochondrial and apoptosis-related protein expression, while treatment with fasudil and ROCK1 si-RNA significantly attenuated these responses. Conclusion The Rho-kinase signaling pathway participates in tubular mitochondrial oxidative injury and apoptosis via regulating mitochondrial dyneins/biogenic genes in UA nephropathy, which suggests that the mitochondrial pathway might be a potential therapeutic target for hyperuricemia nephropathy.

The high expression of miR-564 in patients with systemic lupus erythematosus promotes differentiation and maturation of DC cells by negatively regulating TP53 expression in vitro

Background miRNA is involved in the occurrence and progression of systemic lupus erythematosus (SLE), but the regulatory effect of miRNA on dendritic cells in SLE patients is still unclear. Material and methods Bioinformatics methods were used to analyze the differentially expressed miRNA and its target genes in SLE patients. In vitro experiments were conducted to explore the effects and mechanisms of differentially expressed miRNAs in SLE patients on the differentiation and maturation of monocyte-derived dendritic cells. Results Bioinformatics analysis showed that miR-564 was up-regulated in SLE patients, and TP53 was the core target gene of miR-564. The expression level of miR-564 showed a rising trend during the differentiation and maturation of monocytes into Mo-DC cells. The differentiation, maturation and proliferation of Mo-DC cells were significantly inhibited by transfection with miR-564 antagomir. The expression of TP53 is negatively regulated by miR-564. In rescue experiments, the proliferation and migration of DC cells were significantly restored by co-transfection of miR-564 antagomir and TP53 si-RNA. Conclusion Highly expressed miR-564 promotes the maturation, proliferation of Mo-DC cells by negatively regulating the expression of TP53.

Hypoxia-induced Preadipocyte factor 1 expression in human lung fibroblasts through ERK/PEA3/c-Jun pathway

Background Several studies have reported that hypoxia plays a pathological role in severe asthma and tissue fibrosis. Our previous study showed that hypoxia induces A disintegrin and metalloproteinase 17 (ADAM17) expression in human lung fibroblasts. Moreover, preadipocyte factor 1 (Pref-1) is cleaved by ADAM17, which participates in adipocyte differentiation. Furthermore, Pref­1 overexpression is involved in liver and heart fibrosis. Studies have demonstrated that polyoma enhancer activator 3 (PEA3) and activator protein 1 (AP-1) play crucial roles in lung fibrosis, and the Pref-1 promoter region contains PEA3 and AP-1 binding sites, as predicted. However, the roles of extracellular signal-regulated kinase (ERK), PEA3, and AP-1 in hypoxia-stimulated Pref-1 expression in human lung fibroblasts remain unknown. Methods The protein expression in ovalbumin (OVA)-induced asthmatic mice was performed by immunohistochemistry and immunofluorescence. The protein expression or the mRNA level in human lung fibroblasts (WI-38) was detected by western blot or quantitative PCR. Small interfering (si) RNA was used to knockdown gene expression. The collaboration with PEA3 and c-Jun were determined by coimmunoprecipitation. Translocation of PEA3 from the cytosol to the nucleus was observed by immunocytochemistry. The binding ability of PEA3 and AP-1 to Pref-1 promoter was assessed by chromatin immunoprecipitation. Results Pref-1, PEA3, and hypoxia-inducible factor 1α (HIF-1α) were expressed in the lung sections of ovalbumin-treated mice. Hypoxia induced Pref­1 protein upregulation and mRNA expression in human lung fibroblasts (WI­38 cells). Moreover, PEA3 small interfering (si) RNA decreased the expression of hypoxia-induced Pref­1 in WI­38 cells. Hypoxia induced PEA3 phosphorylation, translocation of PEA3 from the cytosol to the nucleus, PEA3 recruitment and AP-1 binding to the Pref­1 promoter region, and PEA3-luciferase activity. Furthermore, hypoxia induced c-Jun-PEA3 complex formation, and U0126 (an ERK inhibitor) or curcumin (an AP­1 inhibitor) downregulated hypoxia-induced Pref-1 expression. Conclusions These results implied that ERK, PEA3, and AP­1 participate in hypoxia­induced Pref­1 expression in human lung fibroblasts.