Identification and analysis of differently expressed transcription factors in aristolochic acid nephropathy.

BACKGROUND: Aristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephropathy caused by Aristolochic acid (AA). AAN is associated with the development of nephropathy and urothelial carcinoma. It is estimated that more than 100 million people worldwide are at risk of developing AAN. However, the underlying mechanisms driving renal deterioration in AAN remain poorly understood, and the treatment options are limited. METHODS: We obtained GSE27168 and GSE136276 series matrix data from the Gene Expression Omnibus (GEO) related to AAN. Using the R Studio environment, we applied the limma package and WGCNA package to identify co-differently expressed genes (co-DEGs). By GO/KEGG/GSVA analysis, we revealed common biological pathways. Subsequently, co-DEGs were subjected to the String database to construct a protein-protein interaction (PPI) network. The MCC algorithms implemented in the Cytohubba plugin were employed to identify hub genes. The hub genes were cross-referenced with the transcription factor (TF) database to identify hub TFs. Immune infiltration analysis was performed to identify key immune cell groups by utilizing CIBERSORT. The expressions of AAN-associated hub TFs were verified in vivo and in vitro. Finally, siRNA intervention was performed on the two TFs to verify their regulatory effect in AAN. RESULTS: Our analysis identified 88 co-DEGs through the "limma" and "WGCNA" R packages. A PPI network comprising 53 nodes and 34 edges was constructed with a confidence level >0.4. ATF3 and c-JUN were identified as hub TFs potentially linked to AAN. Additionally, expressions of ATF3 and c-JUN positively correlated with monocytes, basophils, and vessels, and negatively correlated with eosinophils and endothelial cells. We observed a significant increase in protein and mRNA levels of these two hub TFs. Furthermore, it was found that siRNA intervention targeting ATF3, but not c-JUN, alleviated cell damage induced by AA. The knockdown of ATF3 protects against oxidative stress and inflammation in the AAN cell model. CONCLUSION: This study provides novel insights into the role of ATF3 in AAN. The comprehensive analysis sheds light on the molecular mechanisms and identifies potential biomarkers and drug targets for AAN treatment.

Layer-by-Layer Assembly of Renal-Targeted Polymeric Nanoparticles for Robust Arginase-2 Knockdown and Contrast-Induced Acute Kidney Injury Prevention.

The mitochondrial enzyme arginase-2 (Arg-2) is implicated in the pathophysiology of contrast-induced acute kidney injury (CI-AKI). Therefore, Arg-2 represents a candid target for CI-AKI prevention. Here, layer-by-layer (LbL) assembled renal-targeting polymeric nanoparticles are developed to efficiently deliver small interfering RNA (siRNA), knockdown Arg-2 expression in renal tubules, and prevention of CI-AKI is evaluated. First, near-infrared dye-loaded poly(lactic-co-glycolic acid) (PLGA) anionic cores are electrostatically coated with cationic chitosan (CS) to facilitate the adsorption and stabilization of Arg-2 siRNA. Next, nanoparticles are coated with anionic hyaluronan (HA) to provide protection against siRNA leakage and shielding against early clearance. Sequential electrostatic layering of CS and HA improves loading capacity of Arg-2 siRNA and yields LbL-assembled nanoparticles. Renal targeting and accumulation is enhanced by modifying the outermost layer of HA with a kidney targeting peptide (HA-KTP). The resultant kidney-targeting and siRNA loaded nanoparticles (PLGA/CS/HA-KTP siRNA) exhibit proprietary accumulation in kidneys and proximal tubular cells at 24 h post-tail vein injection. In iohexol-induced in vitro and in vivo CI-AKI models, PLGA/CS/HA-KTP siRNA delivery alleviates oxidative and nitrification stress, and rescues mitochondrial dysfunction while reducing apoptosis, thereby demonstrating a robust and satisfactory therapeutic effect. Thus, PLGA/CS/HA-KTP siRNA nanoparticles offer a promising candidate therapy to protect against CI-AKI.

MMP13 promotes the osteogenic potential of BMP9 by enhancing Wnt/ß-catenin signaling via HIF-1α upregulation in mouse embryonic fibroblasts.

Bone morphogenetic protein 9 (BMP9) has been validated as one of the most potent osteoinduction factors, but its underlying mechanism remains unclear. As a member of the matrix metalloproteinase (MMP) family, MMP13 may be involved in regulating the lineage-specific differentiation of mouse embryonic fibroblasts (MEFs). The goal of this study was to determine whether MMP13 regulates the osteoinduction potential of BMP9 in MEFs, which are multipotent progenitor cells widely used for stem cell biology research. In vitro and in vivo experiments showed that BMP9-induced osteogenic markers and/or bone were enhanced by exogenous MMP13 in MEFs, but were reduced by MMP13 knockdown or inhibition. The expression of hypoxia inducible factor 1 alpha (HIF-1α) was induced by BMP9, which was enhanced by MMP13. The protein expression of ß-catenin and phosphorylation level of glycogen synthase kinase-3 beta (GSK-3ß) were increased by BMP9 in MEFs, as was the translocation of ß-catenin from the cytoplasm to the nucleus; all these effects of BMP9 were enhanced by MMP13. Furthermore, the MMP13 effects of increasing BMP9-induced ß-catenin protein expression and GSK-3ß phosphorylation level were partially reversed by HIF-1α knockdown. These results suggest that MMP13 can enhance the osteoinduction potential of BMP9, which may be mediated, at least in part, through the HIF-1α/ß-catenin axis. Our findings demonstrate a novel role of MMP13 in the lineage decision of progenitor cells and provide a promising strategy to speed up bone regeneration.

Identification and validation of hub genes in drug induced acute kidney injury basing on integrated transcriptomic analysis.

Background: Drug-induced acute kidney damage (DI-AKI) is a clinical phenomenon of rapid loss of kidney function over a brief period of time as a consequence of the using of medicines. The lack of a specialized treatment and the instability of traditional kidney injury markers to detect DI-AKI frequently result in the development of chronic kidney disease. Thus, it is crucial to continue screening for DI-AKI hub genes and specific biomarkers. Methods: Differentially expressed genes (DEGs) of group iohexol, cisplatin, and vancomycin's were analyzed using Limma package, and the intersection was calculated. DEGs were then put into String database to create a network of protein-protein interactions (PPI). Ten algorithms are used in the Cytohubba plugin to find the common hub genes. Three DI-AKI models' hub gene expression was verified in vivo and in vitro using PCR and western blot. To investigate the hub gene's potential as a biomarker, protein levels of mouse serum and urine were measured by ELISA kits. The UUO, IRI and aristolochic acid I-induced nephrotoxicity (AAN) datasets in the GEO database were utilized for external data verification by WGCNA and Limma package. Finally, the Elisa kit was used to identify DI-AKI patient samples. Results: 95 up-regulated common DEGs and 32 down-regulated common DEGs were obtained using Limma package. A PPI network with 84 nodes and 24 edges was built with confidence >0.4. Four hub genes were obtained by Algorithms of Cytohubba plugin, including TLR4, AOC3, IRF4 and TNFAIP6. Then, we discovered that the protein and mRNA levels of four hub genes were significantly changed in the DI-AKI model in vivo and in vitro. External data validation revealed that only the AAN model, which also belonged to DI-AKI model, had significant difference in these hub genes, whereas IRI and UUO did not. Finally, we found that plasma TLR4 levels were higher in patients with DI-AKI, especially in vancomycin-induced AKI. Conclusion: The immune system and inflammation are key factors in DI-AKI. We discovered the immunological and inflammatory-related genes TLR4, AOC3, IRF4, and TNFAIP6, which may be promising specific biomarkers and essential hub genes for the prevention and identification of DI-AKI.

Wnt/ß-Catenin Promotes the Osteoblastic Potential of BMP9 Through Down-Regulating Cyp26b1 in Mesenchymal Stem Cells.

BACKGROUND: All-trans retinoic acid (ATRA) promotes the osteogenic differentiation induced by bone morphogenetic protein 9 (BMP9), but the intrinsic relationship between BMP9 and ATRA keeps unknown. Herein, we investigated the effect of Cyp26b1, a critical enzyme of ATRA degradation, on the BMP9-induced osteogenic differentiation in mesenchymal stem cells (MSCs), and unveiled possible mechanism through which BMP9 regulates the expression of Cyp26b1. METHODS: ATRA content was detected with ELISA and HPLC-MS/MS. PCR, Western blot, and histochemical staining were used to assay the osteogenic markers. Fetal limbs culture, cranial defect repair model, and micro-computed tomographic were used to evaluate the quality of bone formation. IP and ChIP assay were used to explore possible mechanism. RESULTS: We found that the protein level of Cyp26b1 was increased with age, whereas the ATRA content decreased. The osteogenic markers induced by BMP9 were increased by inhibiting or silencing Cyp26b1 but reduced by exogenous Cyp26b1. The BMP9-induced bone formation was enhanced by inhibiting Cyp26b1. The cranial defect repair was promoted by BMP9, which was strengthened by silencing Cyp26b1 and reduced by exogenous Cyp26b1. Mechanically, Cyp26b1 was reduced by BMP9, which was enhanced by activating Wnt/ß-catenin, and reduced by inhibiting this pathway. ß-catenin interacts with Smad1/5/9, and both were recruited at the promoter of Cyp26b1. CONCLUSIONS: Our findings suggested the BMP9-induced osteoblastic differentiation was mediated by activating retinoic acid signalling, viadown-regulating Cyp26b1. Meanwhile, Cyp26b1 may be a novel potential therapeutic target for the treatment of bone-related diseases or accelerating bone-tissue engineering.

lncRNA MEG3 Promotes PDK4/GSK-3ß/ß-Catenin Axis in MEFs by Targeting miR-532-5p.

Studies reported the positive and negative osteogenic effects of MEG3 in mesenchymal stem cells (MSCs). This study aims at clarifying the osteogenic potential of MEG3 and the underlying mechanism. Bone morphogenetic protein 9- (BMP9-) transfected MSCs were recruited as an osteogenic model in vitro, and ectopic bone formation were used in vivo to explore the effect of MEG3 on osteogenesis. We found that overexpression of MEG3 facilitated BMP9-induced osteogenic markers, ALP activities, and matrix mineralization. However, knockdown of MEG3 attenuated BMP9-induced osteogenic markers. MEG3 increased the phosphorylation of GSK-3ß and the protein level of ß-catenin. Pyruvate dehydrogenase kinase 4 (PDK4) can also combine with GSK-3ß and increase the latter phosphorylation. Moreover, MEG3 increased the mRNA level of PDK4. The ceRNA analysis showed that MEG3 may regulate the expression of PDK4 via microRNA 532-5p (miR-532-5p). The MEG3-enhanced GSK-3ß/ß-catenin axis can be attenuated by miR-532-5p, and miR-532-5p inhibitor partly rescued endogenous PDK4 and MEG3-mediated expression of PDK4. MEG3 may potentiate PDK4 and GSK-3ß/ß-catenin by inhibiting miR-532-5p.

Matricellular Protein SMOC2 Potentiates BMP9-Induced Osteogenic Differentiation in Mesenchymal Stem Cells through the Enhancement of FAK/PI3K/AKT Signaling.

Mesenchymal stem cells (MSCs) can self-renew and differentiate into multiple lineages, making MSC transplantation a promising option for bone regeneration. Both matricellular proteins and growth factors play an important role in regulating stem cell fate. In this study, we investigated the effects of matricellular protein SMOC2 (secreted modular calcium-binding protein 2) on bone morphogenetic protein 9 (BMP9) in mouse embryonic fibroblasts (MEFs) and revealed a possible molecular mechanism underlying this process. We found that SMOC2 was detectable in MEFs and that exogenous SMOC2 expression potentiated BMP9-induced osteogenic markers, matrix mineralization, and ectopic bone formation, whereas SMOC2 knockdown inhibited these effects. BMP9 increased the levels of p-FAK and p-AKT, which were either enhanced or reduced by SMOC2 and FAK silencing, respectively. BMP9-induced osteogenic markers were increased by SMOC2, and this increase was partially abolished by silencing FAK or LY290042. Furthermore, we found that general transcription factor 2I (GTF2I) was enriched at the promoter region of SMOC2 and that integrin ß1 interacted with SMOC2 in BMP9-treated MEFs. Our findings demonstrate that SMOC2 can promote BMP9-induced osteogenic differentiation by enhancing the FAK/PI3K/AKT pathway, which may be triggered by facilitating the interaction between SMOC2 and integrin ß1.

Pyruvate dehydrogenase kinase 4 promotes osteoblastic potential of BMP9 by boosting Wnt/ß-catenin signaling in mesenchymal stem cells.

Bone morphogenetic protein 9 (BMP9) is an effective osteogenic factor and a promising candidate for bone tissue engineering. The osteoblastic potential of BMP9 needs to be further increased to overcome its shortcomings. However, the details of how BMP9 triggers osteogenic differentiation in mesenchymal stem cells (MSCs) are unclear. In this study, we used real-time PCR, western blot, histochemical staining, mouse ectopic bone formation model, immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation to investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in BMP9-induced osteogenic differentiation of C3H10T1/2 cells, as well as the underlying mechanism. We found that PDK4 was upregulated by BMP9 in C3H10T1/2 cells. BMP9-induced osteogenic markers and bone mass were increased by PDK4 overexpression, but decreased by PDK4 silencing. ß-catenin protein level was increased by BMP9, which was enhanced by PDK overexpression and decreased by PDK4 silencing. BMP9-induced osteogenic markers were reduced by PDK4 silencing, which was almost reversed by ß-catenin overexpression. PDK4 increased the BMP9-induced osteogenic markers, which was almost eliminated by ß-catenin silencing. Sclerostin was mildly decreased by BMP9 or PDK4, and significantly decreased by combined BMP9 and PDK4. In contrast, sclerostin increased significantly when BMP9 was combined with PDK4 silencing. BMP9-induced p-SMAD1/5/9 was increased by PDK4 overexpression, but was reduced by PDK4 silencing. PDK4 interacts with p-SMAD1/5/9 and regulates the sclerostin promoter. These findings suggest that PDK4 can increase the osteogenic potential of BMP9 by enhancing Wnt/ß-catenin signaling via the downregulation of sclerostin. PDK4 may be an effective target to strengthen BMP9-induced osteogenesis.

LncRNA MIR17HG promotes the proliferation, migration, and invasion of retinoblastoma cells by up-regulating HIF-1α expression via sponging miR-155-5p.

Long non-coding RNA (lncRNA) miR-17-92a-1 cluster host gene (MIR17HG) is oncogenic in several cancers. This study was aimed to probe into its expression characteristics and biological functions in retinoblastoma (RB) and to explore its role in regulating microRNA-155-5p (miR-155-5p) and hypoxia-inducible factor-1α (HIF-1α). In this study, paired RB samples were collected, and expression levels of MIR17HG, miR-155-5p, and HIF-1α were examined by quantitative real-time polymerase chain reaction (qRT-PCR); the proliferation, migration, and invasion of RB cells were detected by cell counting kit-8 (CCK-8) and transwell assays; qRT-PCR and Western blot were used to analyze the changes of miR-155-5p expression and HIF-1α expression. We found that MIR17HG expression was significantly up-regulated in RB samples, which was negatively correlated with miR-155-5p expression. The proliferation, migration, and invasion of RB cells were promoted by MIR17HG overexpression but inhibited by MIR17HG knockdown. MiR-155-5p suppressed the proliferation, migration, and invasion of RB cells. MIR17HG positively regulated the expression of HIF-1α on both mRNA and protein levels in RB cells. Additionally, miR-155-5p was identified as a target of MIR17HG. The data in this study suggest that MIR17HG exerts oncogenic effects in RB via the miR-155-5p/HIF-1α axis.

The role of Serpina3n in the reversal effect of ATRA on dexamethasone-inhibited osteogenic differentiation in mesenchymal stem cells.

BACKGROUND: Glucocorticoid-induced osteoporosis (GIOP) is the most common secondary osteoporosis. Patients with GIOP are susceptible to fractures and the subsequent delayed bone union or nonunion. Thus, effective drugs and targets need to be explored. In this regard, the present study aims to reveal the possible mechanism of the anti-GIOP effect of all-trans retinoic acid (ATRA). METHODS: Bone morphogenetic protein 9 (BMP9)-transfected mesenchymal stem cells (MSCs) were used as an in vitro osteogenic model to deduce the relationship between ATRA and dexamethasone (DEX). The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin were detected using real-time quantitative polymerase chain reaction, Western blot, and immunofluorescent staining assay. ALP activities and matrix mineralization were evaluated using ALP staining and Alizarin Red S staining assay, respectively. The novel genes associated with ATRA and DEX were detected using RNA sequencing (RNA-seq). The binding of the protein-DNA complex was validated using chromatin immunoprecipitation (ChIP) assay. Rat GIOP models were constructed using intraperitoneal injection of dexamethasone at a dose of 1 mg/kg, while ATRA intragastric administration was applied to prevent and treat GIOP. These effects were evaluated based on the serum detection of the osteogenic markers osteocalcin and tartrate-resistant acid phosphatase 5b, histological staining, and micro-computed tomography analysis. RESULTS: ATRA enhanced BMP9-induced ALP, RUNX2 expressions, ALP activities, and matrix mineralization in mouse embryonic fibroblasts as well as C3H10T1/2 and C2C12 cells, while a high concentration of DEX attenuated these markers. When DEX was combined with ATRA, the latter reversed DEX-inhibited ALP activities and osteogenic markers. In vivo analysis showed that ATRA reversed DEX-inhibited bone volume, bone trabecular number, and thickness. During the reversal process of ATRA, the expression of retinoic acid receptor beta (RARß) was elevated. RARß inhibitor Le135 partly blocked the reversal effect of ATRA. Meanwhile, RNA-seq demonstrated that serine protease inhibitor, clade A, member 3N (Serpina3n) was remarkably upregulated by DEX but downregulated when combined with ATRA. Overexpression of Serpina3n attenuated ATRA-promoted osteogenic differentiation, whereas knockdown of Serpina3n blocked DEX-inhibited osteogenic differentiation. Furthermore, ChIP assay revealed that RARß can regulate the expression of Serpina3n. CONCLUSION: ATRA can reverse DEX-inhibited osteogenic differentiation both in vitro and in vivo, which may be closely related to the downregulation of DEX-promoted Serpina3n. Hence, ATRA may be viewed as a novel therapeutic agent, and Serpina3n may act as a new target for GIOP.

COX-2 promotes the osteogenic potential of BMP9 through TGF-ß1/p38 signaling in mesenchymal stem cells.

This study investigated the effects of transforming growth factor-ß1 (TGF-ß1) and cyclooxygenase-2 (COX-2) on bone morphogenetic protein 9 (BMP9) in mesenchymal stem cells (MSCs). We found that BMP9 increased mRNA levels of TGF-ß1 and COX-2 in C3H10T1/2 cells. BMP9-induced osteogenic markers were enhanced by TGF-ß1 and reduced by TGF-ßRI-specific inhibitor LY364947. BMP9 increased level of p-Smad2/3, which were either enhanced or reduced by COX-2 and its inhibitor NS398. BMP9-induced osteogenic markers were decreased by NS398 and it was partially reversed by TGF-ß1. COX-2 increased BMP9-induced osteogenic marker levels, which almost abolished by LY364947. BMP9-induced bone formation was enhanced by TGF-ß1 but reduced by silencing TGF-ß1 or COX-2. BMP9's osteogenic ability was inhibited by silencing COX-2 but partially reversed by TGF-ß1. TGF-ß1 and COX-2 enhanced activation of p38 signaling, which was induced by BMP9 and reduced by LY364947. The ability of TGF-ß1 to increase the BMP9-induced osteogenic markers was reduced by p38-specific inhibitor, while BMP9-induced TGF-ß1 expression was reduced by NS398, but enhanced by COX-2. Furthermore, CREB interacted with Smad1/5/8 to regulate TGF-ß1 expression in MSCs. These findings suggest that COX-2 overexpression leads to increase BMP9's osteogenic ability, resulting from TGF-ß1 upregulation which then activates p38 signaling in MSCs.

Analysis and Validation of Hub Genes in Blood Monocytes of Postmenopausal Osteoporosis Patients.

Osteoporosis is a common systemic bone disease caused by the imbalance between osteogenic activity and osteoclastic activity. Aged women are at higher risk of osteoporosis, partly because of estrogen deficiency. However, the underlying mechanism of how estrogen deficiency affects osteoclast activity has not yet been well elucidated. In this study, GSE2208 and GSE56815 datasets were downloaded from GEO database with 25 PreH BMD women and 25 PostL BMD women in total. The RRA algorithm determined 38 downregulated DEGs and 30 upregulated DEGs. Through GO analysis, we found that downregulated DEGs were mainly enriched in myeloid cell differentiation, cytokine-related functions while upregulated DEGs enriched in immune-related biological processes; pathways like Notch signaling and MAPK activation were found in KEGG/Rectome pathway database; a PPI network which contains 66 nodes and 91 edges was constructed and three Modules were obtained by Mcode; Correlation analysis helped us to find highly correlated genes in each module. Moreover, three hub genes FOS, PTPN6, and CTSD were captured by Cytohubba. Finally, the hub genes were further confirmed in blood monocytes of ovariectomy (OVX) rats by real-time PCR assay. In conclusion, the integrative bioinformatics analysis and real-time PCR analysis were utilized to offer fresh light into the role of monocytes in premenopausal osteoporosis and identified FOS, PTPN6, and CTSD as potential biomarkers for postmenopausal osteoporosis.

Cyclooxygenase-2/sclerostin mediates TGF-ß1-induced calcification in vascular smooth muscle cells and rats undergoing renal failure.

In this study, we studied the effect and possible mechanism of TGF-ß1 on vascular calcification. We found that the serum levels of TGF-ß1 and cycloxygenase-2 (COX-2) were significantly increased in patients with chronic kidney disease. Phosphate up regulated TGF-ß1 in vascular smooth muscle cells (VSMCs). TGF-ß1 decreased the markers of VSMCs, but increased osteogenic markers and calcification in aortic segments. The phosphate-induced osteogenic markers were reduced by the TGFßR I inhibitor (LY364947), which also attenuated the potential of phosphate to reduce VSMC markers in VSMCs. Both phosphate and TGF-ß1 increased the protein level of ß-catenin, which was partially mitigated by LY364947. TGF-ß1 decreased sclerostin, and exogenous sclerostin decreased the mineralization induced by TGF-ß1. LY364947 reduced the phosphate and TGF-ß1 induced COX-2. Meanwhile, the effects of TGF-ß1 on osteogenic markers, ß-catenin, and sclerostin, were partially reversed by the COX-2 inhibitor. Mechanistically, we found that p-Smad2/3 and p-CREB were both enriched at the promoter regions of sclerostin and ß-catenin. TGF-ß1 and COX-2 were significantly elevated in serum and aorta of rats undergoing renal failure. Therapeutic administration of meloxicam effectively ameliorated the renal lesion. Our results suggested that COX-2 may mediate the effect of TGF-ß1 on vascular calcification through down-regulating sclerostin in VMSCs.