siRNA Assays Research Articles

Development and experimental verification of novel angiogenesis related prognostic model and immune infiltration characterization in osteosarcoma

BackgroundAs the most common primary bone cancer, osteosarcoma (OS) still lacks satisfactory therapeutic outcomes. Therefore, it is crucial to further evaluate OS at different risk levels and identify new intervention targets. Many evidences suggest the important role of angiogenesis in OS, but further exploration is needed.MethodsWe utilized public databases TARGET and GEO and employed bioinformatics algorithms such as LASSO, univariate and multivariate Cox regression analyses, and unsupervised consensus clustering to explore the role of angiogenesis-related genes (AGRGs) in OS. By calculating AGRG scores, we further analyzed OS molecular subtypes based on AGRGs. The correlation between AGRG scores and immune infiltration was subsequently examined. In vitro experiments, including WB, PCR, siRNA, migration, and invasion assays, were used to determine the value of the selected targets for OS.ResultsUltimately, we established an OS prognosis model based on five AGRGs (COL5A2, CXCL6, FSTL1, NRP1, and TNFRSF21) that can independently validate prognosis levels. In vitro experiments confirmed the aberrant expression of CXCL6 in OS and its potential role in migration and invasion.ConclusionOur study reveals the impact of angiogenesis on OS from a novel perspective and provides potential intervention targets.

MiR-106b-5p protects against drug-induced liver injury by targeting vimentin to stimulate liver regeneration.

Understanding the endogenous mechanism of adaptive response to drug-induced liver injury (arDILI) may discover innovative strategies to manage DILI. To gain mechanistic insight into arDILI, we investigated exosomal miRNAs in the adaptive response to toosendanin-induced liver injury (TILI) of mice. Exosomal miR-106b-5p was identified as a specific regulator of arDILI by comprehensive miRNA profiling. Outstandingly, miR-106b-5p agomir treatment alleviated TILI and other DILI by inhibiting apoptosis and promoting hepatocyte proliferation. Conversely, antagomir treatments had opposite effects, indicating that miR-106b-5p protects mice from liver injury. Injured hepatocytes released miR-106b-5p-enriched exosomes taken up by surrounding hepatocytes. Vim (encodes vimentin) was identified as an important target of miR-106b-5p by dual luciferase reporter and siRNA assays. Furthermore, single-cell RNA-sequencing analysis of toosendanin-injured mouse liver revealed a cluster of Vim + hepatocytes; nonetheless declined following miR-106b-5p cotreatment. More importantly, Vim knockout protected mice from acetaminophen poisoning and TILI. In the clinic, serum miR-106b-5p expression levels correlated with the severity of DILI. Indeed, liver biopsies of clinical cases exposed to different DILI causing drugs revealed marked vimentin expression among harmed hepatocytes, confirming clinical relevance. Together, we report mechanisms of arDILI whereby miR-106b-5p safeguards restorative tissue repair by targeting vimentin.

Palmitic Acid Modulation of the Insulin-Like Growth Factor System in Hypothalamic Astrocytes and Neurons

Introduction: Insulin-like growth factor (IGF)1 and IGF2 have neuroprotective effects, but less is known regarding how other members of the IGF system, including IGF binding proteins (IGFBPs) and the regulatory proteinase pappalysin-1 (PAPP-A) and its endogenous inhibitor stanniocalcin-2 (STC2) participate in this process. Here, we analyzed whether these members of the IGF system are modified in neurons and astrocytes in response to palmitic acid (PA), a fatty acid that induces cell stress when increased centrally. Methods: Primary hypothalamic astrocyte cultures from male and female PND2 rats and the pro-opiomelanocortin (POMC) neuronal cell line, mHypoA-POMC/GFP-2, were treated with PA, IGF1 or both. To analyze the role of STC2 in astrocytes, siRNA assays were employed. Results: In astrocytes of both sexes, PA rapidly increased cell stress factors followed by increased Pappa and Stc2 mRNA levels and then a decrease in Igf1, Igf2, and Igfbp2 expression and cell number. Exogenous IGF1 did not revert these effects. In mHypoA-POMC/GFP-2 neurons, PA reduced cell number and Pomc and Igf1 mRNA levels, and increased Igfbp2 and Stc2, again with no effect of exogenous IGF1. PA increased STC2 expression, but no effects of decreasing its levels by interference assays or exogenous STC2 treatment in astrocytes were found. Conclusions: The response of the IGF system to PA was cell and sex specific, but no protective effects of the IGFs were found. However, the modifications in hypothalamic PAPP-A and STC2 indicate that further studies are required to determine their role in the response to fatty acids and possibly in metabolic control.

Characterization of stmn1a and genetic effects of a 22-bp Indel in its promoter region of bighead carp Hypophthalmichthys nobilis

Stathmin 1 (stmn1) gene, which is involved in the control of cell cycle progression, cell proliferation, differentiation and motility, is a crucial relay in the signal transduction of multiple signaling pathways. Reports on the function of stmn1 in fish are scarce. In this study, the characterization and function of stmn1a gene were explored in an important food fish, namely bighead carp (Hypophthalmichthys nobilis). The bighead carp stmn1a (Hynstmn1a) genomic sequence, which has 2995 bp with an ORF of 447 bp, is evolutionarily conserved in teleost fish. The highest level of expression of Hynstmn1a is in the un-fertilized eggs and spleen of adult bighead carp. The overexpression of Hynstmn1a facilitates cell cycle progression and suppresses apoptosis in EPC cells. In an siRNA assay, disruption of stmn1a in EPC cells resulted in the inhibition of cell cycle progression and promotion of apoptosis. Analysis of promoter activity indicated that an upstream transcription factor (foxo4) regulates Hynstmn1a. Zebrafish mutants with stmn1a knockout by CRISPR/Cas9 exhibited a significant retardation of body length (p ​< ​0.05). There was no significant difference in body weight between stmn1a-mutant zebrafish and wild-type (p ​= ​0.053). A 22-bp Indel in the promoter region of Hynstmn1a with an observed heterozygosity (Ho) of 0.136 in a bighead carp population (n ​= ​191). The polymorphism of this 22-bp Indel genotype is significantly associated with body length (BL), head length (HL) (p ​< ​0.05), body weight (BW), head height (HH), and head width (HW) (p ​< ​0.01), showing its genetic effects on growth promotion. This study sheds light on the function of stmn1a in fish and demonstrates the potential of the 22-bp Indel of Hynstmn1a as a gene marker when selecting breeding individuals for faster growth and a bigger head of bighead carp in aquaculture.

Loss-of-function variants in GLMN are associated with generalized skin hyperpigmentation with or without glomuvenous malformation.

Inherited hyperpigmented skin disorders comprise a group of entities with considerable clinical and genetic heterogenicity. The genetic basis of a majority of these disorders remains to be elucidated. This study aimed to identify the underlying gene for an unclarified disorder of autosomal-dominant generalized skin hyperpigmentation with or without glomuvenous malformation. Whole-exome sequencing was performed in five unrelated families with autosomal-dominant generalized skin hyperpigmentation. Variants were confirmed using Sanger sequencing and a minigene assay was employed to evaluate the splicing alteration. Immunofluorescence and transmission electron microscopy (TEM) were used to determine the quantity of melanocytes and melanosomes in hyperpigmented skin lesions. GLMN knockdown by small interfering RNA assays was performed in human MNT-1 cells to examine melanin concentration and the underlying molecular mechanism. We identified five variants in GLMN in five unrelated families, including c.995_996insAACA(p.Ser333Thrfs*11), c.632 + 4delA, c.1470_1473dup(p.Thr492fs*12), c.1319G > A(p.Trp440*) and c.1613_1614insTA(Thr540*). The minigene assay confirmed that the c.632 + 4delA mutant resulted in abolishment of the canonical donor splice site. Although the number of melanocytes remained unchanged in skin lesions, as demonstrated by immunofluorescent staining of tyrosinase and premelanosome protein, TEM revealed an increased number of melanosomes in the skin lesion of a patient. The GLMN knockdown MNT-1 cells demonstrated a higher melanin concentration, a higher proportion of stage III and IV melanosomes, upregulation of microphthalmia-associated transcription factor and tyrosinase, and downregulation of phosphorylated p70S6 K vs. mock-transfected cells. We found that loss-of-function variants in GLMN are associated with generalized skin hyperpigmentation with or without glomuvenous malformation. Our study implicates a potential role of glomulin in human skin melanogenesis, in addition to vascular morphogenesis.

βAR-mTOR-lipin1 pathway mediates PKA-RIIβ deficiency-induced adipose browning.

Background: Enhancing white adipose tissue (WAT) browning combats obesity. The RIIβ subunit of cAMP-dependent protein kinase (PKA) is primarily expressed in the brain and adipose tissue. Deletion of the hypothalamic RIIβ gene centrally induces WAT browning, yet the peripheral mechanisms mediating this process remain unexplored. Methods: This study investigates the mechanisms underlying WAT browning in RIIβ-KO mice. Genetic approaches such as β3-adrenergic receptors (β3ARs) deletion and sympathetic denervation of WAT were utilized. Genome-wide transcriptomic sequencing and bioinformatic analysis were employed to identify potential mediators of WAT browning. siRNA assays were employed to knock down mTOR and lipin1 in vitro, while AAV-shRNAs were used for the same purpose in vivo. Results: We found that WAT browning substantially contributes to the lean and obesity-resistant phenotypes of RIIβ-KO mice. The WAT browning can be dampened by β3ARs deletion or WAT sympathetic denervation. We identified that adipocytic mTOR and lipin1 may act as mediators of the WAT browning. Inhibition of mTOR or lipin1 abrogates WAT browning and hinders the lean phenotype of RIIβ-KO mice. In human subcutaneous white adipocytes and mouse white adipocytes, β3AR stimulation can activate mTOR and causes lipin1 nuclear translocation; knockdown of mTOR and Lipin1 mitigates WAT browning-associated gene expression, impedes mitochondrial activity. Moreover, mTOR knockdown reduces lipin1 level and nuclear translocation, indicating that lipin1 may act downstream of mTOR. Additionally, in vivo knockdown of mTOR and Lipin1 diminished WAT browning and increased adiposity. Conclusions: The β3AR-activated mTOR-lipin1 axis mediates WAT browning, offering new insights into the molecular basis of PKA-regulated WAT browning. These findings provide potential adipose target candidates for the development of drugs to treat obesity.

Astragaloside IV Treats Parkinson's Disease by Regulating the Proliferation and Differentiation of NSCs through the SHH-Nurr1 Pathway.

Recently, there has been a surge of interest in enhancing the differentiation of neural stem cells (NSCs) and supplementing dopamine neurons as a potential treatment for Parkinson's disease, the second most prevalent neurodegenerative disorder. Two factors, sonic hedgehog (SHH) and nuclear receptor-related 1 protein (Nurr1), have been identified as influential in NSCs differentiation. Additionally, Astragaloside IV (AS-IV), an active compound derived from Astragalus, has also been discovered to impact NSCs differentiation. To assess the effects of AS-IV on cell activity, CCK-8 and flow cytometry techniques were employed. Meanwhile, western blotting, immunofluorescence, and real-time PCR were utilized to detect protein expression both in vivo and in vitro. Furthermore, siRNA assay was used to verify the association between SHH and Nurr1 and to investigate whether AS-IV exerts its effects through this pathway. The experimental findings revealed that AS-IV enhances cell activity and promotes the expression of differentiation proteins related to NSCs. Furthermore, the relationship between the SHH-Nurr1 pathway was confirmed, demonstrating that AS-IV induces NSCs differentiation via this pathway. Consequently, SHH, acting as the upstream signaling pathway of Nurr1, influences its expression, while AS-IV regulates the proliferation and differentiation of NSCs by modulating the SHH-Nurr1 pathway.

Identification of epithelial-mesenchymal transition-related biomarkers in lung adenocarcinoma using bioinformatics and lab experiments.

Lung adenocarcinoma accounts for approximately 40% of lung cancer cases and poses a serious threat to human health. Therefore, there is an urgent need to identify central biomarkers in lung adenocarcinoma. We first identified the EMT-associated genes in LUAD based on the TCGA cohort. Then we screened these 90 EMT-associated genes using univariate Cox regression analysis and LASSO regression analysis to develop a prognostic gene signature in the training set. The predictive performance of the gene signature was assessed in the validation set and multiple external test sets using the ROC cure, C index and log-rank tests. RT-PCR, western blot, wound healing assays, and siRNA methods were further used to investigate the role of PLEK2 in tumor behaviors. Eight genes (CCNB1, PLEK2, DERL3, C1QTNF6, DLGAP5, HMMR, GJB3, and SPOCK1) were eventually selected to develop an eight-gene signature. The 5-year AUC of the gene signature has a robust predictive ability both for predicting overall survival (0.774, 0.756, and 0.669 in the external test sets, respectively), and for progression free survival (0.774, 0.746, and 0.755 in the external test sets, respectively). C-index of the gene signature was 0.961 ± 0.005, 0.916 ± 0.011, and 0.868 ± 0.234 in the external test sets, respectively. Four genes (C1QTNF6, DLGAP5, HMMR, and PLEK2) were identified as key genes in LUAD progression, which were upregulated in the cancerous tissue compared with in the normal tissue (P < 0.001), and correlated with an unwanted prognosis in lung cancer (P < 0.05). PLEK2 was used as an example to explore its effect on LUAD progression in vitro using RT-PCR, western blot, CCK8, si-RNA and wound healing assay. Silencing of PLEK2 was shown to reduce proliferative and migrated ability of lung cancer cells via prohibition of autophagy. This study developed a novel EMT-related gene signature benefiting precision medicine, and identified four pivotal genes which can serve as therapeutic targets in LUAD. Four key genes can serve as molecular targets for patients with LUAD; silencing of PLEK2 was shown to reduce proliferative and migrated ability of lung cancer cells via prohibition of autophagy.

Preferential FGF18/FGFR activity in pseudoglandular versus canalicular stage human lung fibroblasts.

Fibroblast growth factor (FGF) signaling is necessary for proper lung branching morphogenesis, alveolarization, and vascular development. Dysregulation of FGF activity has been implicated in various lung diseases. Recently, we showed that FGF18 promotes human lung branching morphogenesis by regulating mesenchymal progenitor cells. However, the underlying mechanisms remain unclear. Thus, we aimed to determine the role of FGF18 and its receptors (FGFR) in regulating mesenchymal cell proliferation, migration, and differentiation from pseudoglandular to canalicular stage. We performed siRNA assays to identify the specific FGFR(s) associated with FGF18-induced biological processes. We found that FGF18 increased proliferation and migration in human fetal lung fibroblasts (HFLF) from both stages. FGFR2/FGFR4 played a significant role in pseudoglandular stage. HFLF proliferation, while FGFR3/FGFR4 were involved in canalicular stage. FGF18 enhanced HFLF migration through FGFR2 and FGFR4 in pseudoglandular and canalicular stage, respectively. Finally, we provide evidence that FGF18 treatment leads to reduced expression of myofibroblast markers (ACTA2 and COL1A1) and increased expression of lipofibroblast markers (ADRP and PPARγ) in both stages HFLF. However, the specific FGF18/FGFR complex involved in this process varies depending on the stage. Our findings suggest that in context of human lung development, FGF18 tends to associate with distinct FGFRs to initiate specific biological processes on mesenchymal cells.

Tetrahydropalmatine ameliorates hepatic steatosis in nonalcoholic fatty liver disease by switching lipid metabolism via AMPK-SREBP-1c-Sirt1 signaling axis

BackgroundNonalcoholic fatty liver disease (NAFLD) is becoming a global epidemic without effective treatment currently available. NAFLD is characterized by an increase in hepatic de novo lipogenesis (DNL) and inadequate compensatory enhancement in fatty acid oxidation (FAO), which disturbs lipid homeostasis. In NAFLD, lipid metabolism relies heavily on metabolic reprogramming. Moreover, lipid metabolism plays an essential role in switching between lipogenesis and FAO, which is beneficial for the anti-NAFLD therapy. Our recent study demonstrated that the phytochemical tetrahydropalmatine (THP) has positive efficacy in hepatocellular carcinoma (HCC). However, it remains unclear whether the therapeutic benefits of THP are primarily due to delaying the progression of hepatic steatosis to HCC. PurposeThis work aimed to systemically evaluate the pharmacological functions and underlying mechanisms of THP in NAFLD using both in vitro and in vivo models. MethodsNAFLD models were established using high-fat diet (HFD)-fed mice in vivo and palmitic acid- and oleic acid-challenged hepatocytes in vitro. Metabonomics analysis concomitant with biochemical indices and computational biology assays were performed comprehensively to reveal the key link between the treatment of NAFLD and the AMPK-SREBP-1c-Sirt1 signaling axis. ResultsHepatic metabolomics analysis revealed that THP altered lipid metabolism by enhancing FAO and inhibiting glycolysis, tricarboxylic acid cycle, and urea cycle in HFD-fed mice. Analysis of gene expression showed that THP profoundly suppressed hepatic DNL and promoted FAO. THP supplementation not only significantly decreased body/liver weight gain and serum indices but also ameliorated hepatic steatosis. Simultaneously, impaired lipotoxicity was observed in vivo and in vitro after THP supplementation, protecting against steatosis-driven injury. Metabolic phenotype assays showed that THP promoted switching from glycolysis inhibition to FAO enhancement in steatotic cells, resulting in reprogramming lipid metabolism. Mechanistically, THP accelerated lipid oxidation by activating AMPK-SREBP-1c-Sirt1 axis signaling. Applying molecular docking combined with surface plasmon resonance and cellular thermal shift assay target engagement, as well as siRNA assays, AMPKα was confirmed as a direct molecular target of THP. ConclusionIn summary, THP ameliorates hepatic steatosis in NAFLD by switching lipid metabolism via the AMPK-SREBP-1c-Sirt1 pathway. This work provides an attractive phytochemical component for therapy against hepatic steatosis in NAFLD.

Endogenous retrotransposons cause catastrophic deoxyribonucleic acid damage in human trophoblasts

To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts. Experimental ex vivo study SETTING: Hospital-affiliated University PATIENT(S): Trophoblasts from a patient with unexplained recurrent pregnancy loss and patients with spontaneous and elective abortions (n=10) INTERVENTION(S): Biochemical and genetic analysis and modification of primary human trophoblasts. To phenotype and systematically evaluate the underlying pathogenic mechanism for elevated DNA damage observed in trophoblasts derived from a patient with unexplained recurrent pregnancy loss, transcervical embryoscopy, G-band karyotyping, RNASeq, qPCR, immunoblotting, biochemical and siRNA assays, and whole-genome sequencing were performed. Transcervical embryoscopy revealed a severely dysmorphic embryo that was euploid on G-band karyotyping. RNASeq was notable for markedly elevated LINE-1 expression, confirmed with qPCR and that resulted in elevated expression of LINE-1-encoded proteins, as shown by Immunoblotting. Immunofluorescence, biochemical and genetic approaches demonstrated that overexpression of LINE-1 caused reversible widespread genomic damage and apoptosis. De-repression of LINE-1 elements in early trophoblasts results in reversible but widespread DNA damage.

Trichostatin D as a Novel KLF2 Activator Attenuates TNFα-Induced Endothelial Inflammation.

Krüppel-like factor 2 (KLF2) is an atherosclerotic protective transcription factor that maintains endothelial cell homeostasis through its anti-inflammatory, anti-oxidant, and antithrombotic properties. The aim of this study was to discover KLF2 activators from microbial secondary metabolites and explore their potential molecular mechanisms. By using a high-throughput screening model based on a KLF2 promoter luciferase reporter assay, column chromatography, electrospray ionization mass spectrometry (ESI-MS), and nuclear magnetic resonance (NMR) spectra, trichostatin D (TSD) was isolated from the rice fermentation of Streptomyces sp. CPCC203909 and identified as a novel KLF2 activator. Real-time-quantitative polymerase chain reaction (RT-qPCR) results showed that TSD upregulated the mRNA level of KLF2 in endothelial cells. Functional assays showed that TSD attenuated monocyte adhesion to endothelial cells, decreased vascular cell adhesion protein 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) expression, and exhibited an anti-inflammatory effect in tumor necrosis factor alpha (TNFα)-induced endothelial cells. We further demonstrated through siRNA and western blot assays that the effects of TSD on monocyte adhesion and inflammation in endothelial cells were partly dependent on upregulating KLF2 expression and then inhibiting the NOD-like receptor protein 3 (NLRP3)/Caspase-1/interleukin-1beta (IL-1β) signaling pathway. Furthermore, histone deacetylase (HDAC) overexpression and molecular docking analysis results showed that TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 activities. Taken together, TSD was isolated from the fermentation of Streptomyces sp. CPCC203909 and first reported as a potential activator of KLF2 in this study. Furthermore, TSD upregulated KLF2 expression by inhibiting HDAC 4, 5, and 7 and attenuated endothelial inflammation via regulation of the KLF2/NLRP3/Caspase-1/IL-1β signaling pathway.

Baicalin improved hepatic injury of NASH by regulating NRF2/HO-1/NRLP3 pathway

Being at the important pathological stage and the critical treatment period of non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH) which is associated with fibrosis, hepatic and liver cancer has become a serious medical problem. As one of the major effective components in Scutellaria baicalensis, baicalin takes on anti-oxidant and anti-inflammatory activities. Nevertheless, its effects on NASH and its underlying molecular mechanism have not been thoroughly understood yet. In previous study, we have clarified baicalin could inhibit pyroptosis of hepatocytes mediated by NLRP3 in vitro, but the verification in vivo and upstream mechanism still need further work. Here the NASH mouse model was induced by feeding with a high fat diet (HFD) for 8–12 weeks. Thereafter, in the following weeks, NASH mice were given with HFD plus baicalin. We, subsequently, examined their hepatic function and inflammatory response and conducted the HE staining of liver samples. Furthermore, the underlying molecular mechanism was revealed through diverse molecular biological experiments including quantitative real-time PCR (qRT-PCR), Western blotting (WB), siRNA and CCK8 assays in HepG2 cells incubated with free fatty acid, and was verified in NASH mice. The in vivo findings indicated that baicalin decreased lipid accumulation and inflammation in the liver tissues of NASH mice, as evidenced by the enhanced NRF2/HO-1 expression and the reduced NLRP3/Caspase1/GSDMD levels, and these factors were involved in the pyroptosis pathway. Meanwhile, baicalin also contributed greatly against oxidative injury. The anti-inflammatory effect of baicalin was confirmed by experiments in vitro. For another, knockdown of NRF2 obviously weakened the protective effects of baicalin and reduced the NLRP3/Caspase1/GSDMD-mediated pyroptosis.This study indicates that baicalin is able to attenuate hepatic cell pyroptosis in vivo and in vitro in the case of NASH by regulating the NRF2/HO-1/NRLP3 pathway.

Oroxylin A ameliorates AKI-to-CKD transition through maintaining PPARα-BNIP3 signaling-mediated mitochondrial homeostasis.

Background: Acute kidney injury (AKI) occurs in approximately 7–18% of all hospitalizations, but there are currently no effective drug therapy for preventing AKI or delaying its progression to chronic kidney disease (CKD). Recent studies have shown that Scutellaria baicalensis, a traditional Chinese herb, could attenuate cisplatin-induced AKI, although the mechanism remains elusive. Further, it is unknown whether its major active component, Oroxylin A (OA), can alleviate kidney injury. Methods: The therapeutic effect of OA was evaluated by using ischemia-reperfusion (IR) and cisplatin mediated-AKI mice and HK-2 cells under hypoxia-reoxygenation (HR) conditions. HE staining, transmission electron microscopy, flow cytometry, immunofluorescence, qPCR, Western blot, PPARα inhibitor, BNIP3 siRNA and ChIP assay were used to explore the role and mechanism of OA in AKI. Results: OA ameliorated tubular damage and dramatically decreased serum creatinine (Scr) and urea nitrogen (BUN), and the expressions of renal injury markers (Kim-1, Ngal) in AKI mice induced by both IR injury and cisplatin, as well as attenuating AKI-to-CKD transition. In vitro experiments showed that OA alleviated HR-induced mitochondrial homeostasis imbalance in renal tubular epithelial cells. Mechanistically, OA dose-dependently induced the expression of Bcl-2/adenovirus E1B 19-kDa interacting protein (BNIP3), while knockdown of BNIP3 expression reversed the protection of OA against HR-mediated mitochondrial injury. Network pharmacological analysis and experimental validation suggested that OA enhanced BNIP3 expression via upregulating the expression of peroxisome proliferator activated receptor alpha (PPARα), which induced the transcription of BNIP3 via directly binding to its promoter region. Both in vitro and in vivo experiments confirmed that the renoprotective effect of OA was dramatically reduced by GW6471, a PPARα antagonist. Conclusion: Our findings revealed that OA ameliorates AKI-to-CKD transition by maintaining mitochondrial homeostasis through inducing PPARα-BNIP3 signaling pathway, indicating that OA may serve as a candidate therapeutic strategy for alleviating AKI and CKD.

Vitamin K2 Improves Osteogenic Differentiation by Inhibiting STAT1 via the Bcl-6 and IL-6/JAK in C3H10 T1/2 Clone 8 Cells

Osteogenic activity of vitamin K2 (VK2), a small molecular nutrient, has been suggested. However, the underlying mechanisms have not been fully elucidated. Therefore, this study aimed to explore the mechanisms by which VK2 promotes osteogenic differentiation. The effects of VK2 on osteogenic differentiation indicators were determined in C3H10 T1/2 clone 8 cells. The RNA-seq analysis was used to explore the hypothesis that VK2 promotes osteogenic differentiation. Small interfering RNA (siRNA) assay and plasmid transfection assay were used to determine the potential role of VK2 in the modulation of Bcl-6/STAT axis and IL-6/JAK/STAT signaling pathway. VK2 significantly increased alkaline phosphatase (ALP) activity, ALP, osteocalcin (OCN), and RUNX2 abundance, and RUNX2 protein expression. RNA-seq analysis showed that there were 314 differentially expressed genes (DEGs) upregulated and 1348 DEGs downregulated by VK2. PPI analysis determined the top 10 hub genes upregulated or downregulated by VK2. Overexpression of Bcl-6 increased osteogenic differentiation and decreased expression of STAT1. Administration with VK2 restored the inhibition by siBcl-6 in osteogenic differentiation. Knockdown of IL-6 decreased the mRNA levels of genes associated with the JAK/STAT signaling pathway, and increased markers of osteoblast differentiation. Furthermore, treatment with VK2 improved inhibition in osteogenic differentiation and decreased enhancement of JAK/STAT signaling pathway related genes by overexpression of IL-6. Our study suggests that VK2 could improve osteogenic differentiation via the Bcl-6/STAT axis and IL-6/JAK/STAT signaling pathway.

Tofacitinib May Inhibit Myofibroblast Differentiation from Rheumatoid-Fibroblast-like Synoviocytes Induced by TGF-β and IL-6

During rheumatoid arthritis (RA), the pathogenic role of resident cells within the synovial membrane is suggested, especially for a population frequently referred to as fibroblast-like synoviocytes (FLSs). In this study, we assess the markers of myofibroblast differentiation of RA-FLSs by ex vivo observations and in vitro evaluations following the stimulation with both TGF-β and IL-6. Furthermore, we investigated the possible inhibiting role of tofacitinib, a JAK inhibitor, in this context. Myofibroblast differentiation markers were evaluated on RA synovial tissues by immune-fluorescence or immune-histochemistry. RA-FLSs, stimulated with transforming growth factor (TGF-β) and interleukin-6 (IL-6) with/without tofacitinib, were assessed for myofibroblast differentiation markers expression by qRT-PCR and Western blot. The same markers were evaluated following JAK-1 silencing by siRNA assay. The presence of myofibroblast differentiation markers in RA synovial tissue was significantly higher than healthy controls. Ex vivo, α-SMA was increased, whereas E-Cadherin decreased. In vitro, TGF-β and IL-6 stimulation of RA-FLSs promoted a significant increased mRNA expression of collagen I and α-SMA, whereas E-Cadherin mRNA expression was decreased. In the same conditions, the stimulation with tofacitinib significantly reduced the mRNA expression of collagen I and α-SMA, even if the Western blot did not confirm this finding. JAK-1 gene silencing did not fully prevent the effects of stimulation with TGF-β and IL-6 on these features. TGF-β and IL-6 stimulation may play a role in mediating myofibroblast differentiation from RA-FLSs, promoting collagen I and α-SMA while decreasing E-Cadherin. Following the same stimulation, tofacitinib reduced the increases of both collagen I and α-SMA on RA-FLSs, although further studies are needed to fully evaluate this issue and confirm our results.

AKAP12 ameliorates liver injury via targeting PI3K/AKT/PCSK6 pathway

A kinase anchor protein 12（AKAP12）is a scaffold protein that is critical for cell structure maintenance and signal transduction. However, the role of AKAP12 in liver injury remains unclear. Here, we attempt to explore the potential contribution of AKAP12 in liver injury and elucidate its underlying molecular mechanism. We found that AKAP12 deletion in acute liver injury (ALI) activates the PI3K/AKT phosphorylation signaling pathway, induces the increased expression of PCSK6 and its downstream inflammation-related genes, and prompts macrophages to produce a large number of inflammatory factors. And knockdown of PCSK6 by in vivo siRNA assay reversed in liver injury AKAP12Δhep mice, demonstrating that PCSK6 has an important role in ALI. Furthermore, we found that signal transducer and activator of transcription 3 (STAT3) and serine/threonine kinase Akt (AKT) were upregulated in AKAP12Δhep mice of chronic liver injury. To sum up, our study here demonstrates that AKAP12 has a protective role in ALI and chronic liver fibrosis, at least in part through inhibition of the PI3K/AKT/PCSK6 pathway. Our findings provide a new potential treatment for liver injury with important clinical implications.

Expression and Clinical Significance of High‐Mobility Group AT‐hook 2 (HMGA2) in Osteosarcoma

ObjectiveAlthough high‐mobility group AT‐hook 2 (HMGA2) has been shown to have crucial roles in the pathogenesis and metastasis of various malignancies, its expression and significance in osteosarcoma remain unknown. Here we evaluate the expression, clinical prognostic value, and overall function of HMGA2 in osteosarcoma.MethodsSixty‐nine osteosarcoma patient specimens within a tissue microarray (TMA) were analyzed by immunohistochemistry for HMGA2 expression. Demographics and clinicopathological information including age, gender, tumor location, metastasis, recurrence, chemotherapy response, follow‐up time, and disease status were also collected. After validation of expression, we determined whether there was a correlation between HMGA2 expression and patient clinicopathology. HMGA2 expression was also evaluated in osteosarcoma cell lines and patient tissues by Western blot, we analyzed the expression of HMGA2 in the human osteosarcoma cell lines MG63, 143B, U2OS, Saos‐2, MNNG/HOS, and KHOS. HMGA2‐specific siRNA and clonogenic assays were then used to determine the effect of HMGA2 inhibition on osteosarcoma cell proliferation, growth, and chemosensitivity.ResultsHMGA2 expression was elevated in the osteosarcoma patient specimens and human osteosarcoma cell lines. HMGA2 was differentially expressed in human osteosarcoma cell lines. Specifically, a relatively high expression of HMGA2 was present in KHOS, MNNG/HOS, 143B and a relatively low expression was in MG63, U2OS as well as Saos‐2. HMGA2 expression is correlated with metastasis and shorter overall survival. High HMGA2 expression is an independent predictor of poor osteosarcoma prognosis. There was no significant correlation between HMGA2 expression and the age, gender, or tumor site of the patient. HMGA2 expression is predominantly within the nucleus. The expression of HMGA2 also directly correlated to neoadjuvant chemoresistance. There was a significant reduction of HMGA2 expression in the siRNA transfection group. After the use of siRNA, the proliferation of osteosarcoma cells is decreased and the chemosensitivity of osteosarcoma cells is significantly increased.ConclusionOur study supports HMGA2 as a potential prognostic biomarker and therapeutic target in osteosarcoma.

Isolation, Identification, and Immunomodulatory Mechanism of Peptides from Lepidium meyenii (Maca) Protein Hydrolysate.

Maca is a protein-enriched edible plant with immunomodulatory activity. However, the role of proteins in the immunomodulatory activity of maca is unclear. In this study, peptide products of maca proteins obtained through in vitro gastrointestinal digestion were isolated and purified, and the immunomodulatory activities of these peptides were assessed in macrophages (RAW 264.7 cells). The results show that the maca protein hydrolysate enhanced the phagocytic capacity and NO, TNF-α, and IL-6 secretion of RAW 264.7 cells. Forty-five peptides from known proteins of maca or the cruciferous family were identified by ultraperformance liquid chromatography-tandem mass spectrometry in the hydrolysate, and the peptide RNPFLP exhibited the strongest immunomodulatory activity. Antibody blocking, siRNA, pathway inhibitors, and western blot assays showed that RNPFLP-activated RAW 264.7 cells through the NF-κB and MAPK signaling pathways mediated by TLR2 and TLR4 receptors. An analysis of the structure-activity relationship showed that the N9-H60 active site in arginine plays an important role in the immunomodulatory activity of RNPFLP. This study provides a new understanding of the immunomodulatory activity of maca.

The c-Myc/TBX3 Axis Promotes Cellular Transformation of Sarcoma-Initiating Cells.

Sarcomas are highly aggressive cancers of mesenchymal origin whose clinical management is highly complex. This is partly due to a lack of understanding of the molecular mechanisms underpinning the transformation of mesenchymal stromal/stem cells (MSCs) which are presumed to be the sarcoma-initiating cells. c-Myc is amplified/overexpressed in a range of sarcomas where it has an established oncogenic role and there is evidence that it contributes to the malignant transformation of MSCs. T-box transcription factor 3 (TBX3) is upregulated by c-Myc in a host of sarcoma subtypes where it promotes proliferation, tumor formation, migration, and invasion. This study investigated whether TBX3 is a c-Myc target in human MSCs (hMSCs) and whether overexpressing TBX3 in hMSCs can phenocopy c-Myc overexpression to promote malignant transformation. Using siRNA, qRT-PCR, luciferase reporter and chromatin-immunoprecipitation assays, we show that c-Myc binds and directly activates TBX3 transcription in hMSCs at a conserved E-box motif. When hMSCs were engineered to stably overexpress TBX3 using lentiviral gene transfer and the resulting cells characterised in 2D and 3D, the overexpression of TBX3 was shown to promote self-renewal, bypass senescence, and enhance proliferation which corresponded with increased levels of cell cycle progression markers (cyclin A, cyclin B1, CDK2) and downregulation of the p14ARF/MDM2/p53 tumor suppressor pathway. Furthermore, TBX3 promoted the migratory and invasive ability of hMSCs which associated with increased levels of markers of migration (Vimentin, SLUG, SNAIL, TWIST1) and invasion (MMP2, MMP9). Transcriptomic analysis revealed that genes upregulated upon TBX3 overexpression overlapped with c-myc targets, were involved in cell cycle progression, and were associated with sarcomagenesis. Together, the data described indicate that the c-Myc/TBX3 oncogenic molecular pathway may be a key mechanism that transforms hMSCs into sarcomas.