Serum lipidomic profiling by UHPLC-MS/MS may be able to detect early-stage endometrial cancer.

Nowadays, screening for endometrial cancer (EC) primarily relies on clinical symptoms and imaging, which makes it difficult to detect early-stage disease. Here, we conducted a widely targeted lipidomic analysis of 38 human serum samples in a discovery set and 40 human serum samples in a validation set to profile the dysregulated lipid species and establish lipid biomarkers for early-stage EC. This comprehensive lipidomic determination of 616 serum lipids indicated significant differences between early-stage EC patients and healthy controls. Three phases of lipid biomarker investigation (discovery, validation, and determination of the lipid biomarker panel) were performed, which revealed the upregulation of some sphingolipid, glycerophospholipid, and glycerolipids and downregulation of some carnitine. Consistently, the perturbation of sphingolipid and glycerophospholipid metabolism was also observed from pathway enrichment analysis. Moreover, a lipid biomarker panel, including ursodeoxycholic acid, PC(O-14:0_20:4), and Cer(d18:1/18:0), was established. This panel was assessed as an effective diagnostic model to distinguish early-stage EC patients from healthy controls and atypical endometrial hyperplasia patients within the area under the receiver operating characteristic curve (AUC) reaching 0.903 and 0.928, respectively. In particular, the comparison results of the diagnostic efficacy indicated that the lipid biomarker panel was superior to clinically established indicators for EC diagnosis, including HE4, CA125, CA153, and CA199, suggesting that it could be used as an excellent supplementary method for the diagnosis of early-stage EC. In conclusion, we established a novel and non-invasive lipid biomarker for early-stage EC detection and these findings may provide new insight into the pathological mechanisms of EC.

Single-cell transcriptomic data reveal the increase in extracellular matrix organization and antigen presentation abilities of fibroblasts and smooth muscle cells in patients with pelvic organ prolapse.

INTRODUCTION AND HYPOTHESIS: We aimed to explore the cellular properties of fibroblasts and smooth muscle cells (SMCs), the two major cell types of the vagina wall, in pelvic organ prolapse (POP) to improve the knowledge of the underlying molecular mechanisms of POP. METHODS: The single-cell RNA sequencing (scRNA-seq) profile GSE151202 was downloaded from NCBI Gene Expression Omnibus, in which vaginal wall tissues were harvested from patients with anterior vaginal wall prolapse and control subjects respectively. The scRNA-seq data of samples (5 POP and 5 controls) were adopted for analysis. Cluster analysis was performed to identify the cell subclusters. Trajectory analysis was applied to construct the differentiation trajectories of fibroblasts and SMCs. Cellular communication analysis was carried out to explore the ligand-receptor interactions between fibroblasts/SMCs and immune cells. RESULTS: Ten subclusters were determined in both groups, among which fibroblasts and SMCs were the most abundant cell types. Compared with controls, fibroblasts increased whereas SMCs declined in POP. During the transition of fibroblasts and SMCs from a normal into a disease state, extracellular matrix organization and antigen presentation were heightened. The intercellular communications were altered in POP. Interactions between fibroblasts/SMCs and macrophages/natural killer/T cells were strengthened as more ligand-receptor pairs involved in antigen presentation pathways were gained in POP. CONCLUSION: Extracellular matrix organization and antigen presentation abilities of fibroblasts and SMCs were enhanced in POP.

Exosomal miR-374c-5p derived from mesenchymal stem cells suppresses epithelial-mesenchymal transition of hepatocellular carcinoma via the LIMK1-Wnt/ß-catenin axis.

Metastasis is a leading cause to treatment failure in hepatocellular carcinoma (HCC) patients. Exosomes act as pivotal mediators in communication between different cells and exert effects on recipient cells by delivering bioactive cargoes, such as microRNAs (miRNAs). MiRNAs function in multiple steps of HCC development, including metastasis. MiR-374c-5p was previously identified as a tumor suppressor in some malignancies, while the current knowledge of its role in HCC metastasis is still limited. Herein, miR-374c-5p was found to be downregulated in HCC cell lines and clinical samples, and positively related with favorable prognosis in HCC patients. MiR-374c-5p transferred by exosomes derived from bone marrow mesenchymal stem cell (BMSC) suppressed migration, invasion and proliferation of HCC cells. LIMK1 was verified as downstream target gene of miR-374c-5p. Knockdown of LIMK1 reduced invasion, migration and proliferation of HCC cells, whereas overexpression functioned oppositely. The miR-374c-5p/LIMK1 axis suppressed epithelial-mesenchymal transition (EMT) by inactivating Wnt/ß-catenin pathway. In addition, miR-374c-5p was downregulated and LIMK1 upregulated in TGF-ß1 induced EMT. This EMT model could be reversed by LIMK1 silencing or miR-374c-5p overexpression. These results suggest that exo-miR-374c-5p suppresses EMT via targeting LIMK1-Wnt/ß-catenin axis and the axis is involved in TGF-ß1 induced metastasis of HCC, thereby identifying miR-374c-5p as a potential target for HCC treatment.

XIAP, commonly targeted by tumor suppressive miR-3607-5p and miR-3607-3p, promotes proliferation and inhibits apoptosis in hepatocellular carcinoma.

MicroRNAs (miRNAs) are frequently aberrantly expressed in hepatocellular carcinoma (HCC) and are involved in its development. However, their role and mechanism in HCC are still not fully elucidated. Differential expression analysis and survival analysis were performed to identify potential miRNAs in HCC and miR-3607 was identified as a candidate therapeutic target and prognostic biomarker. RT-qPCR confirmed the low expression of mature miR-3607-3p and miR-3607-5p in HCC. Functional experiments suggested that both miR-3607-3p and miR-3607-5p significantly inhibited HCC proliferation and induced apoptosis. Next, the detailed mechanism of miR-3607-3p and miR-3607-5p in HCC was explored by combination of bioinformatic analysis and experimental validation, and uncovered that XIAP, a common target gene of miR-3607-3p and miR-3607-5p, was involoved in their tumor suppressive effects. Finally, a XIAP-associated protein-protein interaction network, consisting of 10 positively correlated genes, was established. Collectively, we for the first time suggest that miR-3607-3p and miR-3607-5p inhibit HCC by acting one common target XIAP.

The value of the apparent diffusion coefficient in differentiating type II from type I endometrial carcinoma.

BACKGROUND: Diagnostic type II endometrial carcinoma (EC) is considered more aggressive and has a poorer prognosis than type I EC; differentiation between them is helpful for preoperative clinical decision-making. However, the diagnostic value of the apparent diffusion coefficient (ADC) in differentiating them remains unclear. PURPOSE: To investigate the value of ADC in differentiating type II EC from type I EC. MATERIAL AND METHODS: Ninety-four patients with EC who underwent diffusion-weighted imaging (DWI) were retrospectively included and divided into type I and type II subgroups, based on the postoperative pathologic results. We analyzed the clinical characteristics, conventional magnetic resonance imaging manifestations, and ADC mean values (ADCmean), ADC minimum values (ADCmin), and ADC max values (ADCmax). Receiver operating characteristic (ROC) curve analysis was further used to assess the predictive performance. RESULTS: The ADCmean, ADCmin, and tumor size differed significantly between the two subtypes. The area under the ROC curve (AUC) for ADCmean and ADCmin was 0.787 (95% confidence interval [CI] = 0.692-0.88) and 0.835 (95% CI = 0.751-0.919) for predicting type II EC, respectively. The optimal cut-off value of ADCmean for prediction was 0.757 × 10-3 mm2/s with a sensitivity of 91%, a specificity of 58%, and an accuracy of 74%, while for ADCmin was 0.637 × 10-3 mm2/s with a sensitivity of 82%, a specificity of 73%, and an accuracy of 75%. CONCLUSION: EC with lower ADCmean and ADCmin values derived from DWI, and a larger size, are indicative of type II EC.

Peptidomic analysis on synovial tissue reveals galectin-1 derived peptide as a potential bioactive molecule against rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease that leads to small joints irreversible destruction. Despite intense efforts, the pathophysiology of RA currently remains unclear. We aimed to gain insight into the pathophysiology process in peptidomic perspective and to identify bioactive peptides for RA treatment. METHODS: The endogenous peptides in synovial tissue between control and rheumatoid arthritis group were identified by liquid chromatography-mass spectrometry (LC-MS/MS). Since the biological function of peptides were always associated with precursor proteins, the potential function of the differentially peptides were predicted by GO and pathway analysis of their precursors. Besides, peptides located in the domains of their precursors were identified. Finally, we determined the impact of galectin-1 derived peptide by administration on the damage to MH7A cells caused by TNF-α. RESULTS: Totally, 141 down-regulated peptides and 10 up-regulated peptides were identified (Fold change > 1.5 and P < 0.05). It indicated that these differentially peptides were tightly involved in the pathophysiology process of RA preliminarily. Finally, we identified a peptide derived from the domain of galectin-1 could inhibit the abnormal proliferation induced by TNF-α and promoted apoptosis of MH7A. CONCLUSION: In summary, our study provided a better understanding of endogenous peptides in RA. We found a peptide that might be used in anti-RA treatment.

Sinomenine Inhibited Interleukin-1ß-Induced Matrix Metalloproteinases Levels via SOCS3 Up-Regulation in SW1353 Cells.

Matrix metalloproteinases (MMPs) are required for collagen degradation which play a key pathological role in arthritis progression. Herein, the effect of sinomenine (SN) on Interleukin 1 beta (IL-1ß)-induced MMPs production and its underlying mechanism were explored in SW1353 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that 200 and 400 µM SN significantly inhibited SW1353 cell proliferation, thus the lower dose of SN (25-100 µM) were used in the subsequent experiments. Notably, the increased mRNA and protein levels of suppressor of cytokine signaling (SOCS) 3 were dose-dependently induced by SN. SN significantly suppressed mRNA and protein levels of MMPs in IL-1ß-induced SW1353 cells. Through Western blot analysis, SN showed inhibitory effect on IL-1ß-induced TAK1 and p65 phosphorylation. Moreover, SN blocked the interaction of TRAF6 and TAK1 resulting in inactivation of IL-1ß pathway. Mechanistically, the inhibitory effect of SN on MMPs levels alongside TRAF6 and TAK1 interactions was abrogated by silencing SOCS3. Moreover, SN did not inhibit TAK1 kinase activity. In TAK1 silencing cells, the levels of MMPs and p65 phosphorylation of SN-treatedcells were lower than dimethyl sulfoxide (DMSO)-treated cells, indicating that blocking interaction was not a unique way for SN to inhibit MMPs levels. Finally, SN significantly inhibited IL-6-induced Janus tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in SW1353 cells. The levels of JAK2 phosphorylation and MMPs did not show a significant difference between IL-6 + SOCS3-small interfering RNA (siRNA) + SN group and IL-6 + SOCS3-siRNA + DMSO group. These findings demonstrated that SOCS3 expression was increased by SN blocked IL-1ß-induced interaction between TRAF6 and TAK1 as well as IL-6 pathway activation, thereby culminating in the inhibition of MMPs levels.

Identification of potential miRNA-mRNA regulatory network contributing to pathogenesis of HBV-related HCC.

BACKGROUND: Hepatitis B virus (HBV) is one of the major risk factors of hepatocellular carcinoma (HCC). Increasing evidence indicates that microRNA (miRNA)-mRNA axis is involved in HCC. However, a comprehensive miRNA-mRNA regulatory network in HBV-related HCC is still absent. This study aims to identify potential miRNA-mRNA regulatory pathways contributing to pathogenesis of HBV-related HCC. METHODS: Microarray GSE69580 was downloaded from Gene Expression Omnibus (GEO) database. GEO2R and 'R-limma' were used to conduct differential expression analysis. The common miRNAs appeared in the two analytic sets were screened as potential differentially expressed miRNAs (DE-miRNAs). The prognostic roles of screened DE-miRNAs in HCC were further evaluated using Kaplan-Meier plotter database. Target genes of DE-miRNAs were predicted by miRNet. Then, protein-protein interaction (PPI) networks were established for these targets via the STRING database, after which hub genes in the networks were identified by Cytoscape. Functional annotation and pathway enrichment analyses for the target genes were performed through DAVID database. Three enriched pathways related to HBV-related HCC were selected for further analysis and potential target genes commonly appeared in all three pathways were screened. Cytoscape was employed to construct miRNA-hub gene network. The expression and correlation of potential miRNAs and targets were further detected in clinical HBV-related HCC samples by qRT-PCR. RESULTS: 7 upregulated and 9 downregulated DE-miRNAs were accessed. 5 of 7 upregulated DE-miRNAs and 5 of 7 downregulated DE-miRNAs indicated significant prognostic roles in HCC. 2312 and 1175 target genes were predicted for the upregulated and downregulated DE-miRNAs, respectively. TP53 was identified as the hub gene in the PPI networks. Pathway enrichment analysis suggested that these predicted targets were linked to hepatitis B, pathways in cancer, microRNAs in cancer and viral carcinogenesis. Further analysis of these pathways screened 20 and 16 target genes for upregulated and downregulated DE-miRNAs, respectively. By detecting the expression of 36 target genes, six candidate target genes were identified. Finally, a potential miRNA-mRNA regulatory network was established based on the results of qRT-PCR and expression correlation analysis. CONCLUSIONS: In the study, potential miRNA-mRNA regulatory pathways were identified, exploring the underlying pathogenesis and effective therapy strategy of HBV-related HCC.

In silico analysis excavates potential biomarkers by constructing miRNA-mRNA networks between non-cirrhotic HCC and cirrhotic HCC.

BACKGROUND: Mounting evidences have demonstrated that HCC patients with or without cirrhosis possess different clinical characteristics, tumor development and prognosis. However, few studies directly investigated the underlying molecular mechanisms between non-cirrhotic HCC and cirrhotic HCC. METHODS: The clinical information and RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) database. Differentially expressed genes (DEGs) of HCC with or without cirrhosis were obtained by R software. Functional annotation and pathway enrichment analysis were performed by Enrichr. Protein-protein interaction (PPI) network was established through STRING and mapped to Cytoscape to identify hub genes. MicroRNAs were predicted through miRDB database. Furthermore, correlation analysis between selected genes and miRNAs were conducted via starBase database. MiRNAs expression levels between HCC with or without cirrhosis and corresponding normal liver tissues were further validated through GEO datasets. Finally, expression levels of key miRNAs and target genes were validated through qRT-PCR. RESULTS: Between 132 non-cirrhotic HCC and 79 cirrhotic HCC in TCGA, 768 DEGs were acquired, mainly involved in neuroactive ligand-receptor interaction pathway. According to the result from gene expression analysis in TCGA, CCL19, CCL25, CNR1, PF4 and PPBP were renamed as key genes and selected for further investigation. Survival analysis indicated that upregulated CNR1 correlated with worse OS in cirrhotic HCC. Furthermore, ROC analysis revealed the significant diagnostic values of PF4 and PPBP in cirrhotic HCC, and CCL19, CCL25 in non-cirrhotic HCC. Next, 517 miRNAs were predicted to target the 5 key genes. Correlation analysis confirmed that 16 of 517 miRNAs were negatively regulated the key genes. By detecting the expression levels of these key miRNAs from GEO database, we found 4 miRNAs have high research values. Finally, potential miRNA-mRNA networks were constructed based on the results of qRT-PCR. CONCLUSION: In silico analysis, we first constructed the miRNA-mRNA regulatory networks in non-cirrhotic HCC and cirrhotic HCC.

The Critical Role of PTEN/PI3K/AKT Signaling Pathway in Shikonin-Induced Apoptosis and Proliferation Inhibition of Chronic Myeloid Leukemia.

BACKGROUND/AIMS: Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm. Tyrosine kinase inhibitors (TKIs) are commonly used to treat CML; however, drug resistance of CML cells to TKIs has limited their clinical application. Shikonin, a traditional Chinese herb, has long been used to treat leukemia in China, but the roles and related molecular mechanisms of shikonin treatment in CML remain unclear. Here, we aimed to evaluate the effects of shikonin on the proliferation, apoptosis, and migration of K562 cells, a CML cell line. METHODS: Firstly, K562 cell proliferation and apoptosis were tested by CCK8 assay and flow cytometry with Annexin V-FITC/PI staining. Cell migration was measured by Transwell migration assay. In addition, western blot was performed to determine the proteins (PI3K, Bax, Bcl-2, cleaved caspase-3, PTEN, p-AKT, AKT, CXCR4, SDF-1, CD44) involved in the mechanism of action of shikonin. Finally, neutrophils from peripheral blood of CML patients were obtained, and cell proliferation and apoptosis were tested by CCK8 assay and flow cytometry. RESULTS: Shikonin reduced the proliferation of K562 cells in a time- and dose-dependent manner and promoted the apoptosis of K562 cells. Moreover, shikonin increased the PTEN level and inactivated the PI3K/AKT signaling pathway, subsequently upregulating BAX in K562 cells. In addition, shikonin could block K562 cell migration via the CXCR4/SDF-1 axis. Finally, shikonin significantly inhibited the proliferation and promoted the apoptosis of neutrophils from CML patients. CONCLUSION: These results demonstrated that shikonin inhibits CML proliferation and migration and induces apoptosis by the PTEN/PI3K/AKT pathway, revealing the effects of shikonin therapy on CML.

Identification of invasion-metastasis-associated microRNAs in hepatocellular carcinoma based on bioinformatic analysis and experimental validation.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most lethal cancer, mainly attributing to its high tendency to metastasis. Vascular invasion provides a direct path for solid tumor metastasis. Mounting evidence has demonstrated that microRNAs (miRNAs) are related to human cancer onset and progression including invasion and metastasis. METHODS: In search of invasion-metastasis-associated miRNAs in HCC, microarray dataset GSE67140 was downloaded from the Gene Expression Omnibus database. Differentially expressed miRNAs (DE-miRNAs) were obtained by R software package and the potential target genes were predicted by miRTarBase. The database for annotation, visualization and integrated discovery (DAVID) was introduced to perform functional annotation and pathway enrichment analysis for these potential targets of DE-miRNAs. Protein-protein interaction (PPI) network was established by STRING database and visualized by Cytoscape software. The effects of the miR-494-3p and miR-126-3p on migration and invasion of HCC cell lines were evaluated by conducting wound healing assay and transwell assay. RESULTS: A total of 138 DE-miRNAs were screened out, including 57 upregulated miRNAs and 81 downregulated miRNAs in human HCC tumors with vascular invasion compared with human HCC tumors without vascular invasion. 762 target genes of the top three upregulated and downregulated miRNAs were predicted, and they were involved in HCC-related pathways, such as pathway in cancer, focal adhesion and MAPK signaling pathway. In the PPI network, the top 10 hub nodes with higher degrees were identified as hub genes, such as TP53 and MYC. Through constructing the miRNA-hub gene network, we found that most of hub genes could be potentially modulated by miR-494-3p and miR-126-3p. Of note, miR-494-3p and miR-126-3p was markedly upregulated and downregulated in HCC cell lines and tissues, respectively. In addition, overexpression of miR-494-3p could significantly promote HCC migration and invasion whereas overexpression of miR-126-3p exerted an opposite effect. CONCLUSIONS: Targeting miR-494-3p and miR-126-3p may provide effective and promising approaches to suppress invasion and metastasis of HCC.

RNASET2 impairs the sperm motility via PKA/PI3K/calcium signal pathways.

Asthenozoospermia is one of the leading causes of male infertility owing to a decline in sperm motility. Herein, we determined if there is a correlation between RNASET2 content on human spermatozoa and sperm motility in 205 semen samples from both asthenozoospermia patients and normozoospermia individuals. RNASET2 content was higher in sperm from asthenozoospermia patients than in normozoospermia individuals. On the other hand, its content was inversely correlated with sperm motility as well as progressive motility. Moreover, the inhibitory effect of RNASET2 on sperm motility was induced by incubating normozoospermic sperm with RNase T2 protein. Such treatment caused significant declines in intracellular spermatozoa PKA activity, PI3K activity and calcium level, which resulted in severely impaired sperm motility, and the sperm motility was largely rescued by cAMP supplementation. Finally, protein immunoprecipitation and mass spectrometry identified proteins whose interactions with RNASET2 were associated with declines in human spermatozoa motility. AKAP4, a protein regulating PKA activity, coimmunoprecipated with RNASET2 and they colocalized with one another in the sperm tail, which might contribute to reduced sperm motility. Thus, RNASET2 may be a novel biomarker of asthenozoospermia. Increases in RNASET2 can interact with AKAP4 in human sperm tail and subsequently reduce sperm motility by suppressing PKA/PI3K/calcium signaling pathways.

Effects of Hesperidin on H2O2-Treated Chondrocytes and Cartilage in a Rat Osteoarthritis Model.

BACKGROUND The purpose of this research was to investigate the effects of hesperidin on hydrogen peroxide (H2O2)-induced chondrocytes injury and cartilage degeneration in a rat model of osteoarthritis (OA). MATERIAL AND METHODS Chondrocytes were isolated from rat knee joints and treated with hesperidin alone or combined with H2O2. Then, Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability. Activity of reactive oxygen species (ROS) and levels of malondialdehyde (MDA) were estimated. Cell apoptosis was assessed by flow cytometry assay. In addition, gene expression levels were measured for caspase 3, tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1ß), collagen type II (Col2a1), aggrecan, (sex-determining region Y)-box 9 (SOX9), matrix metalloproteinase (MMP)-13, and inducible nitric oxide synthase (iNOS) through quantitative real-time polymerase chain reaction (qPCR). To examine the effects on cartilage destruction in vivo, hesperidin or vehicle control were orally administrated in a surgically-induced osteoarthritis (OA) model. RESULTS The results indicated that hesperidin pretreatment of chondrocytes reduce H2O2-induced cytotoxicity and apoptosis. Hesperidin pretreatment decreased the formation of MDA and intracellular ROS, including chondrocyte apoptosis. Hesperidin also reversed the activity of H2O2 on inhibiting the Col2a1, aggrecan, and SOX9 gene expression and increasing the gene expression of caspase 3, IL-1ß, TNFα, iNOS, and MMP13. In addition, hesperidin administration markedly attenuated cartilage destruction and reduced IL-1ß and TNF-α levels in a surgically-induced OA model. CONCLUSIONS Our study suggests that hesperidin can prevent H2O2-induced chondrocytes injury through its antioxidant effects in vitro and reduce cartilage damage in a rat model of OA.

Proteomic Analysis Reveals a New Benefit of Periodic Mechanical Stress on Chondrocytes.

BACKGROUND/AIMS: In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress. In our previous studies, we found that periodic mechanical stress can promote proliferation and matrix synthesis through the integrin beta 1-mediated ERK1/2 pathway, and we used proteomic analysis to detect quantitative changes in chondrocytes under periodic mechanical stress. Despite these results, the effects and mechanisms of periodic mechanical stress are still not fully understood, so in this study we extended our study using phosphoproteomic techniques. METHODS: We used phosphoproteomic techniques to detect phosphorylation changes in chondrocytes under periodic mechanical stress and combined the results with the quantitative proteomic data to further explore the underlying mechanisms. Data were obtained by phosphorylation inhibition, quantitative real-time PCR (qPCR) analysis, western blot analysis and immunofluorescence assay. RESULTS: From phosphoproteomic analysis, a total of 1073 phosphorylated proteins and 2054 phosphopeptides were identified. The number of significant differentially expressed proteins and phosphopeptides was 97 and 108, respectively (ratio >1.20 or <0.83 at p<0.05). Periodic mechanical stress increased glycogen synthase kinase 3-beta (GSK3-beta) phosphorylation at Y216, promoted the phosphorylation of beta-catenin, decreased beta-catenin levels and suppressed the expression of type I collagen. In contrast, inhibition of GSK3-beta by TWS119, which specifically inhibits the phosphorylation of Y216, suppressed the phosphorylation of beta-catenin, which resulted in the accumulation of beta-catenin and an increase in the expression of type I collagen. CONCLUSIONS: We successfully constructed differentially expressed phosphoproteomic profiles of rat chondrocytes under periodic mechanical stress, and discovered a potential new therapeutic benefit in which periodic mechanical stress suppressed the formation of type I collagen in the matrix of chondrocytes via phosphorylation of GSK3-beta and beta-catenin.

Globular Adiponectin Attenuated H2O2-Induced Apoptosis in Rat Chondrocytes by Inducing Autophagy Through the AMPK/ mTOR Pathway.

BACKGROUND/AIMS: Chondrocyte apoptosis is closely related to the development and progression of osteoarthritis. Global adiponectin (gAPN), secreted from adipose tissue, possesses potent anti-inflammatory and antiapoptotic properties in various cell types. This study aimed to investigate the role of autophagy induced by gAPN in the suppression of H2O2-induced apoptosis and the potential mechanism of gAPN-induced autophagy in chondrocytes. METHODS: H2O2 was used to induce apoptotic injury in rat chondrocytes. CCK-8 assay was performed to determine the viability of cells treated with different concentrations of gAPN with or without H2O2. Cell apoptosis was detected by flow cytometry and TUNEL staining. Mitochondrial membrane potential was examined using JC-1 fluorescence staining assay. The autophagy inhibitors 3-MA and Bafilomycin A1 were used to treat cells and then evaluate the effect of gAPN-induced autophagy. To determine the downstream pathway, chondrocytes were preincubated with the AMPK inhibitor Compound C. Beclin-1, LC3B, P62 and apoptosis-related proteins were identified by Western blot analysis. RESULTS: H2O2 (400 µM)-induced chondrocytes apoptosis and caspase-3 activation were attenuated by gAPN (0.5 µg/mL). gAPN increased Bcl-2 expression and decreased Bax expression. The loss of mitochondrial membrane potential induced by H2O2 was also abolished by gAPN. Furthermore, the antiapoptotic effect of gAPN was related to gAPN-induced autophagy by increased formation of Beclin-1 and LC3B and P62 degradation. In particular, the inhibition of gAPN-induced autophagy by 3-MA prevented the protective effect of gAPN on apoptosis induced by H2O2. Moreover, gAPN increased p-AMPK expression and decreased p-mTOR expression. Compound C partly suppressed the expression of autophagy-related proteins and restored the expression of p-mTOR suppressed by gAPN. Thus, the AMPK/mTOR pathway played an important role in the induction of autophagy and protection of H2O2-induced chondrocytes apoptosis by gAPN. CONCLUSIONS: gAPN protected chondrocytes from H2O2-induced apoptosis by inducing autophagy possibly associated with AMPK/mTOR signal-pathway activation.

Proteomic Analysis Reveals Grb2 as a Key Regulator of Periodic Mechanical Stress Transduction in Chondrocytes.

BACKGROUND/AIMS: Periodic mechanical stress could significantly promote chondrocyte proliferation and matrix synthesis. However, the mechanisms underlying the ability of chondrocyte detecting and responding to periodic mechanical stimuli have not been well delineated. METHODS: Quantitative proteomic analysis was performed to construct the differently expressed proteome profiles of chondrocyte under pressure. Then a combination of Western blot, quantitative real-time PCR, lentiviral vector and histological methods were used to confirm the proteomic results and investigate the mechanoseing mechanism. RESULTS: Growth factor receptor-bound protein 2 (Grb2), a component of integrin adhesome, was found a 1.49-fold increase in dynamic stress group. This process was mediated through integrin ß1, leading to increased phosphorylation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase 1/2 (ERK1/2) respectively and then produce the corresponding biological effects. CONCLUSION: This was the first time to demonstrate Grb2 has such an important role in periodic mechanotransduction, and the proteomic data could facilitate the further investigation of chondrocytes mechanosensing.

Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling.

BACKGROUND/AIMS: Periodic mechanical stress can promote chondrocyte proliferation and matrix synthesis to improve the quality of tissue-engineered cartilage. Although the integrin ß1-ERK1/2 signal cascade has been implicated in periodic mechanical stress-induced mitogenic effects in chondrocytes, the precise mechanisms have not been fully established. The current study was designed to probe the roles of CaMKII and Pyk2 signaling in periodic mechanical stress-mediated chondrocyte proliferation and matrix synthesis. METHODS: Chondrocytes were subjected to periodic mechanical stress, proliferation was assessed by direct cell counting and CCK-8 assay; gene expressions were analyzed using quantitative real-time PCR, protein abundance by Western blotting. RESULTS: Mechanical stress, markedly enhanced the phosphorylation levels of Pyk2 at Tyr402 and CaMKII at Thr286. Both suppression of Pyk2 with Pyk2 inhibitor PF431396 or Pyk2 shRNA and suppression of CaMKII with CaMKII inhibitor KN-93 or CaMKII shRNA blocked periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Additionally, either pretreatment with KN-93 or shRNA targeted to CaMKII prevented the activation of ERK1/2 and Pyk2 under conditions of periodic mechanical stress. Interestingly, in relation to periodic mechanical stress, in the context of Pyk2 inhibition with PF431396 or its targeted shRNA, only the phosphorylation levels of ERK1/2 were abrogated, while CaMKII signal activation was not affected. Moreover, the phosphorylation levels of CaMKII- Thr286 and Pyk2- Tyr402 were abolished after pretreatment with blocking antibody against integrinß1 exposed to periodic mechanical stress. CONCLUSION: Our results collectively indicate that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through the integrinß1-CaMKII-Pyk2-ERK1/2 signaling cascade.

Circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis.

Circular RNAs (circRNAs), a class of noncoding RNAs generated from pre-mRNAs, participate in regulation of genes. The mechanism for regulation, however, is unknown. Here, to determine if, in human keratinocyte (HaCaT) cells, circular RNAs are involved in arsenite-induced acceleration of the cell cycle, a circRNA microarray was performed to analyze the variability of circRNAs in arsenite-treated HaCaT (As-HaCaT) cells and in arsenite-transformed (T-HaCaT) cells in comparison to control HaCaT cells. Among the circRNAs up-regulated in both As-HaCaT cells and T-HaCaT cells, hsa:circRNA_100284 (circ100284) had the greatest increase and was chosen for further research. The presence of circ100284 was confirmed in HaCaT cells. In these cells, arsenite induced increases of EZH2 and cyclin D1 and accelerated the cell cycle. MicroRNA (miR)-217 suppressed the expression of EZH2 was involved in regulation of the cell cycle. Further, in HaCaT cells exposed to arsenite, EZH2 regulated the cell cycle by binding to the promoter of CCND1, which codes for cyclin D1. Moreover, knockdown of circ100284 with siRNA inhibited the cell cycle acceleration induced by arsenite, but this inhibition was reversed by co-transfection with circ100284 siRNA and by a miR-217 inhibitor. Knockdown of circ100284 with siRNA or transfected with miR-217 mimic inhibited the capacity of T-HaCaT cells for colony formation, invasion, and migration, effects that were reversed by co-transfection with a miR-217 inhibitor or by epigenetic expression of EZH2. These results suggest that, in HaCaT cells, arsenite increases circ100284 levels, which act as a sponge for miR-217 and up-regulate the miR-217 target, EZH2, which, in turn, up-regulates cyclin D1and CDK4, and thus accelerates the cell cycle and leads to malignant transformation. Thus, circ100284, via miR-217 regulation of EZH2, is involved in the arsenite-accelerated cell cycle of human keratinocytes in carcinogenesis. This establishes a previously unknown mechanism between arsenite-induced acceleration of the cell cycle and carcinogenesis.

Down-regulation of RBP-J mediated by microRNA-133a suppresses dendritic cells and functions as a potential tumor suppressor in osteosarcoma.

BACKGROUND AND OBJECTIVE: In recent years, immunotherapy for the treatment of tumors have been established. Dendritic cells (DCs) are extremely efficient and professional antigen presenting cells (APCs), which are an important target for immune therapeutic interventions in cancer. In present study, we investigated whether RBP-J signaling regulated by miR-133a was involved in the DCs mediated tumor suppressor in osteosarcoma. METHODS: DCs were isolated from 30 osteosarcoma patients and 30 healthy subjects. Mouse macrophage-like cell line RAW264.7 were cultured and osteosarcoma mouse model with injection of murine osteosarcoma cell line S180 were established. RESULTS: In osteosarcoma patients, miR-133a expression level of DCs was increased, and RBP-J expression in mRNA and protein levels were decreased. MiR-133a inhibitor promoted maturation and activation of DCs in osteosarcoma patients. In osteosarcoma mouse model, miR-133a mimic suppressed the maturation and activation of spleen DCs, while miR-133a inhibitor promoted them. Overexpression of miR-133a decreased therapeutic effect of DCs on osteosarcoma mice. In RAW264.7 cells, miR-133a was observed to target RBP-J and regulate its expression. MiR-133a mimic inhibited the maturation of DCs in cells exposed to LPS, the effect of which was reversed by overexpression of RBP-J. CONCLUSION: RBP-J mediated by miR-133a probably contributed to the regulation of DCs maturation and activation in osteosarcoma, which functioned as a therapeutic target for the immunotherapy in cancers.

A novel role for integrin-linked kinase in periodic mechanical stress-mediated ERK1/2 mitogenic signaling in rat chondrocytes.

In recent years, a variety of studies have been performed to investigate the cellular responses of periodic mechanical stress on chondrocytes. Integrin ß1-mediated ERK1/2 activation was proven to be indispensable in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. However, other signal proteins responsible for the mitogenesis of chondrocytes under periodic mechanical stress remain incompletely understood. In the current investigation, we probed the roles of integrin-linked kinase (ILK) signaling in periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. We found that upon periodic mechanical stress induction, ILK activity increased significantly. Depletion of ILK with targeted shRNA strongly inhibited periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. In addition, pretreatment with a blocking antibody against integrin ß1 resulted in a remarkable decrease in ILK activity in cells exposed to periodic mechanical stress. Furthermore, inhibition of ILK with its target shRNA significantly suppressed ERK1/2 activation in relation to periodic mechanical stress. Based on the above results, we identified ILK as a crucial regulator involved in the integrin ß1-ERK1/2 signal cascade responsible for periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis.