Omentin-1 is associated with atrial fibrillation in patients with cardiac valve disease.

BACKGROUND: Epicardial adipose tissue (EAT) remodeling and adipocytokines are associated with structural remodeling in atrial fibrillation (AF). However, the role of omentin-1, a novel adipocytokine, in structural remodeling remains unknown. METHODS: Hematoxylin and eosin (H&E) and Masson's trichrome stains were used to investigate the histology of EAT and right atrial appendages. The expression levels of adipocytokines in these human samples were determined by immunohistochemical assay and western blotting. Models of transforming growth factor (TGF)-ß1-induced activation of cardiac fibroblasts (CFs) and TGF-ß1-induced endothelial-mesenchymal transition (EndMT) of human umbilical vein endothelial cell (HUVEC) were established to explore roles of omentin-1 in these processes. To determine changes in adipocytokines secretion under hypoxia conditions, adipocytes were treated with 5% O2 and 95% N2, and then CFs and HUVECs were co-cultured with the conditioned medium of adipocytes to determine the effects of hypoxia-treated adipocytes on these cells. RESULTS: Expression of omentin-1 was downregulated in the EAT and right atrial appendages from patients with AF compared to samples from patients without AF, while the TGF-ß1 level was upregulated in EAT from patients with AF. EAT from patients with AF exhibited adipocyte hypertrophy and severe interstitial fibrosis. Omentin-1 inhibited TGF-ß1-induced CF activation and reversed TGF-ß1-induced HUVEC EndMT. Adipocytes treated with hypoxia exhibited downregulation of omentin-1 and partly activated CFs. CONCLUSIONS: This study demonstrated that omentin-1 was an antifibrotic adipocytokine and was downregulated in patients with AF, which was partly mediated by hypoxia.

Endogenous reduction of miR-185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K.

Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR-185-5p were differently expressed in plasma from patients with ACS by high-throughput RNA sequencing. The expressional levels of miR-185-5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT-PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR-185-5p. In HUVECs, miR-185-5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR-185-5p inhibitors performed the opposites. Further, the inhibitory effects of miR-185-5p up-regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus-mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain-function of miR-185-5p by agomir infusion down-regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR-185-5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR-185-5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.

[Expression change of SH2B1, SOCS3, PTP1B and NPY in mice hypothalamus and its relation with obesity].

OBJECTIVE: To investigate the expression pattern of adapter protein with a Src-homology 2 domain (SH2B1), the suppressor of cytokine signaling-3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B) and neturopetide Y (NPY) in obese and normal mice hypothalamus and its relation with serum leptin and insulin levels. METHODS: The obesity animal model was prepared with healthy C57/bl6 mice. Lee's index and Homeostasis model assessment-insulin resistance (HOMA-IR) were calculated. The mRNA levels of SH2B1, SOCS3, PTP1B and NPY were measured by fluorescent quantitation RT-PCR. The SH2B1 and NPY protein expressions were detected by Western blot. RESULTS: Compared with the normal mice of the same age, SH2B1 mRNA expression in the obese mice hypothalamus decreased. SOCS3 and PTP1B mRNA expression increased. Western blot showed that SH2B1 protein expression decreased, while NPY protein expression increased in the obese mice. Linear correlation analysis showed that the serum leptin and fasting insulin levels were negatively correlated with SH2B1mRNA expression and positively correlated with SOCS3 and PTP1B mRNA expression. CONCLUSION: SH2B1, SOCS3, PTP1B and NPY are key factors for obesity development.

[Effect of DJ-1 siRNA on biological behavior of human lung squamous carcinoma SK-MES-1 cells].

OBJECTIVE: RNA interference technology (siRNA) was used to inhibit the expression of DJ-1 gene in lung squamous cell carcinoma SK-MES-1 cells, and the cell biological behaviors were investigated to explore the function of DJ-1 gene. METHODS: A targeted DJ-1 siRNA lentiviral vector with a green fluorescent protein (GFP) as a reporter was constructed. The constructed DJ-1 siRNA and control-siRNA vectors were infected into SK-MES-1 cells as experimental (DJ-1 siRNA) and control (Control siRNA) groups, respectively. The DJ-1 protein expression was determined by Western blot. The cell proliferation capability was measured with methyl thiazolyl tetrazolium (MTT). The cell cycle was analyzed by flow cytometry. The capability of cell migration was determined by Transwell method. RESULTS: Compared with control-siRNA and blank-control groups, the protein expression of DJ-1 gene was down-regulated, the capability of cell proliferation was obviously inhibited (P<0.01), the cell cycle was arrested with increased number of G1- and G2-phase cells and reduced number of S-phase cells, and the capability of cell migration was significantly decreased (P<0.01) in the DJ-1 siRNA-infected cells. CONCLUSION: DJ-1 gene might play a role in promoting cell proliferation and cell migration capability in vitro in lung cancer SK-MES-1 cells.

The prognosis of patients with postoperative hyperglycemia after Stanford type A aortic dissection surgery and construction of prediction model for postoperative hyperglycemia.

Objective: The mortality of type A aortic dissection (TAAD) is extremely high. The effect of postoperative hyperglycemia (PHG) on the prognosis of TAAD surgery is unclear. This study aims to investigate the prognosis of patients with PHG after TAAD surgery and construct prediction model for PHG. Methods: Patients underwent TAAD surgery from January 2016 to December 2020 in Xiangya Hospital were collected. A total of 203 patients were included and patients were divided into non PHG group and PHG group. The occurrence of postoperative delirium, cardiac complications, spinal cord complication, cerebral complications, acute kidney injury (AKI), hepatic dysfunction, hypoxemia, and in-hospital mortality were compared between two groups. Data from MIMIC-IV database were further applied to validate the relationship between PHG and clinical outcomes. The prediction model for PHG was then constructed using Extreme Gradient Boosting (XGBoost) analysis. The predictive value of selected features was further validated using patient data from MIMIC-IV database. Finally, the 28-days survival rate of patient with PHG was analyzed using data from MIMIC-IV database. Results: There were 86 patients developed PHG. The incidences of postoperative AKI, hepatic dysfunction, and in-hospital mortality were significant higher in PHG group. The ventilation time after surgery was significant longer in PHG group. Data from MIMIC-IV database validated these results. Neutrophil, platelet, lactic acid, weight, and lymphocyte were selected as features for prediction model. The values of AUC in training and testing set were 0.8697 and 0.8286 respectively. Then, five features were applied to construct another prediction model using data from MIMIC-IV database and the value of AUC in the new model was 0.8185. Finally, 28-days survival rate of patients with PHG was significantly lower and PHG was an independent risk factor for 28-days mortality after TAAD surgery. Conclusion: PHG was significantly associated with the occurrence of AKI, hepatic dysfunction, increased ventilation time, and in-hospital mortality after TAAD surgery. The feature combination of neutrophil, platelet, lactic acid, weight, and lymphocyte could effectively predict PHG. The 28-days survival rate of patients with PHG was significantly lower. Moreover, PHG was an independent risk factor for 28-days mortality after TAAD surgery.

Identification of immune-related hub genes and analysis of infiltrated immune cells of idiopathic pulmonary artery hypertension.

Objectives: Idiopathic pulmonary artery hypertension (IPAH) is a rare but life-threaten disease. However, the mechanism underlying IPAH is unclear. In this study, underlying mechanism, infiltration of immune cells, and immune-related hub genes of IPAH were analyzed via bioinformatics. Methods: GSE15197, GSE48149, GSE113439, and GSE117261 were merged as lung dataset. Weighted gene correlation network analysis (WGCNA) was used to construct the co-expression gene networks of IPAH. Gene Ontology and pathway enrichment analysis were performed using DAVID, gene set enrichment analysis (GSEA), and gene set variation analysis (GSVA). Infiltration of immune cells in lung samples was analyzed using CIBERSORT. GSE22356 and GSE33463 were merged as peripheral blood mononuclear cells (PBMCs) dataset. Immune-related differentially expressed genes (IRDEGs) of lung and PBMCs dataset were analyzed. Based on the intersection between two sets of IRDEGs, hub genes were screened using machine learning algorithms and validated by RT-qPCR. Finally, competing endogenous RNA (ceRNA) networks of hub genes were constructed. Results: The gray module was the most relevant module and genes in the module enriched in terms like inflammatory and immune responses. The results of GSEA and GSVA indicated that increasement in cytosolic calcium ion, and metabolism dysregulation play important roles in IPAH. The proportions of T cells CD4 memory resting and macrophage M1 were significantly greater in IPAH group, while the proportions of monocytes and neutrophils were significantly lower in IPAH group. IRDEGs of two datasets were analyzed and the intersection between two set of IRDEGs were identified as candidate hub genes. Predictive models for IPAH were constructed using data from PBMCs dataset with candidate hub genes as potential features via LASSO regression and XGBoost algorithm, respectively. CXCL10 and VIPR1 were identified as hub genes and ceRNA networks of CXCL10 was constructed. Conclusion: Inflammatory response, increasement in cytosolic calcium ion, and metabolism dysregulation play important roles in IPAH. T cells CD4 memory resting and macrophage M1 were significantly infiltrated in lung samples from patients with IPAH. IRDEGs of lung dataset and PBMCs dataset were analyzed, and CXCL10 and VIPR1 were identified as hub genes.

The predictive values of monocyte-lymphocyte ratio in postoperative acute kidney injury and prognosis of patients with Stanford type A aortic dissection.

Objectives: Postoperative acute kidney injury (pAKI) is a serious complication of Stanford type A aortic dissection (TAAD) surgery, which is significantly associated with the inflammatory response. This study aimed to explore the relationship between blood count-derived inflammatory markers (BCDIMs) and pAKI and to construct a predictive model for pAKI. Methods: Patients who underwent TAAD surgery were obtained from our center and the Medical Information Mart for Intensive Care (MIMIC)-IV database. The differences in preoperative BCDIMs and clinical outcomes of patients with and without pAKI were analyzed. Logistic regression was used to construct predictive models based on preoperative BCDIMs or white cell counts (WCCs). The performance of the BCDIMs and WCCs models was evaluated and compared using the receiver operating characteristic (ROC) curve, area under the ROC curve (AUC), Hosmer-Lemeshow test, calibration plot, net reclassification index (NRI), integrated discrimination improvement index (IDI), and decision curve analysis (DCA). The Kaplan-Meier curves were applied to compare the survival rate between different groups. Results: The overall incidence of pAKI in patients who underwent TAAD surgery from our center was 48.63% (124/255). The presence of pAKI was associated with longer ventilation time, higher incidence of cerebral complications and postoperative hepatic dysfunction, and higher in-hospital mortality. The results of the logistic regression indicated that the monocyte-lymphocyte ratio (MLR) was an independent risk factor for pAKI. The BCDIMs model had good discriminating ability, predictive ability, and clinical utility. In addition, the performance of the BCDIMs model was significantly better than that of the WCCs model. Analysis of data from the MIMIC-IV database validated that MLR was an independent risk factor for pAKI and had predictive value for pAKI. Finally, data from the MIMIC-IV database demonstrated that patients with a high MLR had a significantly poor 28-day survival rate when compared to patients with a low MLR. Conclusion: Our study suggested that the MLR is an independent risk factor for pAKI. A predictive model based on BCDIMs had good discriminating ability, predictive ability, and clinical utility. Moreover, the performance of the BCDIMs model was significantly better than that of the WCCs model. Finally, a high MLR was significantly associated with poor short-term survival of patients who underwent TAAD surgery.

Unique DUOX2+ACE2+ small cholangiocytes are pathogenic targets for primary biliary cholangitis.

Cholangiocytes play a crucial role in bile formation. Cholangiocyte injury causes cholestasis, including primary biliary cholangitis (PBC). However, the etiology of PBC remains unclear despite being characterized as an autoimmune disease. Using single-cell RNA sequencing (scRNA-seq), fluorescence-activated-cell-sorting, multiplex immunofluorescence (IF) and RNAscope analyses, we identified unique DUOX2+ACE2+ small cholangiocytes in human and mouse livers. Their selective decrease in PBC patients was associated with the severity of disease. Moreover, proteomics, scRNA-seq, and qPCR analyses indicated that polymeric immunoglobulin receptor (pIgR) was highly expressed in DUOX2+ACE2+ cholangiocytes. Serum anti-pIgR autoantibody levels were significantly increased in PBC patients, regardless of positive and negative AMA-M2. Spatial transcriptomics and multiplex IF revealed that CD27+ memory B and plasma cells accumulated in the hepatic portal tracts of PBC patients. Collectively, DUOX2+ACE2+ small cholangiocytes are pathogenic targets in PBC, and preservation of DUOX2+ACE2+ cholangiocytes and targeting anti-pIgR autoantibodies may be valuable strategies for therapeutic interventions in PBC.

Quantitative proteomic study of human lung squamous carcinoma and normal bronchial epithelial acquired by laser capture microdissection.

OBJECTIVE: To investigate the differential protein profile of human lung squamous carcinoma (HLSC) and normal bronchial epithelium (NBE) and provide preliminary results for further study to explore the carcinogenic mechanism of HLSC. METHODS: Laser capture microdissection (LCM) was used to purify the target cells from 10 pairs of HLSC tissues and their matched NHBE, respectively. A stable-isotope labeled strategy using iTRAQ, followed by 2D-LC/Q-STAR mass spectrometry, was performed to separate and identify the differential expression proteins. RESULTS: A total of 96 differential expression proteins in the LCM-purified HLSC and NBE were identified. Compared with NBE, 49 proteins were upregulated and 47 proteins were downregulated in HLSC. Furthermore, the expression levels of the differential proteins including HSPB1, CKB, SCCA1, S100A8, as well as S100A9 were confirmed by western blot and tissue microarray and were consistent with the results of quantitative proteomics. CONCLUSION: The different expression proteins in HLSC will provide scientific foundation for further study to explore the carcinogenic mechanism of HLSC.

[Preliminary study of the prohibitin protein and paclitaxel resistance in ovarian cancer].

OBJECTIVE: To determine the effect of RNA interference with transferred pshRNA/PHB on the biological characteristics of paclitaxel-resistant ovarian cancer cell lines. METHODS: Western blot and real time-PCR were used to assay the expression of PHB protein and mRNA in SKOV3/Taxol-25 and SKOV3 cell lines. The SKOV3/Taxol-25 cell lines were transiently transfected by 3 target-specific small hairpin RNA (shRNA) interference fragments with fluorescent protein named the pshRNA427/PHB1, pshRNA248/PHB2, and pshRNA136/PHB3. The empty plasmid transfection via vehicle Lipofectamine2000 served as a negative control. The expression levels of PHB protein and mRNA were detected by Western blot and real time-PCR after the transfection for 48 h. The silence effect of PHB1 and PHB3 groups was obvious. PHB1, PHB3, and the negative control groups were used for the following experiments. MTT and flow cytometry assay were used to test the cell proliferation, IC50 of paclitaxel, and cell apoptosis in the 3 groups. RESULTS: The expression levels of PHB protein and mRNA (2(-ΔΔCt)) were significantly higher in SKOV3/Taxol-25 cell line than those in SKOV3 cell line (P<0.05). The expression levels of PHB protein and mRNA were significantly lower in the PHB1 and PHB3 groups than those in the negative control group (P<0.05). The cell proliferations in the PHB1 and PHB3 groups were obviously slower than those in the negative control group after transfection for 48 h and 72 h (P<0.05). The IC50 of paclitaxel in the PHB1 and PHB3 groups significantly decreased after transfection for 72 h compared with the negative control group(P<0.05). The cell apoptotic rate in the PHB1 and PHB3 groups significantly increased after transfection for 48 h compared with the negative control group (P<0.05). CONCLUSION: The shRNA/PHB can effectively suppress the expression of PHB gene in paclitaxel-resistant ovarian cancer cell lines. The cell proliferation in paclitaxel-resistant cell lines with removed PHB gene is significantly reduced. The apoptotic rate and the paclitaxel sensitivity of resistant cell lines with removed PHB gene are significantly increased. PHB gene is related to paclitaxel-resistance and interfering PHB gene expression may reduce paclitaxel resistance in ovarian cancer.

Identification of biomarkers and analysis of infiltrated immune cells in stable and ruptured abdominal aortic aneurysms.

Objectives: The mortality rate of abdominal aortic aneurysm (AAA) is extremely high in the older population. This study aimed to identify potential biomarkers of AAA and aortic rupture and analyze infiltration of immune cells in stable and ruptured AAA samples. Methods: Raw data of GSE47472, GSE57691, and GSE98278 were downloaded. After data processing, the co-expression gene networks were constructed. Gene Ontology and pathway enrichment analysis of AAA- and aortic rupture-related gene modules were conducted using the Database for Annotation, Visualization, and Integrated Discovery. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were used for further enrichment analysis. The CIBERSORT tool was used to analyze the relative abundance of immune cells in samples. Differentially expressed immune-related genes were analyzed between different samples. Predictive models were constructed via extreme gradient boosting, and hub genes were identified according to feature importance. Results: Blue and yellow modules were significantly related to AAA, and genes in these modules were associated with the aortic wall and immune response, respectively. In terms of aortic rupture, the most relevant module was significantly enriched in the inflammatory response. The results of GSEA and GSVA suggested that immune cells and the inflammatory response were involved in the development of AAA and aortic rupture. There were significant differences in the infiltration of immune cells and expression levels of immune-related genes among different samples. NFKB1 might be an important transcription factor mediating the inflammatory response of AAA and aortic rupture. After the construction of a predictive model, CD19, SELL, and CCR7 were selected as hub genes for AAA whereas OAS3, IFIT1, and IFI44L were identified as hub genes for aortic rupture. Conclusion: Weakening of the aortic wall and the immune response both contributed to the development of AAA, and the inflammatory response was closely associated with aortic rupture. The infiltration of immune cells was significantly different between different samples. NFKB1 might be an important transcription factor in AAA and aortic rupture. CD19, SELL, and CCR7 had potential diagnostic value for AAA. OAS3, IFIT1, and IFI44L might be predictive factors for aortic rupture.

Analysis of infiltrated immune cells in left atriums from patients with atrial fibrillation and identification of circRNA biomarkers for postoperative atrial fibrillation.

Background: Atrial fibrillation (AF) increases the risk of stroke and heart failure. Postoperative AF (POAF) increases the risk of mortality after cardiac surgery. This study aims to explore mechanisms underlying AF, analyze infiltration of immune cells in left atrium (LA) from patients with AF, and identify potential circular RNA (circRNA) biomarkers for POAF. Methods: Raw data of GSE797689, GSE115574, and GSE97455 were downloaded and processed. AF-related gene co-expression network was constructed using weighted gene correlation network analysis and enrichment analysis of genes in relevant module was conducted. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) were applied to investigate pathways significantly enriched in AF group. Infiltration of immune cells was analyzed using single-sample GSEA. Differentially expressed genes (DEGs) between patients with or without AF were identified and competing endogenous RNA (ceRNA) networks of DEGs were constructed. To screen biomarkers for POAF, differentially expressed circRNAs (DEcircRNAs) between patients with or without POAF were identified. Intersection between DEcircRNAs and circRNAs in ceRNA networks of DEGs were extracted and circRNAs in the intersection were further screened using support vector machine, random forest, and neural network to identify biomarkers for POAF. Results: Three modules were found to be relevant with AF and enrichment analysis indicated that genes in these modules were enriched in synthesis of extracellular matrix and inflammatory response. The results of GSEA and GSVA suggested that inflammatory response-related pathways were significantly enriched in AF group. Immune cells like macrophages, mast cells, and neutrophils were significantly infiltrated in LA tissues from patients with AF. The expression levels of immune genes such as CHGB, HLA-DRA, LYZ, IGKV1-17 and TYROBP were significantly upregulated in patients with AF, which were correlated with infiltration of immune cells. ceRNA networks of DEGs were constructed and has_circ_0006314 and hsa_circ_0055387 were found to have potential predictive values for POAF. Conclusion: Synthesis of extracellular matrix and inflammatory response were main processes involved in development and progression of AF. Infiltration of immune cells was significantly different between patients with or without AF. Has_circ_0006314 and hsa_circ_0055387 were found to have potential predictive values for POAF.

Enhancing the stability of ¹8O-labeled peptides through removal of immobilized trypsin by ZipTips.

Trypsin-catalyzed ¹8O labeling is increasingly used in shotgun proteomics for relative peptide/protein quantitation. However, precise quantitative measurements are often complicated by the instability of ¹8O-labeled peptides caused mainly by oxygen back-exchange. Although a number of attempts have been made to reduce or prevent oxygen back-exchange, there is still room for improvement. Here we demonstrate that the removal of immobilized trypsin by filtration using ZipTips can efficiently minimize oxygen back-exchange and enhance the stability of ¹8O-labeled peptides under various pH conditions. The ¹8O-labeled peptides processed by the approach were successfully separated by immobilized pH gradient-isoelectric focusing (IPG-IEF), and no marked decrease in the extent of labeling was observed. The results also demonstrated that there was no correlation between the extent of ¹8O labeling and molecular weight or isoelectric point (pI). The approach presented here is especially applicable to microscale samples. Its ability to generate stably ¹8O-labeled samples without back-exchange should expand the application scope of the ¹8O-labeling technique.

Insulin Receptor Substrate p53 Ameliorates High-Glucose-Induced Activation of NF-κB and Impaired Mobility of HUVECs.

Diabetes-related macrovascular and microvascular complications lead to poor prognosis. Insulin receptor substrate p53 (IRSp53) is known to act as a substrate for the insulin receptor tyrosine kinase, but its role in endothelial dysfunction remains unclear. Human umbilical vein endothelial cells (HUVECs) treated with D-glucose at different concentrations and a streptozocin-induced rat diabetes mellitus (DM) model were used to investigate the effects of hyperglycemia on the expression levels of IRSp53 and galectin-3 (gal-3) and the inflammatory state and mobility of HUVECs. Thereafter, IRSp53-overexpressing HUVECs and IRSp53-knockdown HUVECs were established using IRSp53-overexpressing lentivirus or IRSp53-siRNA to explore the role of IRSp53 in the HUVEC inflammatory state and HUVEC mobility. D-glucose at high concentration (HG) and hyperglycemia were found to induce downregulation of IRSp53 and upregulation of gal-3 in vitro and in vivo. Treatment with HG resulted in activation of NF-κB in HUVECs and impaired HUVEC mobility. Insulin restored HG-induced changes in the expression levels of IRSp53 and gal-3 in HUVECs and protected the cells from NF-κB activation and impaired mobility. Overexpression of IRSp53 inhibited the activation of NF-κB in HUVECs and strengthened HUVEC migration. Knockdown of IRSp53 facilitated the activation of NF-κB in HUVECs and decreased HUVEC migration. However, neither overexpression nor knockdown of IRSp53 altered the effects of insulin on HG-induced detrimental changes in HUVECs. HG and hyperglycemia resulted in downregulation of IRSp53 in vitro and in vivo. IRSp53 is concluded to inhibit the activation of NF-κB in HUVECs and to strengthen HUVEC migration.

Galectin­3 facilitates the proliferation and migration of nasopharyngeal carcinoma cells via activation of the ERK1/2 and Akt signaling pathways, and is positively correlated with the inflammatory state of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an epithelial carcinoma originating from the nasopharyngeal mucosal tissue and is highly prevalent in southeast Asia. Galectin­3 (gal­3) serves crucial roles in many cancers but its role in NPC remains to be elucidated. The aim of the present study was to investigate the role of gal­3 in NPC. Immunohistochemistry and ELISA were used to determine the expression level of gal­3 in patients with NPC or chronic rhinitis (CR). Gal­3 short hairpin (sh)RNA was established to knockdown gal­3 in 5­8F and 6­10B cells, allowing for the evaluation of the roles of gal­3 in proliferation, migration and apoptosis in NPC cell lines. Immunohistochemistry staining of IL­6 and IL­8 was applied to access the inflammatory state of tumor tissues, and the correlation between the inflammatory state and gal­3 was analyzed. The results demonstrated that gal­3 was upregulated in patients with NPC compared with patients with CR. Knockdown of gal­3 inhibited proliferation and migration in 5­8F and 6­10B cells, as well as promoted apoptosis in these cells. The expression levels of MMP­9 and IL­8 were also decreased in 5­8F and 6­10B cells after transfection with gal­3 shRNA. A positive correlation was identified between the expression level of gal­3 and the inflammatory state of NPC. The phosphorylation levels of ERK1/2 and Akt were downregulated after knockdown of gal­3 in 5­8F and 6­10B cells. In conclusion, the expression level of gal­3 was upregulated in patients with NPC and was positively correlated with the inflammatory state of NPC. The results suggested that gal­3 promoted the proliferation and migration of 5­8F and 6­10B cells, while inhibiting the apoptosis of these cells. Moreover, activation of ERK1/2 and Akt may be the underlying mechanism of the effects of gal­3 on NPC.

Identification of the amyloid ß-protein precursor and cystatin C as novel epidermal growth factor receptor regulated secretory proteins in nasopharyngeal carcinoma by proteomics.

The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid ß-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy.

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5-8F with low RKIP expression and non-metastatic 6-10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5-8F cells to radiation-induced cell death, G(2)-M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6-10B cells protected cells from radiation-induced cell death, G(2)-M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf-1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC.

[Molecular mechanism of SH2B1 in regulating JAK2/IRS2 during obesity development].

OBJECTIVE: In order to investigate the effect of SH2B1 on leptin signal transduction JAK2/IRS2 and its biological function. METHODS: Vitro kinase assay and Western blot were used to analyse tyrosine phosphorylatin of key molecule JAK2 and insulin receptor substrate-2 (IRS2). ELISA was used to measure the plasma leptin levels in mice. The postnatal growth of mice was monitored over 27 weeks. RESULTS: SH2B1 dramatically enhanced the leptin-stimulated tyrosine phosphorylation of JAK2 and IRS2 in HEK293 cells stably expressing LRb (HEK239(LRb)). Leptin-stimulated activation of hypothalamic JAK2 and phosphorylation of hyphothalamic IRS2 were significantly impaired in SH2B1(-/-) mice. The deletion of SH2B1 led to leptin resistance,and fasting and randomly fed plasma leptin levels were respectively 3.2 times and 5.1 times higher in SH2B1(-/-) males than wild-type littermates at 15 weeks of age. SH2B1(-/-) males gained body weight rapidly and exceeded wild-type littermates from 5th week. SH2B1(-/-) (at 21 weeks) was approximately twice heavier than wild-type littermates. CONCLUSION: SH2B1 is an endogenous enhancer of leptin sensitivity and required for maintaining normal bodyweight in mice via leptin JAK2/IRS2 pathway.

Screening for methylation-silenced genes in acute myeloid leukemia HL-60 cell line by a quantitative proteomic approach.

OBJECTIVE: To screen for new methylation association genes in HL-60 to reveal the pathogenesis of leukemia, and provide important theoretical and scientific basis for the prevention and cure of leukemia. METHODS: Two-dimensional fluorescence difference gel electrophoresis (F-2D-DIGE) was performed to separate the total proteins from acute myelogenous leukemia (AML) cell line HL-60 cells with or without 5-aza-2-deoxycytidine (5-aza-2-dC) treatment. Imaging software Decyder 6.5 and PDQuest were used to detect the differential expression protein spots, and matrix-assisted laser desorption/ionizaion time-of-flight mas spectrometer (MALDI-TOF MS) was adopted to identify the differential expression proteins. RESULTS: F-2D-DIGE maps of 5-aza-2-dC-untreated HL-60 and-treated HL-60 cells were established. A total of 53 differential protein spots were detected, and 35 differential proteins were successfully identified. Of the identified proteins, 32 proteins were up-regulated, and 3 proteins were down-regulated in HL-60 cells after 5-aza-2-dC treatment. CONCLUSION: Thirty-five differential proteins may be associated with methylation in HL-60 cell line, which provides the important clues for epigenetic study of leukemia.

Identification of microRNAs enriched in exosomes in human pericardial fluid of patients with atrial fibrillation based on bioinformatic analysis.

BACKGROUND: Atrial fibrillation (AF) is related to structural and electrical atria remodeling. Atrial fibrosis development and progression is characteristic of structural remodeling and is taken as the AF perpetuation substrate. Increasing evidence has confirmed that microRNAs (miRNAs) are associated with AF, including cardiac fibrosis. METHODS: Pericardial fluid (PF) samples were collected from nine adult patients who had congenital heart disease with persistent AF or sinus rhythm (SR) undergoing surgery. Abnormally expressed miRNAs were acquired, and P<0.05 and fold change >2 were taken as the thresholds of differentially expressed miRNAs (DE-miRNAs). The predicted target genes were obtained by miRTarBase. The Database for Annotation, Visualization and Integrated Discovery was used to annotate functions and analyze pathway abundance for latent targets of DE-miRNAs. STRING database was applied to construct a protein-protein interplay (PPI) network, and Cytoscape software was used to visualize the miRNA-hub gene-Kyoto Encyclopedia of Genes and Genomes (KEGG) network. DE-miRNA expressions were evaluated by quantitative polymerase chain reaction (qPCR). RESULTS: Fifty-five exosomal DE-miRNAs were found between the AF and SR samples; these included 24 miRNAs that were upregulated and 31 that were downregulated. For the top 3 downregulated miRNAs (miR-382-3p, miR-3126-5p, and miR-450a-2-3p) 283 predicted target genes were identified, and were implicated in cardiac fibrosis-related pathways, including the hypoxia-inducible factor-1 (HIF1), mitogen-activated protein kinase (MAPK), and adrenergic and insulin pathways. The top 10 hub genes in the PPI network, including mitogen-activated protein kinase 1 (MAPK1) and AKT serine/threonine kinase 1 (AKT1), were identified as hub genes. By establishing the miRNA-hub gene-KEGG network, we observed that these hub genes, which were regulated by miR-382-3p, miR-3126-5p, and miR-450a-2-3p, were involved in many KEGG pathways associated with cardiac fibrosis, such as the AKT1/glycogen synthase kinsase-3ß (GSK-3ß) and transforming growth factor-ß (TGF-ß)/MAPK1 pathways. CONCLUSIONS: The findings of the present study suggest that miR-382-3p, miR-450a-2-3p, and miR-3126-5p contained in exosomes in human PF are pivotal in the progression of AF. The results of qPCR showed that miR-382-3p was consistent with our sequencing data, which indicates its potential value as a therapeutic target for AF.