TGF-ß1-regulated miR-3691-3p targets E2F3 and PRDM1 to inhibit prostate cancer progression.

Transforming growth factor-ß1 (TGF-ß1) acts as a tumor promoter in advanced prostate cancer (PCa). We speculated that microRNAs (miRNAs) that are inhibited by TGF-ß1 might exert anti-tumor effects. To assess this, we identified several miRNAs downregulated by TGF-ß1 in PCa cell lines and selected miR-3691-3p for detailed analysis as a candidate anti-oncogene miRNA. miR-3691-3p was expressed at significantly lower levels in human PCa tissue compared with paired benign prostatic hyperplasia tissue, and its expression level correlated inversely with aggressive clinical pathological features. Overexpression of miR-3691-3p in PCa cell lines inhibited proliferation, migration, and invasion, and promoted apoptosis. The miR-3691-3p target genes E2F transcription factor 3 (E2F3) and PR domain containing 1, with ZNF domain (PRDM1) were upregulated in miR-3691-3p-overexpressing PCa cells, and silencing of E2F3 or PRDM1 suppressed PCa cell proliferation, migration, and invasion. Treatment of mice bearing PCa xenografts with a miR-3691-3p agomir inhibited tumor growth and promoted tumor cell apoptosis. Consistent with the negative regulation of E2F3 and PRDM1 by miR-3691-3p, both proteins were overexpressed in clinical PCa specimens compared with noncancerous prostate tissue. Our results indicate that TGF-ß1-regulated miR-3691-3p acts as an anti-oncogene in PCa by downregulating E2F3 and PRDM1. These results provide novel insights into the mechanisms by which TGF-ß1 contributes to the progression of PCa.

Role of the autophagy-related marker LC3 expression in hepatocellular carcinoma: a meta-analysis.

BACKGROUND: Microtubule-associated protein 1 light chain 3 (LC3), an autophagic gene, has been reported as a vital marker for many diseases and cancers. However, the role of LC3 in hepatocellular carcinoma (HCC) was not still investigated. Therefore, we conducted a meta-analysis to examine the association of LC3 with its clinicopathological and prognostic in HCC. METHODS: We consulted the PubMed, Cochrane Library, Web of Science, EMBASE, China National Knowledge Infrastructure and Wan Fang databases for published studies on LC3 in HCC. Newcastle-Ottawa scale was used to screen the quality of the literature. The statistical analysis was calculated by STATA 14.2. RESULTS: Of the 1329 titles identified, 10 articles involving 949 patients in HCC were included in this meta-analysis. The results of our study show that increased LC3 expression is related to size of tumor, but not to gender, age, number of tumor, liver cirrhosis, HBsAg, TNM stage, alpha fetoprotein, vascular invasion and histological grade. Positive LC3 expression was associated with overall survival by pooled hazard ratio. CONCLUSIONS: This meta-analysis indicated that positive LC3 expression was related to size of tumor, and could predict prognosis in human hepatocellular carcinoma.

Diagnostic value of decoy receptor 3 combined with procalcitonin and soluble urokinase-type plasminogen activator receptor for sepsis.

The levels of decoy receptor 3 (DcR3), soluble urokinase type plasminogen activator receptor (suPAR) and procalcitonin (PCT) are significantly increased in sepsis. We investigated the diagnostic value of DcR3 combined with suPAR and PCT in sepsis. Patients with sepsis, non-infectious systemic inflammatory response comprehensive syndrome (SIRS) and healthy controls were recruited according to the diagnostic standard. We measured DcR3, suPAR, PCT, interleukin-6 (IL-6) and C-reactive protein (CRP), and the diagnostic value was evaluated by receiver operating characteristics (ROC) curves. In our analysis, serum DcR3, suPAR and PCT levels of the sepsis group were significantly higher than those of the SIRS and control groups. However, IL-6, CRP and WBC showed no significant difference between the SIRS group and the sepsis group. The serum DcR3 level was positively correlated with the serum suPAR level (r = 0.37, p = 0.0022) and PCT level (r = 0.37, p = 0.0021). Using DcR3, suPAR and PCT to distinguish SIRS from sepsis, the area under the curve (AUC) values were 0.892, 0.778 and 0.692. When DcR3, suPAR and PCT combined were used for diagnosis of sepsis, the AUC was 0.933, at a cut-off point of 0.342. This combination improved the sensitivity and specificity of diagnosis of sepsis, suggesting that use of the combination of three indexes enhanced the efficiency of sepsis diagnosis.

GTSF1 gene may serve as a novel potential diagnostic biomarker for liver cancer.

The gametocyte-specific factor 1 (GTSF1) gene participates in DNA methylation and retrotransposon activation in germ cells, particularly during cell proliferation. The present study aimed to assess the level of GTSF1 gene expression in liver cancer tumor tissues, and its role in human hepatoma cell lines in vitro and in a nude mouse model in vivo. GTSF1 gene expression was detected in liver cancer tumor tissues, compared with in healthy controls, via reverse transcription quantitative polymerase chain reaction. An adeno-associated virus vector was used to study tumor stem cell proliferation in vivo. A plasmid expressing GTSF1 was constructed and transfected into various human hepatoma cell lines, in order to analyze the cellular proliferation and apoptosis of liver cancer cells using small interfering (si)RNAs in vitro. In the present study, GTSF1 gene expression was detected in 18/24 (75.0%) liver cancer tumor tissues from patients with hepatocellular carcinoma (HCC), and elevated GTSF1 expression was identified in the tissue of one of 32 healthy control samples (3.13%; P<0.05). Notably, the GTSF1 gene was expressed at a higher frequency in AFP-positive HCC samples (14/16, 87.50%) compared with in AFP-negative HCC samples (4/8, 50.0%; P=0.129). In addition, there was no statistical significance between GTSF1 expression in non-HBV-infected (71.42%) and HBV-infected HCC specimens (76.47%), as determined by a χ2 test (P=0.921). It was demonstrated that GTSF1 significantly increased the tumorigenicity of Ad-shNC-transfected (GTSF1-positive) HepG2 cells in the nude mouse xenograft model, whereas the sizes and weights of the tumors in the GTSF1-negative group were dercreased in comparison with the GTSF1-positive group (P<0.05). Reduced levels of GTSF1 mRNA, along with fewer and smaller colonies, were identified in two groups of human liver cancer cells treated with with GTSF1-targeting siRNA, when compared with cells without GTSF1 mRNA interference (P<0.05). In summary, the present study elucidated the GTSF1 mRNA expression pattern in liver cancer, and investigated the potential role of GTSF1 in tumorigenesis. The data suggest an important role for the GTSF1 gene in the molecular etiology of hepatocarcinogenesis, and indicate a potential application of GTSF1 mRNA expression in liver cancer diagnosis and therapy.

Ghrelin protects small intestinal epithelium against sepsis-induced injury by enhancing the autophagy of intestinal epithelial cells.

BACKGROUND: Ghrelin is a hormone that protects against hypoxic injury of cardiac cells by inducing autophagy, but the role of autophagy in sepsis remains unclear. This study aimed to evaluate whether ghrelin could enhance autophagy in rats with intestinal sepsis. METHODS: The cecal ligation and perforation (CLP) method was used to induce sepsis in Sprague-Dawley rats. The rats were assigned to four groups: normal group, sham-operated group, sepsis group, and Ghrelin-treated group. Sera and small intestinal tissues were collected from all groups. The sepsis was evaluated by histological analysis, and autophagy of small intestinal epithelial cells was assessed by electron microscopy, immunofluorescence, and biochemical methods. RESULTS: The expression of autophagy-associated proteins such as LC3, Atg 7 and Beclin 1 increased by 8h post-CLP and declined to basal levels by 12h post-CLP. The expression of LC3, Atg 7 and Beclin 1 in Ghrelin-treated rats was higher than that in rats with sepsis. Furthermore, compared to rats with sepsis, Ghrelin-treated rats showed significantly reduced intestinal mucosa injury at 20h post-CLP. CONCLUSION: Autophagy is induced in the early stages of sepsis. Ghrelin could enhance the autophagy of intestinal epithelial cells in rats with sepsis and protect the small intestinal epithelium against sepsis-induced injury.

Apoptotic Mechanisms of Peroxisome Proliferator-Activated Receptor-γ Activation in Acinar Cells During Acute Pancreatitis.

OBJECTIVE: The objective of this study was to determine the mechanism by which activation of peroxisome proliferator-activated receptor-γ promotes apoptosis of acinar cells in pancreatitis. METHODS: AR42j cells pretreated with the peroxisome proliferator-activated receptor-γ agonist pioglitazone were activated by cerulein as an in vitro model of acute pancreatitis. Inflammatory cytokines and amylase were detected by enzyme-linked immunosorbent assay. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was measured by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining. Activity of caspases was determined. Bax and Bcl-2 levels were assayed by Western blot. RESULTS: Cytokines, amylase, and cellular proliferation decreased in pioglitazone-pretreated cells. Pioglitazone increased the activity of caspases 3, 8, and 9 in cerulein-activated AR42j cells as well as in the pancreas of rats 3 hours after induction of severe acute pancreatitis. Acinar cell apoptosis was induced by reducing the mitochondrial membrane potential in the pioglitazone group. Pioglitazone increased expression of proapoptotic Bax proteins and decreased antiapoptotic Bcl-2 in cerulein-induced AR42j cells and decreased Bcl-2 levels in pancreatic tissue of severe acute pancreatitis rats 1 and 3 hours after induction. CONCLUSION: Pioglitazone may promote apoptosis of acinar cells through both intrinsic and extrinsic apoptotic pathways in acute pancreatitis.

Branched DNA-based Alu quantitative assay for cell-free plasma DNA levels in patients with sepsis or systemic inflammatory response syndrome.

Cell-free circulating DNA (cf-DNA) can be detected by various of laboratory techniques. We described a branched DNA-based Alu assay for measuring cf-DNA in septic patients. Compared to healthy controls and systemic inflammatory response syndrome (SIRS) patients, serum cf-DNA levels were significantly higher in septic patients (1426.54 ± 863.79 vs 692.02 ± 703.06 and 69.66 ± 24.66 ng/mL). The areas under the receiver operating characteristic curve of cf-DNA for normal vs sepsis and SIRS vs sepsis were 0.955 (0.884-1.025), and 0.856 (0.749-0.929), respectively. There was a positive correlation between cf-DNA and interleukin 6 or procalcitonin or Acute Physiology and Chronic Health Evaluation II. The cf-DNA concentration was higher in intensive care unit nonsurviving patients compared to surviving patients (2183.33 ± 615.26 vs 972.46 ± 648.36 ng/mL; P < .05). Branched DNA-based Alu assays are feasible and useful to quantify serum cf-DNA levels. Increased cf-DNA levels in septic patients might complement C-reactive protein and procalcitonin in a multiple marker format. Cell-free circulating DNA might be a new marker in discrimination of sepsis and SIRS.

Hepatocyte nuclear factor 4α induces a tendency of differentiation and activation of rat hepatic stellate cells.

AIM: To investigate the effect of hepatocyte nuclear factor 4α (HNF4α) on the differentiation and transformation of hepatic stellate cells (HSCs). METHODS: By constructing the recombinant adenovirus vector expressing HNF4α and HNF4α shRNA vector, and manipulating HNF4α expression in HSC-T6 cells, we explored the influence of HNF4α and its induction capacity in the differentiation of rat HSCs into hepatocytes. RESULTS: With increased expression of HNF4α mediated by AdHNF4α, the relative expression of Nanog was downregulated in HSC-T6 cells (98.33 ± 12.33 vs 41.33 ± 5.67, P < 0.001). Consequently, the expression of G-P-6 and PEPCK was upregulated (G-P-6: 14.34 ± 3.33 vs 42.53 ± 5.87, P < 0.01; PEPCK: 10.10 ± 4.67 vs 56.56 ± 5.25, P < 0.001), the expression of AFP and ALB was positive, and the expression of Nanog, Type I collagen, α-SMA, and TIMP-1 was significantly decreased. HNF4α also downregulated vimentin expression and enhanced E-cadherin expression. The ultrastructure of HNF4α-induced cells had more mitochondria and ribosomes compared with the parental cells. After silencing HNF4α expression, EPCK, E-cadherin, AFP, and ALB were downregulated and α-SMA and vimentin were upregulated. CONCLUSION: HNF4α can induce a tendency of differentiation of HSCs into hepatocyte-like cells. These findings may provide an effective way for the treatment of liver diseases.

Regulatory roles of the PI3K/Akt signaling pathway in rats with severe acute pancreatitis.

The phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) pathway plays a key role in inflammation. However, the regulatory roles of PI3K/Akt in severe acute pancreatitis (SAP) have not been elucidated. The aim of this study was to investigate the impact of wortmannin, a PI3K/Akt inhibitor, on SAP rats through exposure to sodium taurocholate (STC) after 3 h and 6 h. The SAP group was found to have a significant increase in pancreas Akt expression, along with the activation of serum amylase, TNF-α, IL-1ß, and IL-6, and pancreas histological aggravation. The administration of wortmannin in SAP rats reduced Akt expression, attenuated the level of serum amylase and inflammation factor, and alleviated the damage of pancreatic tissue. Furthermore, the administration of wortmannin led to an obvious reduction in NF-κB and p38MAPK expression in SAP rats. These findings showed that the PI3K/Akt inhibitor wortmannin decreases inflammatory cytokines in SAP rats and suggests its regulatory mechanisms may occur through the suppression on NF-κB and p38MAPK activity.

Effects of peroxisome proliferator-activated receptor-γ activation on apoptosis in rats with acute pancreatitis.

PURPOSE: To investigate the effects and mechanisms of peroxisome proliferator-activated receptor-γ (PPAR-γ) activation on the induction of apoptosis in rats with acute pancreatitis. METHODS: Severe acute pancreatitis (SAP) and mild acute pancreatitis (MAP) were induced and pre-treated with pioglitazone, which is a ligand of PPAR-γ. The expression of inflammatory factors (TNF-α and IL6) of the pancreas was detected by ELISA. The apoptosis in pancreas were detected by TUNEL assay and the activity of caspase 3 was determined. Phosphorylation of p65 in pancreas of SAP or MAP was determined by western-blot. RESULTS: Expression levels of PPAR-γ proteins were elevated in the pancreases of SAP or MAP rats pre-injected with pioglitazone intraperitoneally. Downregulation of the expression TNF-α and IL6 and relief of pathological changes in the pancreas suggested that pioglitazone had protective effects on acute panceatitis. In pioglitazone pre-treated groups, a TUNEL assay indicated a high level of apoptosis in SAP but little apoptosis in MAP, showing pioglitazone could promote taurocholate-induced apoptosis but inhibit ceruleininduced apoptosis in pancraeatic aniniar cells. Furthermore, caspase 3 activity was high in SAP but low in MAP, implying that the apoptotic mechanism in pancreatic acinar cells of AP rats was correlated with caspase 3 activity. Phosphorylation of p65 was reduced in SAP or MAP group pretreated with pioglitazone, indicating that pioglitazone reduced the inflammation reaction by inhibiting the activation of the NF-κB. CONCLUSIONS: These results indicated that activation of PPAR-γ induced apoptosis in pancreatic acinar cells of SAP rats but inhibited apoptosis in pancraeatic acinar cells of MAP rats, which demonstrated that PPAR-γ may be an efficiently therapeutic target in pancreatic inflammation.

Establishment of a leptospirosis model in guinea pigs using an epicutaneous inoculations route.

BACKGROUND: Leptospires are presumed to enter their host via small abrasions or breaches of the skin. The intraperitoneal route, although commonly used in guinea pig and hamster models of leptospirosis, does not reflect conditions encountered during natural infection. The aim of this study is to develop a novel leptospirosis guinea pig model through epicutaneous route and to elucidate the pathogenesis of leptospirosis in experimental guinea pigs by comparing the data from other studies using different infection routes. METHODS: The guinea pigs were inoculated with 5 × 108 Leptospira interrogans strain Lai onto either shaved-only or abraded skin. The guinea pigs were sacrificed at 2, 8, 24, 48, 72, 96 and 144 h post-infection (p.i.) followed by harvest of the lungs, liver, kidneys, spleen, and the skin around the inoculated sites for further examinations. Hematoxylin and eosin (HE) staining and electron microscopy were used to detect the pathologic changes. Real time PCR and immunohistochemistry staining were performed to detect dynamic distribution of leptospires in blood and tissues, respectively. RESULTS: In the guinea pigs with abraded skin inoculations, leptospires were detected in blood as early as 2 h post infection (p.i.) and then disseminated to the liver, lungs and kidneys of almost all animals within 96 h p.i.. Leptospires were also detected engulfed in the swelling vascular endothelial cells and were frequently aggregated around the capillaries in the dermis and subcutaneous tissue under the inoculated site. For the guinea pigs with abraded skin inoculations, hemorrhage at the dermis around the inoculated site was found before the appearance of internal organs hemorrhage, severe lesions such as hemorrhages in the lungs, nephritis, jaundice, haematuria were also observed, and two of seven guinea pigs died at 144 h p.i. while no lesions and leptospires were detected in the shaved-only guinea pigs using the same dose of strain Lai. CONCLUSION: Intact keratinocyte layer is a very efficient barrier against leptospires, and intact skin can prevent the infiltration of leptosipres to the host. Leptospires can penetrate abraded skin and quickly establish a systemic infection by crossing tissue barriers. We have successfully established a novel leptospirosis guinea pig model through epicutaneous inoculations route, which replicates a natural course of infection and appears to be an alternative way to investigate the pathogenesis of leptospirosis, especially in terms of early stage of host-pathogen interactions. This novel model may also be advantageous for studies of the mechanisms involved in cutaneous barriers and epidermal interactions with this organism.

Serum decoy receptor 3, a potential new biomarker for sepsis.

BACKGROUND: Sepsis, a common deadly systemic infection caused by a variety of pathogens, has some clinical symptoms similar to the systemic inflammatory response syndrome (SIRS), a whole-body non-infectious inflammatory reaction to severe insults, such as burn, trauma, hypotensive shock and so on. Treatment of sepsis depends mainly on anti-microbial, while remedy for SIRS might require steroids that could possibly enhance the spread of microbes. Unfortunately, it is very difficult to distinguish these two completely different serious conditions without blood culture, which takes days to grow and identify causative pathogens. We examined a biomarker, serum decoy receptor 3 (DcR3), was evaluated for its utility in the differential diagnosis between sepsis and SIRS. METHODS: Serum DcR3 level in 118 healthy controls, 24 sepsis patients and 43 SIRS patients, was quantitatively measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The serum DcR3 was significantly increased in sepsis patients compared with SIRS patients and healthy controls (6.11±2.58 ng/ml vs 2.62±1.46 ng/ml, and 0.91±0.56 ng/ml, respectively, p<0.001). The areas under the receiver operating characteristic curve of DcR3 for the normal vs. SIRS, normal vs. sepsis and SIRS vs. sepsis were 0.910 (0.870-0.950), 0.992 (0.984-1.000) and 0.896 (0.820-0.973), respectively. In addition, the DcR3 exhibited a positive correlation coefficient with APACHE II score, a most commonly used index for the severity of sepsis (r=0.556, p=0.005). CONCLUSION: The serum DcR3 has a potential to serve as a new biomarker for sepsis with its high specificity and sensitivity.

A cross-sectional survey of participation of asthmatic children in physical activity.

BACKGROUND: Physical exercise has been proven to be beneficial to children with asthma, but the traditional view in China is that asthmatic children should not take part in sports. This study was undertaken to investigate the current status of children with asthma taking part in exercise in China. METHODS: One hundred and twenty-three asthmatic children (7-14 years old) who had visited our asthma control center between February 2009 and June 2009 were enrolled in this cross-sectional study. Each child had a pulmonary function test and his/her health-related quality of life was assessed. The children also finished a questionnaire about their physical activity. As a control group, 109 nonasthmatic children from a primary school were surveyed about their level of activity. RESULTS: Asthmatic children took part in less exercise than their healthy peers, and 62.6% (77/123) of the children with asthma never reached the criteria of exercise prescription for patients with asthma advised by the American College of Sports Medicine. The asthmatic children were divided into two groups based on the level of activity; compared with the group with a higher physical activity level, more children in the group with lower activity believed that exercise could make asthma worse, more parents and teachers restricted the children's exercise, and fewer doctors approved them participating in exercise. All of the parameters of basic lung function were higher in the group with higher activity level. Moreover, the children with a higher exercise level had a higher score on all parts of the pediatric asthma quality-of-life questionnaire. About 78.5% (96/123) of children ever experienced coughing, chest distress, dyspnea, or gasping during exercise, but 49.6% (61/123) had these symptoms occasionally. CONCLUSIONS: Our study reveals that children with asthma do not have enough exercise in China. The concept that children, parents, teachers and doctors have about exercise for patients with asthma is urgent to be updated. We need to prescribe appropriate exercise for children with asthma.

Development of O-antigen gene cluster-specific PCRs for rapid typing six epidemic serogroups of Leptospira in China.

BACKGROUND: Leptospira is the causative agent of leptospirosis. The O-antigen is the distal part of the lipopolysaccharide, which is a key component of outer membrane of Gram-negative bacteria and confers serological specificity. The epidemiology and clinical characteristics of leptospirosis are relative to the serology based taxonomic unit. Identification of Leptospira strains by serotyping is laborious and has several drawbacks. RESULTS: In this study, the O-antigen gene clusters of four epidemic Leptospira serogroups (serogroup Canicola, Autumnalis, Grippotyphosa and Hebdomadis) in China were sequenced and all genes were predicted in silico. Adding published sequences of two serogroups, Icterohaemorrhagiae (strain Lai and Fiocruz L1-130) and Sejroe (strain JB197 and L550), we identified six O-antigen-specific genes for six epidemic serogroups in China. PCR assays using these genes were developed and tested on 75 reference strains and 40 clinical isolates. CONCLUSION: The results show that the PCR-based assays can be reliable and alternative means for rapid typing of these six serogroups of Leptospira.

Factors associated with insulin resistance and fasting plasma ghrelin levels in adolescents with obesity and family history of type 2 diabetes.

OBJECTIVE: To investigate the changes of insulin resistance and fasting plasma ghrelin levels in adolescents with obesity and family history of type 2 diabetes mellitus (FHD) and analyze its related factors. METHODS: The study included 159 adolescents aged 13-15 years. Anthropometric- measurements, including height, weight and waist. Blood samples were collected and fasting plasma glucose (FPG), serum lipids, true insulin (TI) and fasting plasma ghrelin were assayed. They were divided into four groups according to body mass index (BMI) and FHD: 40 non-obese adolescents without FHD in group A; 40 overweight and obese adolescents without FHD in group B; 40 non-obese adolescents with FHD in group C; 39 overweight and obese adolescents with FHD in group D. RESULTS: Group B, C and D had significantly higher levels of HOMA-IR and HOMA-beta index than group A, especially Group D (p<0.01). Group C and D had significantly higher levels of FPG and lower levels of ghrelin than group A (p<0.01). HOMA-IR showed positive correlation with BMI (r=0.445), waist (r=0.435), waist-to-height ratio (WHtR)(r=0.471) (p<0.01). Ghrelin showed negative correlation with FPG (r=-0.339), TI (r=-0.237) and HOMA-IR (r=-0.269) (p<0.01). In multiple linear regression analysis, WHtR (beta=4.925, p=0.000) and FHD (beta=0.492, p=0.000) were significant independent predictors for HOMA-IR. FHD (beta=-289.856, p=0.000) and FPG (beta=-228.203, p=0.001) were significant independent predictors for ghrelin. CONCLUSION: This study showed that the subjects with FHD and obesity who are predisposed to diabetes have insulin resistance in adolescent stage. The lower ghrelin levels in subjects with FHD may be the result of elevated FPG.