MiR-506 suppresses papillary thyroid carcinoma cell proliferation and metastasis via targeting IL17RD.

OBJECTIVE: The aim of this study was to determine the role of microRNA-506-3p (miR-506) in papillary thyroid carcinoma (PTC), and to further explore the underlying mechanism. PATIENTS AND METHODS: The expression level of miR-506 in clinical cases was detected by Real Time-fluorescence quantitative Polymerase Chain Reaction (RT-qPCR). Meanwhile, RT-qPCR was performed to determine miR-506 expression in different PTC cell lines. Bioinformatics software was used to predict the possible target genes of miR-506. Dual-Luciferase reporter gene assay together with Western blot (WB) assay were used to verify the prediction results. Finally, cellular functions such as proliferation and metastasis capacities were detected in vitro. RESULTS: RT-qPCR was used to measure the expression level of miR-506 in 80 paired PTC cases. The results showed that the expression level of miR-506 in PTC tissues was significantly decreased. In vitro, miR-506 expression was also markedly suppressed in four PTC cell lines. TPC-1 cells expressed the lowest level of miR-506. Subsequently, the target gene of miR-506 was predicted by TargetScan, miRBase and miRanda. The prediction results indicated that IL17RD was an alternative target gene of miR-506. Furthermore, miR-506 was found to remarkably inhibit the Luciferase activity of wild-type IL17RD. However, it had no effect on mutant-type. Besides, the protein expression level of IL17RD was significantly reduced in miR-506-overexpressing TPC-1 cells. More importantly, the restored expression of IL17RD could alleviate the blocking effects of miR-506 on cell proliferation, migration and invasion. CONCLUSIONS: In this study, we found that miR-506 could inhibit the proliferation and metastasis of PTC cells. Meanwhile, IL17RD might be a downstream target of the biological process. Our findings provided a new therapeutic direction for the treatment of PTC.

[Antifibrotic effect of pirfenidone on orbital fibroblasts in patients with thyroid-associated ophthalmopathy and its mechanisms].

Objective: To investigate the effects of pirfenidone on orbital fibroblasts (OFs) from patients with thyroid-associated ophthalmopathy (TAO) and its underlying mechanisms. Methods: OFs from patients with TAO were isolated and cultured in DMEM. Cells were divided into four groups and treated with 0, 250, 500 and 1 000 µg/ml pirfenidone for 24, 48 or 72 hours, respectively. Cell proliferation was detected by tetramethyl azo salt (MTT) assay, and cell viability was determined by trypan blue. Transforming growth factor (TGF) ß1 mRNA level was determined by real-time fluorescence quantitative PCR (RT-qPCR). Type Ⅰ and type Ⅲ collagen secreted from cultured cells were measured by enzyme-linked immuno sorbent assay (ELISA). Results: (1) The primary cultured OFs had typical fibroblast spindle-like morphology. (2) MTT assay showed that pirfenidone treatment significantly inhibited the proliferation of OFs in a dose-dependent manner (P<0.05) with the proliferation rates of pirfenidone treated groups of -15.31%, -24.92%, -48.53% from 250, 500, 1 000 µg/ml after 72 h, respectively, in which the inhibition effect of 1 000 µg/ml pirfenidone was significantly different from the other two treated groups (P<0.05). There were no significant differences in the inhibitory effect of the same concentration group among different time points at 24 h, 48 h and 72 h (P>0.05). Trypan blue showed that the survival rate of OFs in different concentrations of pirfenidone from 0,250, 500, 1 000 µg/ml at 72 h were 78.37%, 79.21%, 78.24% and 76.28%, respectively. There were no significant differences between each drug treated and the control group (P>0.05). (3) RT-qPCR results showed that the mRNA expression levels of TGFß1 at 250, 500, 1 000 µg/ml pirfenidone treated groups at 72 h were 0.760±0.010, 0.440±0.006, and 0.290±0.002, respectively. Compared with the control group (0.950±0.014), the differences were statistically significant (all P<0.05). Moreover, TGFß1 mRNA expression level in 1 000 µg/ml pirfenidone treated group was significantly lower than those in the other two treated groups (all P<0.05). The secretion of type Ⅰ collagen (0.633±0.006, 0.527±0.003 and 0.402±0.008) and type Ⅲ collagen (0.511±0.003, 0.439±0.007 and 0.223±0.006) in 250, 500 and 1 000 µg/ml pirfenidone treated groups at 72 h were significantly lower than those in the control group (0.794±0.005, 0.527±0.007, all P<0.05). Type Ⅰ and type Ⅲ collagen secretion in 1 000 µg/ml pirfenidone treated group were significantly lower than those in the other two groups (P<0.05). Conclusions: Pirfenidone inhibits the cell proliferation, TGFß1 expression and collagen secretion of OFs, which may contribute to the anti-fibrotic effect of pirfenidone.

[Analysis of significant microRNA associated with chronic thromboembolic pulmonary hypertension].

Objective: To find key microRNA (miR) associated with chronic thromboembolic pulmonary hypertension (CTEPH). Methods: Affymetrix miR microarray data and GSE56914 data downloaded from GEO database (http: //www.ncbi.nlm.nih.gov/geo/) were obtained and integrated. The microarray data were obtained from peripheral blood samples of CTEPH patients and the matched control. Differentially expressed miRs were screened. Target genes of these miRs were searched. Then, functional enrichment analyses for these miRs were performed. After that, disease network including miRs, target genes and pathways was constructed. Results: Five important miRs including hsa-miR-885-5p, hsa-miR-501-5p, hsa-miR-615-3p, hsa-miR-610, and hsa-miR-346 were identified. Furthermore, hsa-miR-885-5p and hsa-miR-501-5p were significantly enriched in cell cycle pathway. Hsa-miR-615-3p was involved in cytokine-cytokine receptor interaction, axon guidance, focal adhesion and cell cycle pathway. Hsa-miR-610 was significantly enriched in focal adhesion pathway, and hsa-miR-346 was involved in cytokine-cytokine receptor interaction, axon guidance, and focal adhesion pathway. Conclusions: Hsa-miR-885-5p, hsa-miR-501-5p, hsa-miR-615-3p, hsa-miR-610 and hsa-miR-346 are important miRs for the development of CTEPH.

Up-regulation of microRNA-367 promotes liver steatosis through repressing TBL1 in obese mice.

OBJECTIVE: Increasing evidence has demonstrated that microRNAs (miRNAs) play a critical role in the progression of metabolic disorders, including obesity-induced non-alcoholic fatty liver disease (NAFLD). In the present study, the expression and function of miR-367 were investigated. MATERIALS AND METHODS: C57BL/6 male mice aged 8 weeks were fed with a normal diet (ND) or high-fat-diet (HFD). The expression levels of miR-367 were analyzed in livers from two groups of mice by quantitative real-time PCR. Adenovirus containing miR-367 or negative control (NC) were constructed and administrated into C57BL/6 mice by tail vain injection. Potential targets of miR-367 were screened by miRWalk software and luciferase reporter assays. Mutagenesis analysis and Western blots were used to further determine the target of miR-367 in obese mice. RESULTS: We found that the expression of hepatic miR-367 was up-regulated in obese mice. In vitro and in vivo studies further demonstrated that overexpression of miR-367 mimics promoted triglyceride accumulation in cells and lean mice. At the molecular level, transducin beta-like 1 (TBL1), a coactivator of nuclear receptor peroxisome proliferator-activated receptor (PPAR) α, was identified as a direct target of miR-367. As a result, miR-367 overexpression resulted in an inhibition of fatty acid oxidation, leading to hepatosteatosis. CONCLUSIONS: Our data suggest miR-367/TBL1 regulatory pathway might have an important role for in the development of NAFLD.

Feasibility of radical gastrectomy for elderly patients with gastric cancer.

BACKGROUND: The aim of this study was to evaluate the short-term safety and long-term benefits of radical gastrectomy for gastric cancer in elderly patients. METHODS: A total of 729 patients undergoing gastrectomy for adenocarcinoma between December 2008 and December 2011 were enrolled in this retrospective study. Patients were divided into three groups: young group (<65 years), young-old group (65-79 years) and old-old group (≥80 years). RESULTS: Lower albumin levels, higher ASA grades, comorbidities, tumors located in the upper third of the stomach and advanced TNM stages were more common in the young-old and old-old age groups. Overall complications increased significantly with advancing age (15.4%, 24.9%, 48.7%, respectively); respiratory complications largely contributed to the dramatic increase. Severe complications were similar between the young and young-old groups (3.9%, 3.7%), but were significantly increased in the old-old group (12.8%). In multivariate analysis, old-old age, multiple comorbidities and no epidural use were strong predictors for overall complications. Both overall survival and disease-specific survival (DSS) rates declined with advancing age. Multivariate analysis showed that old-old age and TNM stage ≥ II were major independent risk factors for the DSS rate. When adjusted for confounding factors, young-old age was not a risk factor. The median survival time for the old-old patients with stage III tumors was 12.9 months. CONCLUSIONS: It is relatively safe and beneficial for young-old patients to undergo radical gastrectomy as the young patients. However, the decision to perform radical gastrectomy for old-old patients with TNM stage III tumors should be made carefully.

Relationship between a lipoprotein lipase gene polymorphism in placental tissue and insulin resistance in patients with gestational diabetes mellitus.

The aim of this study was to investigate the relationship between a lipoprotein lipase (LPL) gene polymorphism in placental tissue and insulin resistance (IR) in patients with gestational diabetes mellitus. Using polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) analysis, the LPL HindIII RFLP was examined in the placental tissue of 110 patients with gestational diabetes mellitus (observation group) and 110 women with normal gestation (control group). The relationships between fasting plasma glucose (FPG), postprandial plasma glucose (PPG), fasting insulin (FINS), cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), body mass index (BMI), and IR indices and the LPL polymorphism in the two study groups and their offspring were determined. The frequency of the H+ allele was significantly higher in the observation group than in the controls (P < 0.05). There were statistically significant differences in the observation group between the FPG, PPG, LDL, TC, TG, HDL, BMI, FINS, and IR indices of the H+H+ group and those of the non H+H+ type patients (P < 0.05). Correlation analysis showed that the LPL gene polymorphism was positively related to IR. There were statistically significant differences between HDL, BMI, and IR indices between the two study groups (P < 0.05). In conclusion, the LPL gene polymorphism was determined to be the main factor related to IR in women with gestational diabetes, and was also found to be related to the IR of their offspring.

Banding or Burdizzo castration and carprofen administration on peripheral leukocyte inflammatory cytokine transcripts.

The objective was to investigate if Banding or Burdizzo castration of bulls would alter the gene expression profile of a range of peripheral leukocyte inflammatory cytokines (IL-1, IL-6, IL-8, IL-10, interferon-γ and tumor necrosis factor-α) and to determine if the administration of carprofen (C) before castration would affect the expression of these genes. Thirty Holstein-Friesian bulls (5.5 months; Mean 191±(SEM) 3.7 kg) were blocked by weight and randomly assigned to one of five treatments: (1) untreated control (CON); (2) Banding castration at 0 min (BAND); (3) BAND following an i.v. injection of 1.4 mg/kg BW of carprofen (C) at -20 min (BAND+C); (4) Burdizzo castration at 0 min (BURD); or (5) BURD following 1.4 mg/kg BW of carprofen at -20 min (BURD+C). Blood samples were collected at 1 h before castration and 6, 24 and 48 h post-castration for routine hematology and quantitative real-time PCR analysis of cytokine gene expression analysis. Generally, there were no differences (P>0.05) among treatment groups in hematological variables following castration. Cortisol concentrations were unchanged throughout the experimental period in CON bulls. BURD animals had greater cortisol concentrations than BAND and CON animals at 6 h post treatment. Transitory effects were observed only in the expression of IL-6 and TNF-α. The relative expression of IL-6 was greater in the BURD than in the BAND treatment (P<0.05) at 24 h post-castration and was greater in the BURD+C group than in the BURD group (P<0.05) at 48 h. The relative expression of TNF-α was greater in BAND than in the BURD group (P<0.05) at 48 h. In conclusion, these findings indicate that Banding or Burdizzo castration did not have any major effect on peripheral leukocyte inflammatory cytokine gene expression; Banding castration caused a greater pro-inflammatory cytokine gene expression reaction than Burdizzo castration and carprofen administration can affect IL-6 gene expression levels in BURD castrated animals.

Effects of banding or burdizzo castration of bulls on neutrophil phagocytosis and respiratory burst, CD62-L expression, and serum interleukin-8 concentration.

The objective was to investigate measures of neutrophil function in response to banding or burdizzo castration of bulls. Thirty-two Holstein-Friesian bulls (14 mo old, 505 +/- 7.8 kg of BW) were assigned to 1 of 4 treatment groups: 1) sham-handled control (CON); 2) banding castration alone (BAND); 3) burdizzo castration alone (BURD); or 4) cortisol infusion (CORT) as a further control group. For each group on d -14, 8 animals (2 animals/treatment) were tied up in tie stalls (day of treatment = d 0). At -2, 2, 6, 12, 24, 48, 72, and 144 h relative to treatment time, blood samples were collected for analyses of neutrophil phagocytosis and respiratory burst, neutrophil CD62-L expression, and serum IL-8 concentration. Leukocyte counts, phagocytosis activity, and CD62-L expression were similar (P > 0.05) among the 4 treatment groups. The BURD castrates had greater burst activity compared with BAND castrates (P = 0.048) and CON (P = 0.01) at 72 h posttreatment. The BURD castrates had a greater percentage of granulocyte positive leukocytes (Gr%; P < 0.01) at 2 h posttreatment compared with CON and CORT bulls. The BURD castrates had greater (P < 0.05) Gr% compared with BAND, CON, and CORT animals at 24, 48, and 72 h posttreatment. The BURD and BAND castrates had greater Gr% (P < 0.05) compared with CORT bulls at 144 h posttreatment. In general, BAND, BURD, and CORT did not affect serum IL-8 concentration. Banding castration, BURD, and CORT did not induce leukocytosis, whereas BURD induced a modest neutrophilia. Neutrophil functioning in terms of phagocytosis and respiratory burst and serum IL-8 concentration were not compromised by BAND, BURD, and CORT. These findings indicate nonsurgical castration is unlikely to induce a severe acute systemic inflammatory response in terms of neutrophil function.

Effect of carprofen administration during banding or burdizzo castration of bulls on plasma cortisol, in vitro interferon-gamma production, acute-phase proteins, feed intake, and growth.

The objective of this study was to determine the effect of carprofen (C) administration before banding or burdizzo castration of bulls on cortisol, in vitro interferon-gamma (IFN-gamma) production, acute-phase proteins, feed intake, and growth. Fifty Holstein Friesian bulls (5.5 mo old; 191 +/- 3.7 kg) were blocked by weight and assigned randomly to 1 of 5 treatments (n = 10/treatment): 1) untreated control (2) banding castration at 0 min (Band); 3) Band following an i.v. injection of 1.4 mg/kg of BW of C at -20 min (Band+C); 4) Burdizzo castration at 0 min (Burd); or 5) Burd following 1.4 mg/kg of BW of C at -20 min (Burd+C). Castration acutely increased plasma cortisol concentrations compared with control; no significant differences occurred in peak and interval to peak cortisol responses between Band and Band+C or Burd and Burd+C groups. The administration of C in Band+C reduced (P < 0.05) the cortisol concentration between 6 and 12 h postcastration compared with Band animals. Overall, the integrated cortisol response was greater (P < 0.05) in the castrates than in control, whereas C treatments tended to reduce this response compared with Band (P = 0.08) and Burd (P = 0.07), respectively. Plasma fibrinogen was elevated in Band animals on d 14 and in Burd animals on d 3 and 14. Carprofen administration reduced Band- and Burd-induced fibrinogen production on d 14 and 3, respectively. Plasma haptoglobin was elevated in Band animals on d 3 and 35 compared with control, and C administration was effective in reducing the haptoglobin elevation on d 35 in Band+C compared with Band. There were no differences among treatments in in vitro IFN-gamma production induced by concanavalin A and phytohemagglutinin on d 1 and 2. Overall from d -1 to 16, there were no DMI differences among treatments. From d -1 to 35, there were no ADG differences among treatments. In conclusion, banding and burdizzo castration increased plasma cortisol with no change in in vitro IFN-gamma production. Carprofen (1.4 mg/kg of BW) tended to reduce the integrated cortisol response, and it reduced cortisol secretion in banded animals between 6 and 12 h postcastration. There was an increased acute-phase protein production following castration; this response was effectively moderated by the administration of C before castration.