Expression of mir-106b in esophageal squamous cell carcinoma / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the expression of mir-106b in esophageal squamous cell carcinoma (ESCC) tissue and analyze its correlation with the clinicopathologic features of ESCC.</p><p><b>METHODS</b>A total of 200 fresh surgical specimens of ESCC and adjacent tissues collected between 2001 and 2007 were examined for expressions of mir-106b using real-time PCR (RT-PCR). Northern blot analysis for mir-106b was performed in 4 pairs of samples to confirm the RT-PCR results. The relationship between mir-106b expression and clinicopathological features and prognosis of the patients were analyzed.</p><p><b>RESULTS</b>Mir-106b was expressed at significantly higher levels in ESCC tissues than in the paired adjacent tissues. Overexpression of mir-106b was associated with lymph node metastasis, stage of TNM classification and smoking (P<0.05). The survival rate of patients with low mir-106b expression was higher than that of patients with high mir-106b expression (60 vs 37 months, P=0.024). Cox regression analysis indicated that the expression of mir-106b, lymph node metastasis and smoking were independent prognostic factors for ESCC (P<0.05).</p><p><b>CONCLUSION</b>Mir-106b is overexpressed in ESCC tumors, and its overexpression is strongly associated with lymph node metastasis and a poor prognosis. Mir-106b expression is an independent prognostic factor for ESCC and can serve as a biomarker for diagnosis and prognostic evaluation of ESCC.</p>

miR-378 suppresses HBV-related hepatocellular carcinoma tumor growth by directly targeting the insulin-like growth factor 1 receptor / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the impact and mechanism of microRNA (miR)-378 on hepatocellular carcinoma (HCC) cell growth.</p><p><b>METHODS</b>The human hepatoma cell line HepG2 and its derivative HepG2.2.15 (stably expressing hepatitis B virus (HBV)) were transduced with lentiviruses expressing miR-378 or non-expressing controls (nc-Lv). Effects on cell proliferation were assessed by the MTT assay and on colony-formation efficiency by clonogenic assay. Targets of miR-378 were predicted by bioinformatic analysis and validated by luciferase reporter assay in the human embryonic kidney cell line HEK293. Real-time polymerase chain reaction and western blotting were used to monitor expression of the endogenous targets in miR-378- overexpressing HepG2 and HepG2.2.15 cells.</p><p><b>RESULTS</b>The HepG2 and HepG2.2.15 cells transduced with lentivirus expressing miR-378 showed significantly lower cell proliferation and colony formation than the control cells transduced with nc-Lv (P less than 0.01 and P less than 0.05, respectively). The insulin-like growth factor 1 receptor (IGF1R) was predicted as a potential target of miR-378, and luciferase reporter activity of IGF1R was significantly decreased in the HEK293 cells co-transfected with miR-378 (by 41.8% vs. the control cells, P less than 0.01). Moreover, the miRNA-378-mediated effect was narrowed down to the 3'-untranslated region (UTR) of IGF1R. The miRNA-378-mediated reduction of IGF1R specifically involved its protein expression (P less than 0.01) and not its mRNA expression (P more than 0.05).</p><p><b>CONCLUSION</b>miR-378 may suppress growth characteristics of HBV-related HCC by directly targeting the IGF1R 3'-UTR and inhibiting its protein expression.</p>

γδ T lymphocyte function and the polymorphism of T cell receptor V δ chain in lungs of asthmatic patients / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the function of gamma delta T lymphocytes and the polymorphism of T cell receptor V delta chain in the lungs of asthmatic patients and explore the role of gamma delta T cells in airway inflammation.</p><p><b>METHODS</b>Bronchoalveolar lavage fluid BALF was obtained from 7 asthmatic patients and 7 healthy control individuals. The percentage of gamma delta T cell in BALF was measured by flow cytometry. The gamma delta T cell in BALF was purified by magnetic labeled beads. Proliferous activity was examined by MTT assay. Cytokines secreted by gamma delta T cells in medium was assessed by enzyme-linked immunosorbent assay. Polymorphism of T cell receptor V delta chain was detected by RT-PCR and gene scan analysis.</p><p><b>RESULTS</b>The proportion of gamma delta T cell in the BALF of asthmatic patients [(6.39+/-0.71)%] was significantly higher than that in control subjects [(2.62+/-0.37)%] (P<0.01). The proportion of macrophage in the BALF of asthmatic patients [(81+/-4)] was significantly lower than that in control subjects [(86+/-2)] (P<0.05). The proliferation rate of asthmatic patients [(284.2+/-43.6)%] was significantly higher than that of control subjects [(217.5+/-59.5)%] (P<0.05). Interleukin-4 secreted by gamma delta T cells of asthmatic patients [(18.9+/-3.1) pg/ml)] significantly increased when compared with the control subjects [(14.1+/-3.0) pg/ml] (P<0.05). The polymorphism of T cell receptor V delta chain was not significantly different between these two groups.</p><p><b>CONCLUSIONS</b>The increase of gamma delta T cells in the lung of asthmatic patients further exacerbates Th1/Th2 disturbance and airway inflammation. Antigen recognition by gamma delta T cells is non-specific.</p>

Hairpin ribozyme targeted vascular endothelial growth factor (VEGF) inhibited the expression of VEGF and the growth of xenografted tumors / 肿瘤

Objective: Hepatocarcinoma cell line was transfected with anti-vascular endothelial growth factor (VEGF) hairpin ribozyme gene to observe the effect of hairpin ribozyme on VEGF expression and the growth of the xenografted tumors. Methods: The artificial anti-VEGF hairpin ribozyme gene was transfected into hepatocarcinoma SMMC-7721 cells via lipofectin mediation. The blank vector and the cell controls were prepared simultaneously. Then, positive clones were screened by genticin (G418). The transcription of ribozyme was confirmed by RT-PCR. The effects of the ribozyme on VEGF expression of SMMC-7721 cells were detected by semi-quantitative RT-PCR and immunohistochemical method. Cells in each group were inoculated into nude mice. The tumor volume and weight were recorded. The change in microvessel density and expression of VEGF was determined by immunohistochemistry. Results: Ribozyme gene was successfully transferred into tumor cells. The proliferation rate of ribozyme-transfected SMMC-7721 cells was significantly slower (P < 0.01). The expression of VEGF significantly decreased in ribozyme-transfected SMMC-7721 cells. After rebozyme transfection, the tumor formation rate significantly decreased and the growth speed of xenografted hepatocarcinoma markedly slowed down. The microvessel density and angiogenesis of the xenografted hepatocarcinoma were obviously reduced. Conclusion: Anti-VEGF hairpin ribozyme gene significantly inhibited the VEGF expression of hepatocarcinoma in vitro and in vivo by inhibiting angiogenesis of tumor cells. This study provided an experimental evidence for anti-angiogenesis gene therapy for hepatocarcinoma.

Regulation of tyrosylprotein sulfotransferases activity by sulfotyrosine / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases (TPST) 1 and TPST2.</p><p><b>METHODS</b>Constructs of TPST 1 and TPST2 were amplified by polymerase chain reaction (PCR), then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA (shRNA) constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney (HEK) 293T cells were transfected with these plasmids. One day later, cells were split: one part was labeled with 35S-cysteine and methionine or 35S-Na2SO3 overnight, the second part was used for 125I labeled binding experiment, and the third part was retained for binding and flow cytometry.</p><p><b>RESULTS</b>Tyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaCIO3. shRNAs reduced the sulfation of C3a receptor and C5a receptor, and partially blocked the binding of these two receptors to their respective ligands.</p><p><b>CONCLUSIONS</b>The activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3a receptor and C5a receptor to their respective ligands.</p>

Low dose pirfenidone suppresses transforming growth factor beta-1 and tissue inhibitor of metalloproteinase-1, and protects rats from lung fibrosis induced by bleomycina / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the optimal dosage of pirfenidone for the treatment of pulmonary fibrosis induced by bleomycin in Wistar rats, and the alteration of expressions of transforming growth factor beta-1 (TGF-beta 1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-13 (MMP-13) in lung tissue.</p><p><b>METHODS</b>Male Wistar rats were endotracheally instilled with bleomycin or normal saline. Pirfenidone (25-800 mg x kg(-1) x d(-1)), dexamethasone (3 mg/kg), or 1% carboxymethylcellulose sodium were given daily by feed 2 days before instillation of bleomycin. Groups T7 and T14 were fed pirfenidone 50 mg x kg(-1) x d(-1) at 7 days or 14 days after bleomycin instillation. Lungs were harvested at 28 days after bleomycin instillation. Patholological changes in lung tissues were evaluated with HE staining. Lung collagen was stained by sirius red and measured by content of hydroxyproline. Expression of proteins of TGF-beta 1, TIMP-1, and MMP-13 were detected by Western blotting.</p><p><b>RESULTS</b>At doses of 25, 50, and 100 mg x kg(-1) x d(-1), pirfenidone had significant anti-fibrotic effects for bleomycin-induced rat pulmonary fibrosis, and these effects were most significantly attenuated at the dosage of 50 mg x kg(-1) x d(-1) (HE: P < 0.01, P < 0.01, and P = 0.064; sirius red: P < 0.05, P < 0.01, and P < 0.05; hydroxyproline: P = 0.595, P < 0.01, and P = 0.976). Pirfenidone at a dosage of 50 mg x kg(-1) x d(-1) inhibited protein expression of TGF-beta1 and TIMP-1 in lung tissue in the early phase (0.79 and 0.75 times of control group), but had no effect on expression of MMP-13.</p><p><b>CONCLUSION</b>Low dose pirfenidone, especially at dosage of 50 mg x kg(-1) x d(-1), has significant anti-fibrotic effects on bleomycin-induced rat pulmonary fibrosis. Pirfenidone partially inhibits the enhancement of the expression of TGF-beta 1 and TIMP-1 in lung tissue.</p>

CXC chemokine receptor 3 modulates bleomycin-induced pulmonary injury via involving inflammatory process / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the role of CXC chemokine receptor 3 (CXCR3) in bleomycin-induced lung injury by using CXCR3 gene deficient mice.</p><p><b>METHODS</b>Sex-, age-, and weight-matched C57BL/6 CXCR3 gene knockout mice and C57BL/6 wide type mice were challenged by injection of bleomycin via trachea. Lung tissue was stained with HE method. Airway resistance was measured. Bronchoalveolar lavage (BAL) was performed using phosphate buffered saline twice, cell number and differentials were counted by Diff-Quick staining. Interleukin (IL)4, IL-5, IL-12p40, and interfon-y in BAL fluid and lung homogenate were measured by enzyme-linked immunosorbent assay. Unpaired t test was explored to compare the difference between two groups.</p><p><b>RESULTS</b>On day 7 after bleomycin injection via trachea, CXCR3 knockout mice were protected from bleomycin-induced lung injury as evidenced by fewer accumulation of inflammatory cells in the airway and lung interstitium compared with their wild type littermates (P < 0.05). Airway resistance was also lower in CXCR3 knockout mice compared with wild type mice (P < 0.01). Significantly lower level of inflammatory cytokines release, including the altered production of IL-4 and IL-5 both in BAL fluid and lung tissue was seen in CXCR3 knockout mice than in wild type mice (both P<0.05).</p><p><b>CONCLUSION</b>CXCR3 signaling promotes inflammatory cells recruiting and initiates inflammatory cytokines cascade following endotracheal bleomycin administration, indicating that CXCR3 might be a therapeutic target for pulmonary injury.</p>

Inhibition of human lung fibroblast proliferation and the mitogen activated protein kinase pathway by dexamethasone / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the effects of dexamethasone on human lung fibroblast cell proliferation, cell cycles, and cell mitogen-activated protein kinases (MAPKs) passway.</p><p><b>METHODS</b>Dexamethasone was used at various concentration in culture medium. Cell number was counted using a hemacytometer. Whole cell propidium iodide staining and flow cytometric analysis were performed to determine cellular DNA content. MAPK proteins and activation were tested by Western blot analysis with antibodies to c-Jun N-terminal kinase (JNK), phospho-JNK, extracellular signal-regulated kinase (ERK), phospho-ERK, p38 and phospho-p38.</p><p><b>RESULTS</b>1x10(-7) mol/L and 1x10(-6) mol/L dexamethasone suppressed the proliferation of lung fibroblast cells by 34% and 72%, respectively, than that of control. This suppression was dose-dependant. Dexamethasone suppressed cell cycle with accumulation of cells in G1/G0 stage. It increased from 81.9% to 90.1% compared with that of control. We did not find any apoptosis induced by dexamethasone for lung fibroblast cells. Using Western blot analysis, we found that dexamethasone resulted in decreased activity of ERK, but had no effects on JNK and p38.</p><p><b>CONCLUSIONS</b>Dexamethasone may suppresses the proliferation of lung fibroblast cells, which is partly resulted from the facts that it can inhibit ERK activation in MAPK-signaling pathway but has little effect on JNK and p38 pathway. Dexamethasone may not induce lung fibroblast cell apoptosis directly.</p>