Detection of STAT2 in early stage of cervical premalignancy and in cervical cancer

OBJECTIVE@#To measure the expression pattern of STAT2 in cervical cancer initiation and progression in tissue sections from patients with cervicitis, dysplasia, and cervical cancer.@*METHODS@#Antibody against human STAT2 was confirmed by plasmids transient transfection and Western blot. Immunohistochemistry was used to detect STAT2 expression in the cervical biopsies by using the confirmed antibody against STAT2 as the primary antibody.@*RESULTS@#It was found that the overall rate of positive STAT2 expression in the cervicitis, dysplasia and cervical cancer groups were 38.5%, 69.4% and 76.9%, respectively. The STAT2 levels are significantly increased in premalignant dysplasia and cervical cancer, as compared to cervicitis (P< 0.05). Noticeably, STAT2 signals were mainly found in the cytoplasm, implying that STAT2 was not biologically active.@*CONCLUSIONS@#These findings reveal an association between cervical cancer progression and augmented STAT2 expression. In conclusion, STAT2 increase appears to be an early detectable cellular event in cervical cancer development.

Cross-talk between ERK and NF-kappaB signal transduction pathways in the hepatocytes expressing hepatitis C virus nonstructural protein 3 / 中南大学学报(医学版)

OBJECTIVE@#To explore the cross-talk between extracellular signal-regulated kinase (ERK) and nuclear factor (NF-kappaB) signal transduction pathways in the hepatocytes expressing hepatitis C virus nonstructural protein 3 (HCV NS3).@*METHODS@#A cell line QSG7701/NS3, which stably expressed HCV NS3 protein, was constructed by transfecting plasmid pcDNA3.1-NS3 into a human immortalized hepatocyte line QSG7701. Before and after QSG7701/NS3 cells were treated by MEK inhibitor PD98059, the phosphorylation level of ERK and the expression of cyclin D1 protein were detected by Western blot; the DNA binding activities of activator protein 1 (AP-1) and NF-kappaB were evaluated with electrophoretic mobility shift assay (EMSA). Cell cycles were measured by flow cytometry (FCM); the effects of PD98059 on the cell proliferation were determined by MTT assay.@*RESULTS@#HCV NS3 protein could up-regulate the phosphorylation of ERK and the expression level of cyclin D1 in QSG7701 cells. PD98059 could inhibit the cell proliferation mediated by HCV NS3 protein, down-regulate the activities of AP-1 and NF-kappaB, and suppress the expression of cyclin D1.@*CONCLUSION@#The inhibition of ERK can suppress the DNA binding activity of NF-kappaB and the cell proliferation mediated by HCV NS3 protein in a dose-dependent manner. There may be a cross-talk between ERK and NF-kappaB signal transduction pathways, which may exert synergistic action on the proliferation and malignant transformation of hepatocytes.

Mechanism of hepatocyte transformation by HCV NS3 using two-dimensional electrophoresis and mass spectrometry / 中南大学学报(医学版)

OBJECTIVE@#To investigate the proteome of hepatocyte transformation by hepatitis C virus (HCV) nonstructural protein 3 (NS3).@*METHODS@#Human hepatocyte line QSG7701 stably expressing HCV NS3 C-terminal deleted protein was constructed, which was named pRcHCNS3/QSG. Two-dimensional electrophoresis (2-DE) was used to separate the total protein of pRcHCNS3/QSG and pRcCMV transfected cells (pRcCMV/QSG) respectively. Differentially expressed protein spots were identified by mass spectrometry. Western blot confirmed the differentially expressed proteins.@*RESULTS@#2-DE profiles with high resolution and reproducibility were obtained. The average spots of pRcHCNS3/QSG and pRcCMV/QSG were (1183+/-77) and (1095+/-82) respectively, and (920+/-60) spots were matched. Twenty-one differentially expressed protein spots were chosen randomly and 15 were identified by mass spectrometry. Some proteins such as Ras, P38 and HD53 which were involved in signal transduction were increased in pRcHCNS3/QSG cells. Western blot also showed strong expression of phosphorylated P44/42 and P38 in pRcHCNS3/QSG cells. Other differentially expressed proteins were related to cell cycle regulation, immunoreaction, tumor invasion and metastasis, and liver metabolizability.@*CONCLUSION@#HCV NS3 might be involved in cell malignant transformation through affecting protein expression and signal transduction such as MAPK cascade. Further study on the signal transductions and their relationship would not only be helpful to explore the mechanism of HCV related HCC, but also provide a new idea for the molecular treatment of HCC.

AP-1 regulates TGF-beta1-induced secretion of Type I collagen in human lung fibroblasts / 中南大学学报(医学版)

OBJECTIVE@#To investigate the role of AP-1 in the secretion of Type I collagen in TGF-beta1-stimulated human lung fibroblasts.@*METHODS@#Human lung fibroblasts cell line (HLF-02) was cultured, and then stimulated with 10 microg/L TGF-beta1 at different time points. Curcumin was added into the culture medium to inhibit the AP-1 activity before incubating with TGF-beta1. AP-1 DNA binding activity was assayed by electrophoretic mobility shift assay (EMSA), and the expression of Type I collagen was detected by Western blot and RT-PCR.@*RESULTS@#TGF-beta1 could induce the transcription and secretion of Type I collagen in HLF-02 cells(P<0.05). TGF-beta1 could upregulate the AP-1 DNA binding activity ( P<0.05). Curcumin ( 5, 10, 15, and 20 micromol/L) could inhibit the AP-1 DNA binding activity in TGF-beta1-stimulated cells (the inhibition ratio was 17.1%, 17.6%, 24.2%, and 31.3%; P<0.05). Curcumin (5, 10, 15, and 20 micromol/L) could also inhibit the secretion of Type I collagen significantly (the inhibition ratio was 62.1%, 58.8%, 62.1%, and 59.6%; P<0.05).@*CONCLUSION@#AP-1 is responsible for the secretion of TGF-beta1-induced Type I collagen in human lung fibroblasts.

Effect of erigeron breviscapus on the expression of vascular endothelial growth factor in the periodontal tissues of rabbits during orthodontic tooth movement / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To detect the effect of erigeron breviscapus on the expression of vascular endothelial growth factor (VEGF) in the periodontal tissues during orthodontic tooth movement.</p><p><b>METHODS</b>45 rabbits were divided into 3 groups (groups A, B and C). Groups A and B included experimental group of 1, 3, 7 and 14 days respectively. The mandibular first molar of each experimental rabbit was observed. The rabbits of group A and group B received iontophoresis with erigeron breviscapus into the right (group A-R and group B-R) and with normal sodium into the left as the control (group A-L and group B-L). Additionally, the rabbits of group B were designed orthodontic appliance, by which 0.78 N mesial force was applied to pull the mandibular first molars. Group C, group of 0 day, was no iontophoresis and orthodontic appliance as the control. After killed on schedule, the amount of experimental tooth movement was measured and the expression of VEGF was examined by immunohistochemical method.</p><p><b>RESULTS</b>The amount of experimental tooth movement increased successively from 1 to 14 days. The differences among days 3, 7 and 14 were significant in the comparison between group B-R and group B -L (P < 0.01). The expression of VEGF in groups A-R and B-L enhanced apparently compared with that in groups C and A-L (P < 0.01), but that in group B-R was the most apparent (P < 0.01). The expression of VEGF reached the peak level on day 3 in groups A-R and B-R (P < 0.01), but it reached the peak level on day 7 in group B-L (P < 0.01).</p><p><b>CONCLUSION</b>Erigeron breviscapus by iontophoresis can accelerate orthodontic tooth movement, and can meanwhile up-regulate the expression of VEGF in periodontium in the earlier period of orthodontic tooth movement. Thus it can be presumed that one of its mechanisms for erigeron breviscapus to accelerate orthodontic tooth movement is erigeron breviscapus effects the metabolism and differentiation of osteoblast and osteoclast through up-regulating the expression of VEGF in periodontium.</p>

p38/ERK signal pathways regulating the expression of type I collagen and activity of MMP-2 in TGF-beta1-stimulated HLF-02 cells / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of TGF-beta(1)/MAPK signaling pathways in the expression of type I collagen and activity of MMP-2, 9 in human lung fibroblasts.</p><p><b>METHODS</b>Human lung fibroblasts cell line (HLF-02) was cultured and and then stimulated with 10 ng/ml TGF-beta(1) for different time; SB203580 or PD98059 was added into culture medium to block p38 or ERK kinase pathway before incubated with TGF-beta(1); the expression of type I collagen was detected by Western blotting and RT-PCR; zymogram analysis was used to analyze the activity of MMP-2 and MMP-9.</p><p><b>RESULTS</b>(1) In the process of stimulation by TGF-beta(1), the type I collagen mRNA level of 24 h, 48 h and 72 h group was: 1.33 +/- 0.07, 2.46 +/- 0.09 and 2.39 +/- 0.08 respectively; and the type I collagen protein level of 24 h, 48 h and 72 h group was: 114.89 +/- 8.95, 208.16 +/- 6.75 and 211.46 +/- 8.05 respectively; and the activity of MMP-2 of 24 h, 48 h and 72 h group was: 190.33 +/- 5.86, 214.33 +/- 8.39 and 212.67 +/- 11.59 respectively. (2) SB203580 significantly inhibited the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 51%, 24% and 20%); (3) PD98059 also significantly attenuated the TGF-beta(1)-induced expression of type I collagen mRNA, protein and MMP-2 activity (inhibition ratio: 42%, 13% and 16%).</p><p><b>CONCLUSION</b>TGF-beta(1) is capable of inducing the expression of type I collagen mRNA and protein and up-regulating MMP-2 activity in HLF-02 cells. p38 and ERK kinase signaling pathways play important role in regulation and control for this process.</p>

Expression and role of nuclear transcription factor Sp1 in macrophages stimulated by silicon dioxide / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To study the expression and localization of nuclear transcription factor Sp1 in macrophages after stimulated by silicon dioxide in vivo and in vitro.</p><p><b>METHODS</b>Forty Sprague Dawley rats were randomly divided into the control group and the silica exposure group, 20 in each group. The rat silicosis models were established by direct tracheal instillation of silica into rat lung (0.2 g/kg) only once while the control group was instilled with equal amount of saline. Animals were killed at 1st, 7th, 14th, 21st and 28th day after instillation. Dynamic changes of Sp1 protein expression and its cellular localization were detected by immunohistochemistry in pulmonary macrophages. In vitro, Sp1 mRNA and protein expression and their dynamic changes were monitored by RT-PCR and western blotting after stimulated by silicon dioxide in cultured RAW264.7 macrophages respectively. Cellular localization of Sp1 protein was characterized by immunocytochemistry.</p><p><b>RESULTS</b>Compared to the control group, the Sp1 protein expression was increased in pulmonary macrophages and reached the peak at the 14th day in the silica exposure group. In vitro, the Sp1 mRNA level began to rise at 30 minutes after the administration of silicon dioxide and reached the peak at 240 minutes and then decreased to the minimal level at 960 minutes. The Sp1 total protein and nuclear protein also exhibited the similar trend. The former reached the peak at 240 minutes and the latter at 480 minutes. The significant nuclear translocation of Sp1 protein was observed at 120 minutes after the administration of silicon dioxide and became most significant at 480 minutes.</p><p><b>CONCLUSION</b>Silicon dioxide can activate nuclear transcription factor Sp1 in macrophages in vivo and in vitro. Sp1 might play an important pathogenic role in the development of silicosis.</p>

Expression of Ang2 and Tie2 and their relation with the angiogenesis of hepatocellular carcinoma in rats / 中南大学学报(医学版)

OBJECTIVE@#To investigate the relationship between the expression of Ang2, Tie2 and the angiogenesis of hepatocellular carcinoma in rats.@*METHODS@#Thirty-eight healthy male rats were randomly divided into 3 groups: 5 rats in the control group; 25 rats in the experimental group were equally divided into 5-day, 10-day, 15-day, 20-day, and 25-day groups; the other 8 rats were used as the supplement of the experimental group. An allogenic transplanted rat model of CBRH-7919 hepatocellular carcinoma in situ was established by immunosuppression. The expressions of Ang2 and Tie2 were detected by immunohistochemical staining in cancerous tissues of different developmental stages and liver tissues of the control group. At the same time, microvessel density was determined by anti-CD31 immunohistochemical staining.@*RESULTS@#CBRH-7919 hepatocellular carcinoma models were successfully set up in 24 rats. The expression level of Ang2 and Tie2 in cancerous tissues was much higher than that of liver tissues of the control group (P <0.05). The overexpression of Ang2 was pristine and continuous in different developmental stages. The expressions of Ang2 and Tie2 positively correlated with microvessal density in hepatocellular carcinoma (P<0.05).@*CONCLUSION@#The up-regulation of Ang2 and Tie2 may play important roles in the angiogenesis of hepatocellular carcinoma. Ang2 may participate in the start of angiogenesis of hepatocellular carcinoma.

Transforming growth factor-beta1 induced phenotypic differentiation of human lung fibroblasts through mitogen activated protein kinase-dependent pathway / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the role of MAPK signal transduction in TGF-beta1 induced phenotypic differentiation of human lung fibroblasts.</p><p><b>METHOD</b>Human lung fibroblasts cell line (HLF-02) were cultured and then stimulated with 10 ng/ml TGF-beta1 for different time; SB203580 or PD98059 was added into culture medium to prevent p38 or Erk kinase pathway before incubating with TGF-beta1; the expression of alpha-smooth muscle actin (alpha-SMA) was detected by Western blotting and RT-PCR; Western blotting was used to assay phosphorylation of p38, Erk, and JNK kinase.</p><p><b>RESULTS</b>(1) In the process of stimulation by TGF-beta1, the alpha-SMA mRNA expression levels of 24, 48 and 72 h groups were 1.87 +/- 0.11, 2.49 +/- 0.10, 3.02 +/- 0.15 respectively; and the alpha-SMA protein expression levels of 24, 48 and 72 h groups were 3.20 +/- 0.14, 3.96 +/- 0.21, 4.57 +/- 0.13 respectively. (2) TGF-beta1 induced p38, Erk kinase phosphorylation but not JNK kinase. (3) The inhibitors SB203580 and PD98059 suppressed TGF-beta1-induced p38 kinase and Erk phosphorylation respectively. (4) SB203580 significantly attenuated TGF-beta1-induced alpha-SMA mRNA and protein expression (inhibition rate: 30% and 40%); PD98059 also significantly inhibited TGF-beta1-induced alpha-SMA mRNA and protein expression (inhibition rate: 10% and 20%).</p><p><b>CONCLUSION</b>TGF-beta1 is capable of inducing the phenotypic differentiation of HLF-02, which is regulated by p38 and Erk kinase signal pathway.</p>

Effects of silica on the expression of plasminogen activator inhibitor-1 and activator protein-1 in human alveolar epithelial cells type II / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To investigate the mechanism of silicosis by observing the effects of silica on the expression of plasminogen activator inhibitor-1 (PAI-1) and activator protein-1 (AP-1) in human alveolar epithelial cells type II (A549).</p><p><b>METHODS</b>A549 cell and SiO(2) (200 microg /ml) were co-cultured for 0, 4, 8, 16 and 24 h respectively. The reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting and SP immunocytochemistry were used for detections of the PAI-1 mRNA and protein expression. The nucleoprotein and total protein expression of AP-1 were investigated by Western blotting.</p><p><b>RESULTS</b>The expression levels of PAI-1 mRNA and protein were increased in a time-dependent manner(r(mRNA) = 0.911, r(protein) = 0.902, P < 0.05). The expressions of PAI-1 mRNA and protein in experimental groups were higher than that in control group (P < 0.05) and was the highest in 24 h group [(0.73 +/- 0.01) vs (0.36 +/- .03)]. The nucleoprotein expressions of c-jun/c-fos in experimental groups were also higher than in control group (P < 0.05), and the nucleoprotein expression level of c-jun was the highest in 4 h group [(1.54 +/- 0.02) vs (0.56 +/- 0.03)]; the nucleoprotein expression level of c-fos was the highest in 8 h group [(0.36 +/- 0.01) vs (0.15 +/- 0.01)]. Both c-jun and c-fos expression were decreased after 16 h, but the total protein expression of c-jun/c-fos had no difference in all experimental groups. The positive signal of PAI-1 was located in cytoplasm and nucleus.</p><p><b>CONCLUSION</b>SiO(2) could induce PAI-1 expression of A549 in a time-dependent manner, and AP-1 activation can be observed in early time.</p>

Apoptosis and caspase-3 in the model of rat silicosis / 中南大学学报(医学版)

OBJECTIVE@#To explore the changes of apoptosis of cells in the lung tissue of rats with silica instillation and to its significance in silicosis, and to clarify the role of caspase-3 in the apoptosis progress.@*METHODS@#Forty-eight rats were randomly divided into saline control groups and silica instillation groups, and the silicosis model was established in rats. Flow cytometry was used for detecting the rate of apoptosis at various stages. Immunohistochemistry for the expression of cleaved caspase-3.@*RESULTS@#The model of rat silicosis was established successfully. The apoptosis rate in the experimental group was significantly higher than that in the control group, and was increased with time. Caspase-3 was mainly expressed in alveolar epithelium cells, pulmonary macrophages and infiltrated inflammation cells. The expression of caspase-3 in the experimental group was stronger than that in the control group, but its expression intensity was not related to the cell apoptosis (r = 0.215, P > 0.05).@*CONCLUSION@#The apoptosis of the lung cells plays an important role during rat silicosis genesis. Caspase-3 plays an important role in regulating cell apoptosis during rat silicosis genesis.

Regulation of hepatitis C virus core protein on the activity of signal transducers and activators of transcription 3 / 中南大学学报(医学版)

OBJECTIVE@#To investigate the regulation of hepatitis C virus (HCV) core protein on the activity of signal transducers and activators of transcription 3 (stat3).@*METHODS@#A cell line expressing stable HCV core protein-QSG7701-core was constructed by transfecting the pcDNA3. 1-core (expressing HCV core protein) into the human immortalized hepatocyte line QSG7701. The phosphorylation and DNA binding activity of stat3 were detected by immunocytochemistry, Western blot, and electrophoretic mobility shift assay (EMSA).@*RESULTS@#The expression level of phosphorylated stat3 in QSG7701-core cells was significantly lower than that in QSG7701-pcDNA3. 1 cells and untransfected QSG7701 cells, but there were no significant differences in the expression levels of total stat3 among the 3 groups. The positive signal of phosphorylated stat3 in nucleus of QSG7701-core cells was obviously weaker than that in QSG7701-pcDNA3. 1 cells and untransfected QSG7701 cells. EMSA showed that DNA binding activity of stat3 in QSG7701-core cells significantly decreased.@*CONCLUSION@#The expressionof HCV core protein in human hepatocyte line may suppress the phosphorylation and DNA binding activity of stat3, which may be one of the causes for resistance against interferon.

Hepatocyte transformation and tumor development induced by hepatitis C virus NS3 N-terminal protein / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the effect of hepatitis C virus nonstructural protein 3 N-terminal protein (HCV NS3-5') on hepatocyte transformation and tumor development.</p><p><b>METHODS</b>QSG7701 cells were transfected with plasmid pRcHCNS3-5' (expressing HCV NS3 N-terminal protein) by lipofectamine and selected in G418. The expression of HCV NS3 gene and protein was determined by PCR and immunohistochemistry respectively. Biological effect of transfected cells was observed through cell proliferation assay, anchor independent growth, and tumor development in nude mice. The expression of HCV NS3 and c-myc protein in the induced tumor was evaluated by immunohistochemistry.</p><p><b>RESULTS</b>HCV NS3 was strongly expressed in QSG7701 cells transfected with plasmid pRcHCNS3-5' and the positive signal was located in cytoplasm. The HCV NS3 expression and c-myc protein in the induced cytoplasm. Cell proliferation assay showed that the population doubling time in the pRcHCNS3-5' transfected cells was much shorter than that in the pRcCMV and non-transfected cells (24 h, 26 h, 28 h respectively). The cloning efficiencies of transfected cells with pRcHCNS3-5', pRcCMV and non-transfected cells were 33.0%, 1.5%, 1.1% respectively (P < 0.01). Tumor developed in nude mice inoculated with pRcHCNS3-5'transfected cells 15 days after the inoculation. HE staining showed hepatocarcinoma character and immunohistochemistry confirmed HCV NS3 and c-myc expression in the tumor tissue. The positive control group also showed tumor development, while no tumor mass obtained in the nude mice inoculated with pRcCMV and non-transfected cells even 40 days after the injection.</p><p><b>CONCLUSION</b>HCV NS3 N-terminal protein showed cell transformation and tumorigenic features.</p>