Objective To explore the expression and activation of transcription factor E2F1 in cultured human retinal pigment epithelium (RPE) cells. Methods Cultured human RPE cells were divided into two groups after synchronization: one was cultured in Dulbecco′s modified Eagle′s medium (DMEM) without serum; the other was cultured in DMEM supplemented with 20% serum of newborn calf. The expressions of E2F1 protein in two groups were detected by Western blot analysis. The E2F1-DNA binding activities were measured by gel mobility-shift assay(EMSA). Results E2F1 protein of 60 000 molecular weight was detected in the nuclear extract of human RPE cells, and serum stimulation could increase its expression（P＜0.001）. EMSA exhibited the increased binding activity of E2F1 in the serum-stimulated RPE cells with DNA. Conclusions E2F1 is expressed in the nuclei of human RPE cells. Serum stimulation can increase its protein expression as well as binding activity, so as to play a regulation role of gene transcription. (Chin J Ocul Fundus Dis, 2002, 18: 224-226)
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
ObjectiveTo observe the effects of Th9 cell relative factors, including PU.1, interferon regulatory factor 4 (IRF4) and interleukin 4 (IL-4), in rats with pulmonary fibrosis. MethodsNinety SD rats were randomly divided into 3 groups, ie. a normal group, a pulmonary fibrosis group, and a dexamethasone treatment group, with 30 rats in each group. Ten rats in each group were sacrificed respectively on 7th, 14th, 28th days. Model rats were induced by injecting bleomycin into trachea. Real-time PCR was applied to detect mRNA expression of PU.1 and IRF4 in bronchoalveolar lavage fluid. IL-4 in the peripheral blood was measured by ELISA. ResultsIn the normal group, the lung tissue was normal without inflammatory reaction and fibrosis at any time points. In the pulmonary fibrosis group, at the early stage the lung tissue showed alveolar inflammation with a large number of macrophages and other inflammatory cells infiltratation in the pulmonary interstitial and alveolar cavity; on 14th day, part of the alveolar structure disappeared, inflammatory cells infiltrated slightly, while the alveolar septum was mildly widened and fibroblasts proliferated; on 28th day, alveolar structure was destructed, partial alveolar walls were collapsed, alveolar septuml was significantly widened, extracellular matrix was hyperplastic, a wide range of fibrosis occured. In the dexamethasone treatment group, the alveolar structure exsisted completely, and the inflammatory cell infiltration, widened alveolar septum and fibrosis were significantly lighter than those in the pulmonary fibrosis group. PU.1 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, PU.1 mRNA were lower on 14th day and 28th day in the dexamethasone treatment group (P < 0.05). PU.1 mRNA increased from 7th day, reached peak on 14th day, and declined on 28th day. IRF4 mRNA was significantly lower in the pulmonary fibrosis group compared with the normal group. Compared with the pulmonary fibrosis group, IRF4 mRNA was lower on 28th day in the dexamethasone treatment group (P < 0.05). There was a positive correlation between the content of IRF4 mRNA and IL-4 on 14th day in the pulmonary fibrosis group (r=0.044, P < 0.05). ConclusionPU.1 and IRF4 play a role in inflammation leading to pulmonary interstitial fibrosis, and IL-4 may regulate Th9 cells through activating IRF4.
ObjectiveTo investigate the key long non-coding RNAs (lncRNAs) and transcription factors (TFs) in idiopathic pulmonary fibrosis (IPF) by Bioinformatics analysis.MethodsBioinformatics analysis of three gene expression profiles from the Gene Expression Omnibus dataset (GSE2052, GSE44723, and GSE24206), including 42 IPF and 21 normal lung tissues, was performed in this study. Subsequently, differentially expressed genes (DEGs) were filtered, and key genes involved in signaling pathways and the DEG-associated protein-protein interaction network (PPI) were further analyzed. The filtered genes expression was determined by real-time quantitative polymerase chain reaction analysis.ResultsA total of 8483 aberrantly expressed genes were screened, and 29 overlapping genes were identified among these three datasets. A significant enrichment analysis of DEG-associated functions and pathways was further performed. A total of 18 modules were obtained from the DEG PPI network, and most of the modules were involved in polyubiquitination, Golgi vesicle transport, endocytosis and so on. The key genes were obtained through hypergeometric testing, and most of the corresponding genes were closely associated with ubiquitin-mediated proteolysis, the spliceosome, and the cell cycle. These differential expressed genes, such as lncMALAT1, E2F1 and YBX1, were detected in the peripheral blood of IPF patients when compared with those normal control subjects.ConclusionlncMALAT1, E2F1 and YBX1 might be possible regulators for the pathogenesis of idiopathic pulmonary fibrosis.
ObjectiveTo determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. Methods With a change of Decoy oligodeoxynucleotides(ODNs)/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP1 ODNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 μl oligofectamine with different concentrations of ODNs(2.5，5.0，7.5，10.0 and 12.5 μl) were put into 100 μl of DMEM without serum and antibiotics. the (SV-40-PED) cells were transfected after 20 min at room temperature. the final concentration of SP1 decay ODNs were 50，100，150，200 and 250 nmol/L. Transfection effieiency was detected at 26 h after transfection. The intracellular distribution ofSP1 ODNs was determined with a fluorescence microscope. The lactate dehydrogenase (LDH) activity in the supernatant was measured to assess the cytotoxicity.Results The uptake of SP1 ODNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a ber fluorescence intensitywith a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137.12±3.92 U/L in the 72hgroup, which was significantly higher those that in the 26h group（49.61±17.13 U/L）and the 48h group（120.26±8.42 U/L）(Plt;0.01). At 26 h after the transfection, there were no statistical differences at the above LDHlevels in the different-concentration groups（Pgt;0.05）. Conclusion Transfection efficiency is the highest when the final concentration of the SP1 decoy ODNs is 250 nmol/L during the incubation of for 24 h in transfecting the SV-40-PED cells.
Objective To investigate the possible effects of phosphorylated signal transduction and activator of transcription3 (STAT3) in the formation of choroidal neovascuarization (CNV) induced by photocoagulation in rats. Methods The CNV model in rats induced by photocoagulation was established, and the expression of phosphorylated STAT3 at the early stage in CNV were observed by immunofluorescence. To set up the hypoxia model, the specific inhibitor of Janus kinase 2 (JAK2), AG490 was mixed into cell culture fluid and then cultured for 0,1 hour,3,6,12,and 24 hours.Retinal pigment epithelial (RPE) cells proliferation activity were detected by flow cytometry (FCM).the expression of hypoxiainducible factor (HIF)1α and vascular endothelial grow factor (VEGF) mRNA were detected by reverse transcriptase polymerase chain reaction (RT-PCR); the expression of HIF1α protein was detected by Western blot; the content of VEGF in the supernatant of cell culture fluid was measured by enzyme linked immunosorbent assay (ELISA). Results Phosphorylated STAT3 highly expressed in CNV areas in rats 3 days after the photocoagulation. The proliferation activity of human RPE cells under hypoxia condition significantly decreased after inhibition of JAK2/STAT3 signal transduction pathway (t=1.472, 3.566,2.391,6.420; P=0.054,0.038,0.042,0.016). The expression of HIF-1α and VEGF mRNA increased gradually with increasing time of hypoxia;while the expression of HIF1α and VEGF mRNA and the activation of HIF1α protein in cultured human RPE cells with the JAK/STAT3 signal transduction pathway blocked by AG490 were suppressed obviously under hypoxia condition (t=0.07,0.02,0.01, P<0.05); the content of VEGF in RPE cells supernatant decreased significantly (t=1.330,1.106,2.828,7.742,5.610,6.894; P=0.082,0.063,0.014,0.002,0.016,0.011). Conclusion STAT3 may be involved in CNV formation, which may partly dependent on JAK2/STAT3 signal transduction pathway regulating the expression of HIF-1α and VEGF in RPE cells.
Objective To investigate the expression of E26 transformation-specific-1（E26ts-1），matrix metalloproteinase-1（MMP-1）and tissue inhibitor of matrix metalloproteinase-1 （TIMP-1） in choroidal melanoma and the correlation with the tumorprime;s infiltration and metastasis. Methods Immunohistochemistry was used to detect the expression of E26ts-1，MMP-1 and TIMP-1 in 78 cases of choroidal melanoma who were divided into shuttle-cells，paraepithelial-cells and mixed-cells type according to the configuration of tumor cells.The patients were followed up and their average existing time was calculated.The results were statistically computed with statistic SPSS 10.0 package. Results In the 78 cases，shuttle-cells type was found in 21，paraepithelial-cells type in 34，and mixed-cells type in 23. Expression of TIMP-1was low in uveal melanoma，while expression of E26ts-1 and MMP-1 was obviously found in the three types of choroidal melanoma;the sequence of expression intensity was shuttle-cells，mixed-cells and paraepithelial-cells type.Among 37 cases who had been followed up，the shuttle-cells type was in 18 with the average existing time of （78.33plusmn;24.69）months，the mixed-cells type was in 10 with the average existing time of（61.44plusmn;20.46）months，and the paraepithelial-cells type was in 9 with the average existing time of（36.76plusmn;12.19）months.The existing time was negative correlated with the intensity of expresion of E26ts-1 and MMP-1. Conclusion The high expression of E26ts-1 and MMP-1and low expression of TIMP-1may relate to the choroidal melanomaprime;s infiltration and metastasis. (Chin J Ocul Fundus Dis, 2006, 22: 174-176)
Objective To investigate the expression of tumor suppressor gene Lumican mRNA in gastric cancer and its role in the development of gastric cancer. Methods The expressions of Lumican mRNA in gastric cancer tissues, tissues near tumor and normal tissues were detected by using reverse transcription polymerase chain reaction (RT-PCR), clinical pathology for those tissues was studied as well. Results The expression absence rates of Lumican mRNA were 42.4% (28/66), 15.2% (10/66) and 0 in gastric cancer tissues, tissues near tumor and normal tissues respectively. The expression absence rate of Lumican mRNA in patients with lymph node metastasis was 61.1%, which was significantly higher than that of the patients without lymph node metastasis (20.0%, χ2=11.323, P=0.001). The expression absence rate of Lumican mRNA in the gastric cancer at the advanced stages (stages Ⅲ, Ⅳ) was 61.5%, which was significantly higher than that in the gastric cancer at the early stages (stages Ⅰ, Ⅱ, 30.0%, χ2=6.417， P=0.011). The expression absence rates of Lumican mRNA in the high, moderate and poor differentiation tumors were 38.5% (10/26), 38.5% (10/26) and 57.1% (8/14) respectively, the expression absence rate of Lumican mRNA was no significant association with the differentiation degree (χ2=1.576, P=0.455). Conclusion The expression absence of tumor suppressor gene Lumican mRNA may play an important role in the tumorgenesis and influences the prognosis of gastric cancer.
Objective To screen the possible regulatory proteins showing the ability for interaction with serum response factor ( SRF) in the progress of myofibroblast activation, and to see if the proteinprotein interaction is contributing to induce the expression of smooth muscle αactin ( α-SMA) . Methods Phage display cDNA libraries were constructed from the transdifferentiated airway epithelial cells and parental cells. Phage clones were then selectively amplified during the biopanning procedure by using SRF as a bait protein for the two cDNA libraries. Following four rounds of biopanning, recovered cDNAs were sequenced and the obtained sequences were aligned by BLAST tool to select the candidate gene. PAI-RBP1 of the candidate gene was cloned and sub-cloned into pcDNA3. 0 plasmid. Transient transfection and RT-PCR analysis were performed for investigation of the expression of α-SMA. Results Three candidate proteinbinding partners, PAI-RBP1, Nucleolin, and HF1OO, were identified. Among them, PAI-RBP1 pcDNA3. 0 plasmid was subjected to transient co-transfection with SRF, showing up-regulation of α-SMA expression. Conclusions Combined with phage display technique, through protein-protein interaction between core transcription factor and unknown proteins to find a newtranscriptional regulator may serve as an effective strategy. Three novel SRF binding proteins were found from transdifferentiated cells. This study indicates that PAI-RBP1 involves in the activation of myofibroblast by induction of α-SMA expression.
Objectives To investigate the expressions and significance of E2F1, ID1, and Bax protein in gallbladder adenocarcinoma tissues. MethodsThe expressions of E2F1, ID1, and Bax protein in 70 cases of gallbladder adenocarcinoma, 20 cases of high level intraepithelial neoplasia, 30 cases of low level intraepithelial neoplasia, and 20 cases of cholecystitis tissues were tested by using immunohistochemical method. ResultsThe positive expression rates of E2F1, ID1, and Bax protein in gallbladder adenocarcinoma was 84.3%, 70.0%, and 25.7%, respectively; the positive expression rates in high level intraepithelial neoplasia was 75.0%, 65.0%, and 55.0%, respectively; the positive expression rates in low level intraepithelial neoplasia was 16.7%, 23.3%, and 56.7%, respectively; and the positive expression rates in cholecystitis tissues was 10.0%, 20.0%, and 75%, respectively.The positive expression rates of E2F1 and ID1 protein in gallbladder adenocarcinoma were significantly higher than those intraepithelial neoplasia and cholecystitis tissues (P < 0.05), but the positive expression rate of Bax protein in gallbladder adenocarcinoma was lower (P < 0.05).The expressions of E2F1 and ID1 protein were significantly correlated with clinical Nevin staging of gallbladder adenocarcinoma (P < 0.05), but not correlated with the gallbladder adenocarcinoma differentiation degree (P > 0.05).The expression of Bax protein was related to the gallbladder adenocarcinoma differentiation degree (P < 0.05), but not correlated with clinical Nevin staging (P > 0.05).The expression of E2F1 protein was negatively correlated with expression of Bax protein (r=-0.375, P < 0.05), ID1 protein expression has nothing to do with the protein expression of Bax protein (P > 0.05).The expression of E2F1 protein was positively correlated with ID1 protein (r=7.031, P < 0.05). ConclusionsThe E2F1, ID1, and Bax may play an important role in the generation and development of the gallbladder adenocarcinoma.The combined detection of E2F1, ID1, and Bax have important guiding significance for auxiliary diagnosis and clinical staging of gallbladder adenocarcinoma.