ABSTRACT
Objective@#To investigate the expression of myocyte enhancer factor 2B (MEF2B) in mantle cell lymphomas (MCL), and to analyze the correlation between the expression of MEF2B and pathological subtypes, structural subtypes, SOX11 expression and its clinical significance.@*Methods@#Paraffin-embedded tissues were stained with HE, immunohistochemistry (EnVision method) and fluorescence in situ hybridization (FISH) , in addition, the clinical and pathological data of 60 cases of MCL were collected at Sun Yat-sen University Foshan Hospital and Sun Yat-sen University Cancer Center from January,2002 to May, 2019 for analysis.@*Results@#Of the 60 MCLs, males is predominant (M∶F=3∶1). Histologically, the typical MCL is the majority (classical MCL: variant type MCL=48 cases:12 cases) . Fifty cases were classified into non-complete FDC meshwork type MCL, and the remaining 10 cases were classified into the complete-FDC meshwork type MCL group. Patients with classical MCL were more than 60 years old. The coexistent lesion sites both node and extranode in pathological subtype or structural subtype was the most common lesion sites. SOX11(+) MCL was common in classical MCL (P=0.040) and tended to be complete-FDC meshwork type MCL (P=0.086). The expression rate of MEF2B in MCL was 60.0%(36/60). This rate of MEF2B in classical type, complete-FDC meshwork type and SOX11(+) MCL was significantly higher than that variant type, no complete-FDC meshwork type, SOX11(-)MCL (P<0.05), respectively. There was no difference in clinical characteristics of MCL between MEF2B positive and negative groups. Compared with SOX11(-)MCL, the percentage of MEF2B expressed in tumor cells of SOX11(+)MCL was significantly higher (P=0.027). The expression of MEF2B was not related to the proliferation of tumor cells (P=0.341). There was no significant difference in the survival rate between different expression groups of MEF2B and SOX11 (P=0.304 and P=0.819, respectively). Only the mortality of variant type (blastoid/pleomorphic) MCL within 2 years was significantly higher than that of classical type MCL (P<0.05).@*Conclusions@#The expression of MEF2B in MCL is related to the pathological subtypes, structural subtypes and the expression of SOX11, but not to the proliferation and prognosis. The high mortality rate within 2 years is only found in variant MCL. However, the role of MEF2B in MCL needs to be further studied.
ABSTRACT
AIM: To observe the changes in expression and activity of the transcription factor myocyte enhancer factor 2A (MEF2A) during hepatic stellate cells (HSC) activation, and to study the roles of MEF2A in the process of HSC activation. METHODS: Cultured HSC was isolated from male sprague-dawley rat liver on plastic dishes and were used as model of activation. The freshly isolated (0 day) and cultured HSC at time points of 1st, 2nd, 3rd, 4th, 5th, 6th, 7th and 8th day were collected. Expression of MEF2A mRNA was detected by real-time quantitative PCR. MEF2A and α-smooth muscle actin (α-SMA, a marker for activated HSC) were tested by Western blotting. Meanwhile, the MEF2A DNA binding activity was determined by electrophoretic mobility shift assays (EMSA). RESULTS: The expression of MEF2A mRNA was small amounts in the freshly isolated HSC and increased gradually after culture on plastic dishes. Western blotting revealed that the freshly isolated HSC expressed very low levels of MEF2A and α-SMA. The proteins of MEF2A and α-SMA were increased gradually in the process of HSC activation. Increased MEF2A protein was correlated with α-SMA. EMSA revealed that MEF2A DNA binding activity was increased gradually during HSC activation. CONCLUSION: In the process of HSC activation, expression and activity of MEF2A are increased gradually, indicating a role in HSC activation.
ABSTRACT
AIM: To observe the effect of myocyte enhancer factor 2C (MEF2C) on the expression of substance P (Sub P) and neurofilament triplet L (NFL) in rat dorsal root ganglion cells (DRGn cells). METHODS: DRG neurons were dissociated and cultured, and then exposed to different concentrations of nerve growth factor (NGF, 10 μg/L, 30 μg/L, 100 μg/L or 200 μg/L) for 24 h. The neurons cultured in media with the lowest concentration of NGF (10 μg/L) served as control. Real time PCR was used for detecting the mRNA of substance P and NFL in the DRGn cells. Three MEF2C-siRNAs were transfected into PC 12 cell line by the way of chemical mediation. The best siRNA with the highest interference ratio was determined by real time PCR. The DRGn cells knocked out MEF2C gene were also transfected with siRNA, and the expressions of substance P and NFL were measured by real time PCR after stimulated with high concentration of NGF. RESULTS: The expressions of substance P and NFL increased in primary cultured rat DRG neurons in a dose-dependent manner of NGF stimulation. The expression level of MEF2C in experimental group was lower by 50% than that in control group. No change of cyclic AMP response element-binding protein (CREB) was observed. The substance P decreased by 41% in experimental group than that in control group at the same time point. The NFL was decreased by 61%. CONCLUSION: NGF may promote the synthesis of substance P and NFL in rat DRGn cells. MEF2C regulates the expression of substance P and NFL in DRGn cells in rat embryo and MEF2C may be a critical transcriptional factor involved in the airway hyperresponsiveness.
ABSTRACT
Purpose To investigate the effect of small interfering RNA on the increase of myocyte enhancer factor 2A(MEF2A) expression in PC12 cells exposed to hypoxia.Methods PC12 cells were cultured under normal conditions or were exposed to hypoxic conditions.Small interfering RNA targeted MEF2A gene (MEF2A-siRNA) was chemically synthesized. Eukaryocytic expression vector was built and transfected into PC12 cells with liposome. The expression of MEF2A mRNA was detected by real-time PCR. Western blot was used to detect the MEF2A protein.Results Compared with normal control(2~(-△△CT)=1.01±0.02), the mRNA level of MEF2A gene in PC12 cells with the treatment of MEF2A-siRNA was down-regulated significantly by 88%(2~(-△△CT)=0.12±0.03, P＜0.01).The expression of MEF2A protein in hypoxia-treated PC12 cells was markedly higher than that of normal control(98.4±11.7 and 47.5±7.6,P＜0.01).However, MEF2A-siRNA could significantly suppress the increase of MEF2A protein (P＜0.01).Conclusion MEF2A gene silence induced by siRNA might inhibit the increase of MEF2A protein by hypoxia in PC12 cells.
ABSTRACT
Objective To investigate the level of MEF2C phosphorylation (activation) and protein expression, and to further clarify the possible mechanism following ischemia-reperfusion in hippocampal CA1 region of rat. MethodsBrain ischemia was induced by four-vessel occlusion in SD rats. Protein level was determined by Western blotting. Results MEF2C was significantly activated with a peak at 6 h of reperfusion, but its protein expression decreased in late phase of reperfusion (3～5 d). The elevation of activated (17 ku) and the inactivated forms (32 ku) of caspase-3 proteases were remarkable during 1～5 d of reperfusion. In addition, Ac-DEVD-CHO, a specific inhibitor of caspase-3, up-regulated MEF2C protein level of 3 d reperfusion. SB202190 (an inhibitor of P38), but not ERK5-antisense oligonucleotides, not only inhibited MEF2C activation of 6 h reperfusion but also apparently prevented the increase of caspase-3 activation caused by 3 d reperfusion. Conclusion P38/caspase-3 mediated MEF2C pathway may function in the injuries of hippocampal CA1 region of rats following ischemia/reperfusion.
ABSTRACT
Objective To explore the underlying molecular mechanisms of estrogen induced apoptosis of synthetic vascular smooth muscle cells (VSMCs). Methods Cultured VSMCs on passage 3 were divided into three groups: 17?-estradiol (E-2) group, p38 inhibitor group (SB+E-2) and control group. With treatment for 72 hours,the apoptosis of cells was quantified by measuring the intracellular nucleosomes with ELISA; expressions of total p38, phospho-p38 and myocyte enhancer factor-2 (MEF2) were analyzed by Western blotting, with a confirmative immunohistochemical staining for MEF2. Results Compared with that of control cells, the rate of apoptosis in VSMCs grown in E-2 was significantly increased, with up-regulations of total p38, phospho-p38 and MEF2. These effects of E-2 were blocked in SB+E-2 group in which the rate of cell apoptosis and expression of p38 had no obvious change compared with that of control group, while the expression of p-p38 and MEF2 was decreased. Immunohistochemical staining showed that MEF2 was mainly located in cellular nucleus, and its expression was increased in E-2 group but decreased in SB+E-2 group. Conclusion Estrogen may induce apoptosis of synthetic VSMCs through p38 pathway-dependent up-regulation of MEF2.
ABSTRACT
PURPOSE: We have previously cloned three enhancer factor genes encoding proteins that bind to long terminal repeats (LTRs) of Rous sarcoma virus. Among these genes, RSV- EF-I gene is expressed in rat hepatoma tissues and several proliferating cell lines but not in normal rat liver tissues. We have isolated the human homologue of RSV-EF-I gene and examined its expression in human hepatocellular carcinoma tissues. MATERIALS AND METHODS: We have screened the human genomic library and cDNA library of Hep G2 cell line derived from human hepatocellular carcinoma to isolate the human homologue of RSV-EF-I gene. RESULTS: We have isolated one cDNA clone containing about 1.5 kb insert and sequenced. Sequence analysis reveals that this human homologue of RSV-EF-I gene has a high similarities to human YB-1 mRNA, human DNA-binding protein B (dbpB) gene and other Y-box protein genes. It is expressed in human hepatocellular carcinoma but very slightly in normal human liver tissues in Northern blot analysis. CONCLUSION: Our data suggest that the human homologue of RSV-EF-I gene presumably belongs to Y-box protein family genes and plays a role in the transformation of the human hepatoma cells.
Subject(s)
Animals , Humans , Rats , Blotting, Northern , Carcinoma, Hepatocellular , Cell Line , Clone Cells , DNA, Complementary , Gene Library , Genomic Library , Hep G2 Cells , Liver , RNA, Messenger , Rous sarcoma virus , Sarcoma, Avian , Sequence Analysis , Terminal Repeat SequencesABSTRACT
Objective To investigate the inhibition of lymphoid enhancer factor-1 (LEF-1) expression in human malignant melanoma cell line A375 by RNA interference method. Methods Sense and antisense oligonucleotides with hairpin structures, targeted specifically at LEF-1 mRNA, were designed, synthesized, then linked to the expression vector psilencer3.1-H1 neo after annealing. After identification, the re-combinant psilencer3.1-H1/LEF-1 siRNA was used to transfect the cultured A375 cells by a liposome-medi-ated method. The cells expressing the recombinant RNA was detected by G418 screening. The mRNA and protein levels were detected by RT-PCR, Western blotting and immunocytochemistry, respectively. Results The expression vector psilencer3.1-H1/LEF-1 siRNA was successfully constructed, and its stable expression in cell clones was achieved. The mRNA and protein levels of LEF-1 were both down-regulated in the trans-fected cells. Conclusion The recombinant of psilencer3.1-HI/LEF-1 siRNA can inhibit the mRNA and protein expression of LEF-1 in A375 cells.
ABSTRACT
Objective To study the effects of ?-catenin-dependent lymphoid enhancer factor(LEF-1) isoforms on biological behavior of HeLa cells.Methods ?-catenin-dependent LEF-1 genes were obtained by PCR from human lymphoid node cDNA library and inserted into pcDNA3.1/V5-His vector to construct the eukaryotic expression plasmid pcDNA3.1-F-LEF-1.Using lipofectamineTM 2000,the plasmid pcDNA3.1-F-LEF-1 was transfected into Hela cells.Then we screened the stable cell lines that expressed the truncated LEF-1 isoforms by G418 and identified the expression of target gene with Western blot.Then we analyzed the proliferation,apoptosis,cell clone formation and capability of tumor formation in vivo of transfected cell lines.Results We successfully constructed the ?-catenin-dependent LEF-1 eukaryotic expression plasmid and obtained the stable HeLa cell lines that expressed the full-length LEF-1 isoforms.The proliferation and capability of tumor formation in vivo of transfected cells were increased while apoptosis was decreased.Conclusion The overexpression of ?-catenin-dependent isoforms can stimulate the malignant biological behavior of HeLa cells.