共查询到20条相似文献，搜索用时 234 毫秒
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YANG Zu-li KANG Liang HUANG Mei-jin WANG Lei PENG Jun-sheng XIANG Jun LAN Ping WANG Jian-ping 《园艺学报》2008,24(9):1720-1725
AIM:To explore the relationship of methylation status and expression of spleen tyrosine kinase (Syk) gene in colorectal cancer cell lines. METHODS:Bisulfite sodium modification sequencing, methylation specific PCR and Western blotting were used to detect the methylation status and expression of Syk gene in 23 colorectal cancer (CRC) cell lines. Luciferase assay was applied to measure the activity of promoter with or without methylation in CpG islands. Meanwhile, methylation status and expression level were compared in Syk(-) HCT-15 cell line before or after 5-Aza-CdR administration. RESULTS:(1) Among 23 cell lines, loss expression of Syk gene in 9 cell lines due to hypermethylation in promoter, and 14 expression with unmethylation status were observed. The total methylation rate was 39.2%. (2) Microsatellite instability was found in 7 of 9 cell lines with promoter hypermethylation, 4 of 14 were wihtout hypermethylation. The difference between methylation and unmethylation group was significant (P<0.05). (3) 5-Aza-CdR restored methylated-Syk gene promoter activity. Compared to methylated-promoter, luciferase activities increased to 4.5 and 4.7 folds with Syk full size promoter and unmethylated-promoter, respectively. (4) 5-Aza-CdR restored methylated-Syk gene expression and the effect had time-course dependence. CONCLUSION:Hypermethylation of CpG islands in Syk gene promoter silences Syk expression in CRC cell lines, and 5-Aza-CdR restores Syk expression by demethylation. 相似文献
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AIM: To investigate the promoter methylation of secreted frizzled-related protein(SFRP) genes in esophageal squamous cell cancer(ESCC). METHODS: The methods of methylation-specific PCR(MS-PCR) and RT-PCR were applied to examine the CpG methylation of the SFRP promoter and the mRNA expression of SFRP genes,respectively, in 78 samples of ESCC and corresponding adjacent non-cancer tissues. The protein expression of β-catenin was determined by immunohistochemistry.RESULTS: In the ESCC tissues, the frequencies of promoter methylation in SFRP1 , SFRP2 , SFRP4 and SFRP5 genes were 65.4%(51/78), 69.2%(54/78), 62.8%(49/78) and 52.6%(41/78),respectively, significantly higher than those in the adjacent tissues(P<0.01). The hypermethylation of these genes had no correlation with clinical stage and pathological classification in ESCC tissues(P>0.05). The frequency of simultaneous methylation of the 4 genes was correlated with the clinical stage(P<0.05). The positive rates of mRNA expression of the 4 genes in ESCC tissues were 42.3%(33/78), 46.2%(36/78), 50.0%(39/78) and 39.7%(31/78), respectively lower than those in the adjacent tissues(P<0.01). The mRNA expression of SFRP genes and the ectopic expression of β-catenin were correlated with the methylation frequency of SFRP genes(P<0.01).CONCLUSION: Promoter methylation of SFRP1 , SFRP2 , SFRP4 and SFRP5 genes was a frequent event in ESCC, indicating a contribution to the pathogenesis of ESCC through aberrant canonical Wnt/β-catenin signaling pathway. Combination analysis of methylation status in SFRP genes may has definite value on estimating prognosis of ESCC. 相似文献
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AIM: To investigate the characteristic of T-cell acute lymphocytic leukemia 1 (TAL1) gene expression in acute myeloid leukemia (AML) cell lines and in primary AML cells from de novo AML patients with different subtypes. METHODS: Real-time PCR was used to determine the expression of TAL1 mRNA in acute leukemia cell lines (Jurkat, CCRF-CEM, HL-60 and NB4 cell lines) and peripheral blood mononuclear cells from 47 newly diagnosed AML patients. Twelve healthy individuals were served as healthy control group. RESULTS: A significantly increased level in TAL1 mRNA was found in AML cell lines (HL-60 and NB4), T-cell acute lymphacytic leukemia (T-ALL) cell lines (Jurkat, CCRF-CEM) and primary AML cells compared with the healthy controls. Over-expression of TAL1 was found in all detected AML subtypes, the highest level of TAL-1 mRNA was found in AML-M1 and AML-M5 subtype (P<0.05). CONCLUSION: High expression of TAL1 in AML might influence the differentiation and proliferation of myeloid cells, further investigation needs to confirm whether it would be as a biomarker for pathogenesis of AML. 相似文献
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AIM:To study the relation between expression of uPAR and annexinⅡ and fibrinolytic activity in various leukemic cell lines. METHODS:The plasma activity was measured under the reaction between cells of NB4, SHI-1, K562, Jurkat, Raji and plaminogen by chromogenic assay. The protein expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by flow cytometry method. The mRNA expressions of uPAR and annexinⅡin cells of NB4, SHI-1, K562, Jurkat, Raji were detected by RT-PCR. RESULTS:The plasma activity in SHI-1 cells and NB4 cells were higher obviously than that in Raji, K562 and Jurkat cells. The protein expression ratio of uPAR and annexinⅡ in NB4 cells were (13.15±1.61)% and (95.97±1.19)%， respectively, they were (99.00±0.26)%, (90.35±2.15)% respectively in SHI-1 cells, and they were lower in K562, Jurkat, Raji cells. The expression of annexinⅡ mRNA in NB4 cells was higher than that in SHI-1 cells, and they were undectectable in K562 and Jurkat cells. The expression of uPAR mRNA in NB4 and SHI-1 cells were higher than that in Jurkat and K562 cells. The expression of uPAR mRNA in Raji cells was undectectable. CONCLUSION:The primary hyperfibrinolysis in leucocythemia cells was observed, and relation was closely with the expression of annexinⅡ. It might be the main reason for bleeding and disseminated intravascular coagulation in patients with acute promyelocytic leukemia and acute monocytic leukemia. 相似文献
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AIM: To observe the effect of 5Aza-dc on demethylation of TIMP-3 gene promoter in carcinoma cells. METHODS: Both hepatocellular carcinoma cells (H2M) and epidermoid carcinoma cells (A431) with methylation of TIMP-3 promoter gene were treated with 5-Aza-2'-deoxycytidine (5Aza-dc). Invasion ability and motility of the cells were detected by Transwell experiments. Expressions of TIMP-3 protein and mRNA were detected by Western blotting and RT-PCR, respectively. TIMP-3 gene promoter methylation was detected by methylation-specific PCR (MSP). RESULTS: ① Invasion ability and motility of H2M and A431 cells were declined after treatment with 5Aza-dc; ② After treatment with 5Aza-dc, the expression of TIMP-3 protein and mRNA were increased in H2M and A431 cells; ③ After treatment with 5Aza-dc, methylation of TIMP-3 promoter gene was not detectable in the cell lines. CONCLUSIONS: 5Aza-dc induces demethylation in TIMP-3 promoter gene, restores TIMP-3 gene and protein expression, and inhibits invasion ability and motility of the carcinoma cells. 相似文献
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AIM: To study the effects of transforming growth factor-β1 (TGF-β1) on cell apoptosis, cell cycle, production of endogenous TGF-β1, expressions of P27Kip1, cyclin E and bcl-2 mRNA levels in NB4 cells. METHODS: Apoptotic morphological changes were observed by Wright-Giemsa staining. Cell cycle and apoptosis were detected with flow cytometry. Semiquantitative RT-PCR was used to examine the mRNA levels of endogenous TGF-β1, P27Kip1, cyclin E and bcl-2. RESULTS: TGF-β1 significantly restrained the growth and promoted the apoptosis of NB4 cells. The blockage of NB4 cells treated by TGF-β1 at concentration of 5 μg/L was in G1 phase. Endogenous TGF-β1 mRNA expression in NB4 cells was up-regulated when the concentration of exogenous TGF-β1 was <5 μg/L. Meanwhile, the expression of endogenous TGF-β1 mRNA was down-regulated when the concentration of exogenous TGF-β1 was 10 μg/L. After treated with TGF-β1 at concentration of 5 μg/L, P27Kip1 mRNA expression in NB4 cells was up-regulated, cyclin E and bcl-2 were reduced. CONCLUSION: TGF-β1 is able to induce apoptosis and cell cycle distribution abnormally in NB4 cells by (1) Up-regulation of endogenous TGF-β1, so that NB4 cells was induced into apoptosis through consequently high expression of P27Kip1. (2) TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclin E directly, or by inhibiting the activity of cyclin E through the increased expression of P27Kip1. (3) Down-regulation of bcl-2 induces apoptosis of NB4 cells. 相似文献
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MA Peng-jun ZHANG Ming-hao GUO Wei MA Sheng-chao SUN Lei LI Gui-zhong JIANG Yi-deng 《园艺学报》2019,35(11):1951-1956
AIM: To investigate the effect of folic acid and vitamin B12 on homocysteine (Hcy)-induced apoptosis of human umbilical vein endothelial cells (HUVECs) through mammalian sterile 20-like kinase 1 (MST1). ME-THODS: HUVECs were cultured in the absence (control group), or presence of 100 μmol/L Hcy alone (Hcy group) or 100 μmol/L Hcy plus 30 μmol/L folic acid and vitamin B12 (intervention group) for 72 h. The effect of Hcy on the apoptosis of HUVECs was analyzed by flow cytometry. The transfection efficiency of DNA methyltransferase 1 (DNMT1)-overexpressing adenovirus was observed under fluorescence inverted microscope. The mRNA and the protein levels of DNMT1 and MST1 were determined by RT-qPCR and Western blot. The DNA methylation level of MST1 promoter was detected by methylation-specific PCR. RESULTS: Compared with control group, the apoptotic rate (P<0.01) and the expression of MST1 at mRNA (P<0.01) and protein (P<0.05) levels in the HUVECs were significantly increased, while the mRNA levels of DNMT1 was decreased in Hcy group (P<0.01). In addition, folic acid and vitamin B12 treatment significantly inhibited Hcy-mediated apoptosis of HUVECs (P<0.01), increase in MST1 mRNA level (P<0.01) and decrease in DNMT1 mRNA level (P<0.01). Meantime, the mRNA level of MST1 was positively correlated with the apoptotic rate of the HUVECs (r=0.943 9, P<0.001). The expression of DNMT1 at mRNA and protein levels was significantly increased after the transfection of DNMT1-overexpressing adenovirus into HUVECs (P<0.01), and a large amount of green fluorescent protein expression was observed. Meanwhile, the DNA methylation level of MST1 promoter was increased (P<0.01), while the protein level of MST1 was decreased (P<0.01).CONCLUSION: Up-regulation of MST1 promotes Hcy-induced apoptosis of HUVECs, while folic acid and vitamin B12 exert an anti-apoptosis effect, which might be regulated by hypermethylation of MST1 promoter region. 相似文献
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AIM: To investigate the protein expression of DNA methyltransferases (DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L) in gastric cancer and the effects of DNMT inhibitor (5-aza-2’-deoxycytidine) on gastric cancer. METHODS: The method of immunohistochemistry was used to detect DNMT expression in 60 pairs of gastric cancer and corresponding non-cancerous tissues. The expression of DNMT in gastric cancer cells (SGC-7901 and MKN-45) and gastric mucosa cells (GES-1) was detected by immunofluorescence and Western blotting. The proliferation, cell cycle and apoptosis in the cell lines of SGC-7901, MKN-45 and GES-1 were determined by MTT assay and flow cytometry with 5-aza-2’-deoxycytidine treatment. RESULTS: Both gastric cancer and corresponding non-cancerous tissues expressed 5 kinds of DNMT proteins, but the expression levels of DNMT1 and DNMT3A were significantly lower in gastric cancer than those in non-cancerous tissues (P<0.01). The expression levels of DNMT2 and DNMT3L were also lower in the cancer than those in non-cancerous tissues (P<0.05). No difference of DNMT3B expression level between cancer and non-cancerous tissues was observed (P>0.05). On the other hand, the cells lines of SGC-7901, MKN-45 and GES-1 also expressed DNMT proteins. Except DNMT3B, no difference of the other kinds of DNMT between gastric cancer cells and gastric mucosa cells was observed. DNMT inhibitor 5-aza-2’-deoxycytidine did not have any effect on the growth rate, cell cycle distribution or apoptotic rate in gastric cancer cells and gastric mucosa cells. CONCLUSION: Gastric cancer expresses low levels of DNMT proteins than normal gastric mucosa. 5-Aza-2’-deoxycytidine is not useful for treating gastric cancer. 相似文献
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矮生观赏杉木DNA甲基化的水平与模式分析 总被引：1，自引：0，他引：1
为探讨杉木矮化变异与DNA甲基化的关系,以矮生观赏杉木与野生杉木为试验材料,采用基于DNA甲基化敏感扩增多态性分析（Methylation-sensitive amplification polymorphism,MSAP）方法,研究其DNA 序列CCGG 位点的甲基化水平及模式变化特征。应用20个引物组合,在矮生观赏杉木和野生杉木的叶片DNA中均检测出745个CCGG位点,其中甲基化位点数分别为508个和505个,分别占总扩增位点数的68.17%和67.83%;在矮生观赏杉木与野生杉木木质部DNA中分别检测到742个和737个CCGG位点,其中甲基化位点数分别为471个与498个,分别占总扩增位点数的63.52%和67.51%,差异达极显著水平（P < 0.01）。与野生型相比,矮生观赏杉木叶片和木质部DNA甲基化模式发生了一定变化,在叶片DNA中,去甲基化率为17.81%,明显高于超甲基化率15.44%;在木质部DNA中,去甲基化率17.25%,也明显高于超甲基化率14.65%。通过甲基化序列的初步克隆及比对分析发现,矮生观赏杉木中参与MAPK级联途径的蛋白磷酸酶IBR5基因启动子区域的甲基化水平上升。因此推测,植物激素信号转导及其调控基因的甲基化变化可能是矮生观赏杉木形成的原因之一。 相似文献
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AIM：To investigate the effects of staphylococcal enterotoxin A (SEA) on the inhibition of T lymphocyte activation induced by imatinib mesylate (IM). METHODS：Jurkat cells were stimulated with SEA (2 mg/L) and IM (5 nmol/L) for 24 h. The mRNA expression of CD3ε and ζ chains was measured by real-time fluorescence quantitative PCR. The protein levels of CD3ε and ζ chains were detected by Western blotting. RESULTS：The expression of CD3ε and ζ chains at mRNA and protein levels was down-regulated in the Jurkat cells stimulated by IM alone. These down-regulations of CD3ε and ζ chains were reversed by the stimulation of IM combined with SEA. The antagonistic effect of SEA on IM-mediated inhibition of CD3ε mRNA expression was significantly greater than that on CD3ζ mRNA. CONCLUSION:SEA antagonizes imatinib-mediated inhibitory effect on T cell activation. 相似文献
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ZHU Wen-bin JIANG Rui-xue CHEN Shao-ren HU Fang LIU Ji-xin SUN Yan-hong YAO Shu-juan Lü Li-yan ZHANG Chang SUN Yan ZHANG Chun-jing 《园艺学报》2019,35(10):1826-1831
AIM: To observe whether reversing methylation of SARI (suppressor of activator protein-1 regulated by interferon) gene by 5-azacytidine (5-Aza) inhibits the proliferation and invasion of human breast cancer MDA-MB-231 cells. METHODS: MDA-MB-231 cells were treated with 5-Aza at 5 and 10 μmol/L. The methylation of SARI gene promoter was detected by methylation-specific PCR (MSP), and the mRNA expression of SARI was detected by RT-PCR. The protein expression of SARI was determined by Western blot. The cell growth was detected by MTT assay and colony formation assay. The cell invasion ability was detected by Transwell invasion assay. RESULTS: The results of MSP detection showed that the methylation levels of SARI promoter were significantly decreased after 5-Aza treatment (P<0.01). The results of RT-PCR and Western blot showed that the expression of SARI were significantly increased after 5-Aza treatment (P<0.05). The results of MTT and colony formation assays showed that the cell proliferation was significantly decreased after 5-Aza treatment (P<0.05). The results of Transwell invasion test showed that the invasive ability of the cells was significantly decreased after 5-Aza treatment (P<0.05). CONCLUSION: 5-Aza reverses the methylation status of SARI promoter in MDA-MB-231 cells, up-regulates the expression of SARI, and restores its ability to inhibit tumor cell growth and invasion. 相似文献
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AIM: To explore the relationship between hypermethylation of p15INK4B gene and the pathogenesis of hematopoietic malignances. METHODS: The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in bone marrow cells from 54 cases with hematopoietic malignances were detected by RT-PCR, Western blot, and methylation-specific PCR. RESULTS: The p15INK4B gene was methylated more often in high-risk myelodysplastic syndrome (MDS) patients, patients at blast phase of chronic myeloid leukemia (CML-BP) and acute leukemia patients than that in low-risk MDS patients (P<0.01). The expression levels of DNMT3A and DNMT3B in acute leukemia patients, high-risk MDS patients, and CML-BP patients were higher than that in low-risk MDS patients (P<0.05). CONCLUSION: The hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of acute leukemia, high-risk MDS, and blast phase of chronic myeloid leukemia. Furthermore, DNMT3A and DNMT3B are substantially over-expressed in the bone marrow cells of these patients. 相似文献
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WANG Qi-lin LI Rui-qian JIN Cong-guo ZHAO Bin ZHANG Guo-ying BAI Yu LEI Yong-hong 《园艺学报》2014,30(9):1595-1602
AIM：To study the epigenetic mechanisms involved in the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state, and the global difference of histone H3 methylation between androgen-dependent and -independent prostate cancer cells. METHODS：The methylation sites and patterns of histone H3 in androgen-dependent prostate cancer cell line LNCaP and androgen-independent prostate cancer cell line DU145 were analyzed by heavy methyl stable isotope labeling with amino acids in cell culture (SILAC) coupled with 2D LC-MS/MS. Western blotting was used to verify the results from MS. The differential expression of related methylases and demethylases was tested by real-time PCR. RESULTS：Five methylation sites on histone H3 were found in both cell lines, the patterns of which were as follows: H3K14me2, H3R17me1, H3K36me1, H3K36me2, H3K36me3, H3R72me2, H3K79me1 and H3K79me2. There were 2 different peptides both containing methylated H3K36, “KSAPATGGVKKPHR” and “KSAPSTGGVKKPHR”, which were different from the 31th amino acid residue “A” and “S”. The former peptide belonging to histone H3 variants, H31T, H31 and H32, was mainly identified in DU145 cells, the total peptide counts of which were much more than that of the latter peptide belonging to histone H3 variant H31T, suggesting that these 2 cell lines expressed different histone H3 variants. Mono- and dimethylation of H3K36 were not different between these 2 cell lines, but the trimethylation was significantly higher in DU145 cells than that in LNCaP cells. Many H3K36 demethyltransferases were decreased in DU145 cells compared with LNCaP cells. CONCLUSION：The differential expression of histone H3 variants and H3K36 demethyltransferases may result in up-regulation of H3K36 tri-methylation during the evolution of prostate cancer from an androgen-dependent state to an androgen-independent state. 相似文献
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AIM: To study the expression and methylation status of checkpoint with forkhead-associated and ring finger ( CHFR ) in human endometrial cancer cell lines and to explore the effect of demethylation on the colony formation of the cancer cells.METHODS: The expression level of CHFR in endometrial cancer cells was evaluated by semi-quantitative RT-PCR and Western blotting.The methylation status of CHFR was detected by methylation-specific PCR.The proliferation of RL-952 cells was analyzed by colony formation test.RESULTS: Lower expression of CHFR was detected in endometrial cancer cells, especially in RL-952 cells, than that in normal fibroblast (NF).CHFR was weekly expressed in RL-952 cells, the gene of which had aberrant hypermethylation.The unmethylation band of CHFR was observed in NF cells and ISH cells, which had high expression of CHFR.After exposed to 5-azacytidine (5-aza), the methylation band in RL-952 cells disappeared with the recovery of both CHFR mRNA and protein expression, and the colony-forming efficiency was greatly inhibited.CONCLUSION: Aberrant promoter methylation is the main reason for decreasing CHFR expression in endometrial cancer cells.5-aza could restore CHFR expression and inhibit the colony formation effectively. 相似文献
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AIM:To explore the effect of pyrrolidine dithiocarbamate (PDTC),an NF-κB inhibitor,on the proliferation and apoptosis of human multiple myeloma U266 cells and its mechanisms.METHODS:The U266 cells were treated with PDTC at different concentrations (0,25,50,100 and 200 μmol/L)in vitro.The growth inhibitory rate of the U266 cells was detected by CCK-8 assay and cell counting.The cell cycle of the U266 cells was determined by flow cyto-metry,and the apoptosis was examined by flow cytometry with Annexin V-FITC/PI staining.The effect of PDTC on the expression of DNA methyltransferase 1(DNMT1) at mRNA and protein levels was measured by RT-qPCR and Western blot,respectively.The effects of PDTC on the protein levels of NF-κB (P65),DNMT1,Bcl-2,cyclin D1,cleaved caspase-3 and cleaved caspase-8 were determined by Western blot.RESULTS:The protein level of NF-κB (P65) was decreased after treatment with PDTC for 48 h or 72 h.PDTC inhibited the proliferation of U266 cells in both dose-and time-dependent manners.After treatment with PDTC for 48 h,the percentage of U266 cells in G2 phase increased compared with control group (P<0.05).PDTC induced the apoptosis of U266 cells in a dose-dependent manner.The expression of DNMT1 at mRNA and protein levels decreased (P<0.05).The results of Western blot showed that the expression of Bcl-2 in PDTC groups decreased,while the protein levels of cyclin D1,cleaved caspase-3 and cleaved caspase-8 were higher than those in control group (P<0.05).CONCLUSION:The NF-κB inhibitor PDTC inhibits the proliferation of U266 cells by inducing cell apoptosis.It may be related to the down-regulated expression of DNMT1,cell cycle arrest and activation of the apoptotic pathways. 相似文献
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FAN Rui-fang FANG Zhi-gang LIU Xiang-fu ZHENG Yong-jiang LIU Bin-bin LIN Dong-jun 《园艺学报》2010,26(2):293-296
AIM: To detect the protein expression of TIMP3 and RUNX3 in bone marrow mononuclear cells (BMMCs) from acute leukemia (AL) patients and to investigate the relationship between the methylation status of genes and their expressional levels. METHODS: Protein expression of TIMP3 and RUNX3 in 50 samples of BMMCs and 10 samples of peripheral blood mononuclear cells (PBMCs) from healthy volunteers was detected by Western blotting. The prognostic factors related to AL and data from methylation specific polymerase chain reaction were also analyzed. RESULTS: The expression level of RUNX3 with methylation was less than that without methylation in BMMCs from AL patients. The complete remission (CR) rate was related to RUNX3 expression and blasts in bone marrow (BM). BMMCs from patients with silencing of RUNX3 and higher blasts in BM had a lower CR rate. CONCLUSION: Absence of RUNX3 protein expression resulting from methylation of RUNX3 promoter probably plays a role in the pathogenesis of AL and is of value in prognosis. No relationship between methylation of TIMP3 promoter and the pathogenesis of AL is observed. 相似文献
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AIM: To transfer 4 full-length WT1 isoforms cDNA into the leukemia cell line NB4 so as to provide a cell model for studying the WT-1 gene function. METHODS: The eukaryotic expression recombinant vectors for WT1 isoforms (pCB6+/WT1) were introduced into the leukemia cell line NB4 by electroporation. The positive cell clones were screened by G418 culture. The integration of WT1 gene isoforms in NB4 cells as confirmed by PCR. The mRNA and protein of WT1 were detected by RT-PCR and Western blotting. RESULTS: WT1 gene isoforms were successfully transferred into NB4 cells. WT1 mRNA and protein expression in the G418-selected cells increased remarkably compared with the control. CONCLUSION: WT1 gene isoforms were effectively transferred into NB4 cells by electroporation and stably expressed in the transfected cells. 相似文献