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OBJECTIVE@#To explore whether casticin (CAS) suppresses stemness in cancer stem-like cells (CSLCs) obtained from human cervical cancer (CCSLCs) and the underlying mechanism.@*METHODS@#Spheres from HeLa and CaSki cells were used as CCSLCs. DNA methyltransferase 1 (DNMT1) activity and mRNA levels, self-renewal capability (Nanog and Sox2), and cancer stem cell markers (CD133 and CD44), were detected by a colorimetric DNMT activity/inhibition assay kit, quantitative real-time reverse transcription-polymerase chain reaction, sphere and colony formation assays, and immunoblot, respectively. Knockdown and overexpression of DNMT1 by transfection with shRNA and cDNA, respectively, were performed to explore the mechanism for action of CAS (0, 10, 30, and 100 nmol/L).@*RESULTS@#DNMT1 activity was increased in CCSLCs compared with HeLa and CaSki cells (P<0.05). In addition, HeLa-derived CCSLCs transfected with DNMT1 shRNA showed reduced sphere and colony formation abilities, and lower CD133, CD44, Nanog and Sox2 protein expressions (P<0.05). Conversely, overexpression of DNMT1 in HeLa cells exhibited the oppositive effects. Furthermore, CAS significantly reduced DNMT1 activity and transcription levels as well as stemness in HeLa-derived CCSLCs (P<0.05). Interestingly, DNMT1 knockdown enhanced the inhibitory effect of CAS on stemness. As expected, DNMT1 overexpression reversed the inhibitory effect of CAS on stemness in HeLa cells.@*CONCLUSION@#CAS effectively inhibits stemness in CCSLCs through suppression of DNMT1 activation, suggesting that CAS acts as a promising preventive and therapeutic candidate in cervical cancer.
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Female , Humans , Cell Line, Tumor , HeLa Cells , Neoplastic Stem Cells/metabolism , RNA, Small Interfering/metabolism , Uterine Cervical Neoplasms/metabolismABSTRACT
Objective:To investigate the methylation status of O6-methylguanine-DNA methyltransferase (MGMT) in meningioma tissue and its effect on the growth and metastasis of human meningioma cell line IOMM-Lee cells.Methods:The specimens of 34 patients with meningioma were collected. Methylation-specific polymerase chain reaction (MSP) method was used to detect MGMT methylation in meningioma tissue and normal brain tissue; immunohistochemical staining was used to detect the expression of MGMT in meningioma tissue and normal brain tissue. The cultured human glioma cell line IOMM-Lee cells were divided into blank control group (normal cultured IOMM-Lee cells), negative control group (empty vector virus transfected IOMM-Lee cells) and RNAi lentivirus transfection group (transfected with RNAi lentivirus vector to down-regulate the expression of MGMT). The expression of MGMT in IOMM-Lee cells was silenced by RNAi technology. The expression levels of MGMT mRNA and protein in cells were detected by real-time fluorescence quantitative PCR and Western blot. The proliferation activity of cells was detected by cell counting kit-8 (CCK-8) test, the colony forming ability was detected by cell clone formation test, and the invasion and migration ability of cells in each group were detected by Transwell test and cell scratch test.Results:The methylation of MGMT in meningioma tissue reached 88.23% (30/34). MGMT methylation was not detected in normal brain tissue; the staining intensity of MGMT in meningioma tissue was significantly higher than that in normal brain tissue. Compared with the blank control group and the negative control group, the relative expression of MGMT mRNA and protein in IOMM-Lee cells of the RNAi lentiviral transfection group were significantly decreased (all P<0.05). After 24, 48 and 72 hours of transfection, the proliferation activity of IOMM-Lee cells decreased significantly (all P<0.05), with reduced number of cell clone formation and cell invasion (all P<0.05). The rate of scar healing decreased significantly ( P<0.05). Conclusions:MGMT is mostly hypermethylated in human meningiomas. Silencing the expression of MGMT in meningiomas can inhibit the growth and metastasis of meningiomas.
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【Objective】 To investigate the expression and biological role of miR-139-5p in glioblastoma and the regulatory effect of miR-139-5p on DNA methyltransferase 1 (DNMT1). 【Methods】 qRT-PCR was used to detect the expression of miR-139-5p in glioblastoma tumor tissue, paired paracancerous tissue, human normal glioma cell line HEB, and human glioma cell line U251. The expression of miR-139-5p in U251 cells was up-regulated by transfection of miR-139-5p mimetics, and the expression of DNMT1 was down-regulated by transfection of DNMT1-targeted siRNA (DNMT1-siRNA). The expression of DNMT1 and neurofibromatosis type 2 (NF2) in tissues and cells was detected by qRT-PCR, Western blotting, immunohistochemistry and immunofluorescence. The cell counting kit-8 (CCK-8), flow cytometry and Matrigel Transwell were used to evaluate the proliferation, apoptosis and invasion ability of U251 cells. 【Results】 Compared with paracancerous tissues or HEB cells, miR-139-5p expression in glioblastoma tissues and U251 cells was suppressed (P<0.05). Compared with control cells, transfection of miR-139-5p mimic significantly down-regulated the expression of DNMT1 and up-regulated the expression of NF2 (P<0.05). Compared with control cells, transfection of DNMT1-siRNA significantly promoted the expression of NF2 (P<0.05). Transfection of miR-139-5p mimetics or DNMT1-siRNA significantly induced U251 cell apoptosis and inhibited cell invasion (P<0.05). 【Conclusion】 miR-139-5p plays an anti-cancer role in glioblastoma, and it inhibits tumor proliferation and metastasis by targeting negative regulation of DNMT1.
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Objective:To investigate the effects of cell adhesion molecule 1 (CADM1) promoter methylation in ovarian cancer on gene transcription and protein expression levels, and the regulation mechanism of mirNA-148A on CADM1 methylation levels.Methods:A total of 86 patients with ovarian cancer who received surgical treatment in the Affiliated Hospital of Hangzhou Normal University from Jun. 2018 to Jun. 2020 were selected as study subjects. The methylation level of CADM1 gene CpG island in ovarian cancer tissues and adjacent tissues was quantitatively detected. Quantitative real-time polymerase chain reaction was used to detect the mRNA and mirNA-148a expressions of CADM1 gene. The CADM1 gene and DNMT1 protein levels were detected by Western blot. Human ovarian cancer SKOV3 cells were treated with different concentrations of methyltransferase inhibitors (5-Azacytidine, 5-aza) , and CADM1 mRNA expression was detected 72 h later. Human ovarian cancer cell lines SKOV3 were transfected with mir-335-5p mimic, inhibitor and negative control respectively. Then mir-335-5p expression level and CADM1 gene methylation level were detected after transfection.Results:The methylation level of CADM1-1 island in ovarian cancer tissues was 2.89%±0.82%, significantly higher than that of paracancerous normal tissues 1.86%±0.68% ( t=4.936, P<0.001) , and that of CADM1-2 island in ovarian cancer tissues was 3.12%±0.93%, significantly higher than that of paracancerous normal tissues (2.27%±0.69%, t=5.114, P<0.001) . Pearson correlation analysis showed that the methylation level of CADM1-1 island and CADM1-2 island in ovarian cancer tissues was significantly negatively correlated with the relative mRNA expression (r was -0.615 and -0.582, respectively, and both P<0.001) , and with the protein expression level of CADM1 (r was -0.521 and -0.612, respectively, and both P<0.001) . The relative expression level of mirNA-148a in ovarian cancer tissues was 1.53±0.42, significantly lower than that in paracancer tissues (2.59±0.73, t=6.113, P<0.001) . After treatment with different concentrations of 5-AZA, mRNA expression levels of CADM1 gene in SKOV3 cells were significantly higher in the low concentration group and the high concentration group than in the control group (both P<0.05) , and mRNA expression levels in the high concentration group were significantly higher than in the low concentration group ( P<0.05) . After mirNA-148A transfected SKOV3 cells, the relative expression levels of mirNA-148a in the mimic group were significantly increased, while those of inhibitor group were significantly decreased ( P<0.001) . The DNMT1 expression level and CADM1 gene methylation level of mimic group were significantly decreased, while those of inhibitor group were significantly increased (P<0.001) . Conclusion:In ovarian cancer, miRNA-148a can regulate the DNA methylation level of CADM1 gene by acting on the downstream target protein DNMT1, thus affecting the mRNA and protein expression levels of CDM1 gene and participating in the pathogenesis of ovarian cancer.
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Objective:To analyze the relationship between DNA methyltransferase 1 (DNMT1) and forkhead box O3a (FOXO3a) in colon cancer and the diagnostic efficacy of combined detection in predicting the occurrence of colon cancer by detecting the levels of DNMT1 and FOXO3a in serum of colon cancer patients.Methods:A total of 105 patients with colon cancer diagnosed and treated in Xi′an International Medical Center Hospital from September 2019 to September 2020 were selected as the colon cancer group, and 65 patients with colon polyps diagnosed by biopsy during the same period were selected as control group. The levels of DNMT1 and FOXO3a in serum of patients were detected by real-time fluorescence quantitative PCR. Pearson correlation coefficient method was used to analyze the correlation between the levels of DNMT1 and FOXO3a in serum of patients with colon cancer. Subject operating characteristic curve was used to evaluate the diagnostic values of DNMT1 and FOXO3a levels in colon cancer.Results:The serum levels of DNMT1 in the control group and colon cancer group were 0.93±0.28 and 1.34±0.35, compared with the control group, the level of DNMT1 in the colon cancer group was significantly higher, with a statistically significant difference ( t=7.990, P<0.001). The serum levels of FOXO3a were 1.04±0.39 and 0.69±0.18, compared with the control group, the level of FOXO3a in the colon cancer group was significantly lower, with a statistically significant difference ( t=7.940, P=0.001). The serum levels of DNMT1 and FOXO3a in patients with colon cancer were negatively correlated ( r=-0.687, P<0.001). The area under the curve (AUC) of DNMT1 predicting colon cancer was 0.843, the sensitivity was 71.40%, and the specificity was 90.80%. The AUC of FOXO3a predicting colon cancer was 0.812, the sensitivity was 88.60%, and the specificity was 67.70%. The AUC of the two combined predicting colon cancer was 0.859, the sensitivity was 89.50%, and the specificity was 92.30%. Compared with FOXO3a single detection, the predictive value of combined detection of DNMT1 and FOXO3a were higher ( Z=1.982, P=0.047). Conclusion:The level of DNMT1 in the serum of patients with colon cancer is increased, while the level of FOXO3a is decreased. There is a negative correlation between them in the serum of patients with colon cancer. The combined detection of the DNMT1 and FOXO3a can effectively improve the diagnostic value of colon cancer.
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Aim To observe the effect of methylation inhibitors of 5-aza-2'-deoxycytidine (5-Aza-CdR) on SPC-Al - lung cancer cell proliferation, cell scratches and apoptosis, to explore the influence of secretion curl associated protein 1 (SFRPl) and 06-methylguanine-DNA-methyltransferase (MGMT) gene promoter region in which DNA methylation mRNA and protein expression and meaning. Methods The effects of 5-Aza-CdR at different concentrations on the proliferation of human lung cancer SPC-A 1 cells were determined by CCK-8 assay. The effect of 5-Aza-CdR on the migration ability of SPC-A 1 cells was determined by scratch assay. The apoptosis of lung cancer SPC-A 1 cells was detected by Hoechst 33258 staining after treatment with 5-Aza-CdR for 24 h. mRNA and protein expressions of SFRPl and MGMT in SPC-Al cells were detected by RT-PCR and Western blot. Results 5-Aza-CdR could reduce the proliferation of SPC-A 1 cells by concentration gradient,and IC50 was 21.2 jjimol • L
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OBJECTIVE: To investigate the effect of bisphenol A exposure on the expression of DNA methyltransferase(Dnmt) gene in mouse Leydig cell line TM3 cells. METHODS: TM3 cells were randomly divided into 0, 20, 50, 125 and 300 μmol/L dose group(0 μmol/L dose group was the control group). Cells were treated with various concentration of bisphenol A solution for 24 hours.The viability of TM3 cells was determined by CCK-8 method, and the mRNA expression of Dnmt1, Dnmt3 a and Dnmt3 b was detected by real-time fluorescence quantitative polymerase chain reaction. RESULTS:The viability of TM3 cells decreased with the increasing doses of bisphenol A(P<0.01). The relative mRNA expression of Dnmt1 in TM3 cells decreased with the increase of doses of bisphenol A(P<0.01).The relative mRNA expression of Dnmt3 b in TM3 cells in the 20, 50, 125, 300 μmol/L dose group was lower than that in the control group(P<0.05).There was no statistical significant difference in the relative mRNA expression of Dnmt3 a in TM3 cells in these 5 groups(P>0.05). CONCLUSION: Bisphenol exposure is cytotoxic to TM3 cells. This toxic effect may be related to changes in Dnmt mRNA expression.
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Objective@#To investigate the expressions of histone lysine-specific demethylase 1 (LSD1), O6-methylguanine DNA methyltransferase (MGMT) and cell proliferation-associated antigen Ki-67 in high-grade glioma and their influences on prognosis.@*Methods@#Sixty-five cases of grade Ⅲ and Ⅳ glioma confirmed by pathology from January 2011 to June 2017 in the First Affiliated Hospital of Xinjiang Medical University were selected. Immunohistochemistry (SP method) was used to detect the expressions of LSD1, MGMT and Ki-67 in pathological specimens. The therapeutic effect was evaluated by long-term follow-up. The relationships between the three markers and pathological grade, progression-free survival (PFS) and overall survival (OS) were analyzed.@*Results@#The overall positive rates of LSD1, MGMT and Ki-67 in the 65 high-grade glioma specimens were 70.8% (46/65), 60.0% (39/65) and 100.0% (65/65), respectively. There were no significant differences in the expressions of LSD1 and MGMT in grade Ⅲ and Ⅳ glioma (χ2=1.588, P=0.208, χ2=0.013, P=0.908). Ki-67 expression (+ ), (+ + ), (+ + + ) in grade Ⅳ glioma were observed in 18, 19 and 11 cases, respectively. Ki-67 expression (+ ), (+ + ) in grade Ⅲ glioma were observed in 11, 5 cases, and 1 case was (+ + + ), and the difference in expression intensity between the two groups was statistically significant (Z=-2.083, P=0.037). Log-rank test showed that the positive expressions of LSD1, MGMT and Ki-67 were negatively correlated with the PFS of patients with high-grade glioma (χ2=12.217, P=0.007; χ2=4.446, P=0.035; χ2=12.536, P=0.002), also were negatively correlated with OS (χ2=11.708, P=0.008; χ2=6.637, P=0.010; χ2=11.807, P=0.003). Grade Ⅳ patients were more likely to have relapse progression than grade Ⅲ patients (χ2=6.573, P=0.010), and OS was shorter (χ2=3.974, P=0.046). Cox proportional hazards model analysis showed that the expressions of LSD1 (HR=1.361, 95%CI: 1.094-1.694, P=0.006; HR=1.406, 95%CI: 1.117-1.771, P=0.004) and Ki-67 (HR=1.703, 95%CI: 1.175-2.468, P=0.005; HR=1.778, 95%CI: 1.209-2.616, P=0.003) were the independent prognostic risk factors for PFS and OS of patients with high-grade glioma. Correlation analysis results showed that the expression of MGMT was positively correlated with the expression of LSD1 (r=0.406, P=0.001).@*Conclusion@#LSD1, MGMT and Ki-67 have higher positive expression rates in high-grade glioma. MGMT is a prognostic factor for high-grade glioma, and LSD1 and Ki-67 can be used as independent predictors of prognosis for high-grade gliomas.
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@# Objective: To explore the mechanism of long non-coding RNA POU3F3 (lncRNAPOU3F3) affecting temozolomide (TMZ)-resistance in high-grade glioma cells via regulating MGMT expression. Methods: Sixty cases of tissues from patients treated at the Department of Neurosurgery, Peking University International Hospital during January 2016 and January 2018 were collected for this study, including 12 cases from brain trauma patients (normal group), 30 cases from primary high-grade glioma patients (primary onset group) and 18 cases from recurrent high-grade glioma patients (recurrence group, accepted surgery+TMZ already). U251 cells were induced with TMZ at the concentration of 1, 2, 4 and 8 μg/ml and maintained normal growth for a week to construct TMZ-resistant U251cell line (U251 TMZ-resistance, U251-TR); and the normal control group was treated with equal volume of physiological saline. Reverse transcription polymerase chain reaction (qPCR) and Wb were used to detect the mRNA and protein expressions of POU3F3 and MGMT (methylguanine DNA methyltransferase) in normal brain tissues and glioma cells. Lentivirus transfection was used to construct U251 cell line with stable POU3F3 interference (U251-TR siPOU3F3); CCK-8 was used to detect TMZ IC50 value (the half maximal inhibitory concentration) in each group of U251 cells, and Wb was used to detect the expression of MGMT protein in each group of cells. Results: Compared with the normal group and primaryonset group, the expression of POU3F3 in recurrence group was significantly increased (P<0.01). The TMZ IC50 of U251-TR cells was significantly higher than that of U251 cells (P<0.01), and The TMZ IC50 of U251-TR siPOU3F3 cells was significantly lower than that of U251-TR cellsbut higher than that of U251 cells (all P<0.01). The protein and mRNA expressions of POU3F3 and MGMT in U251-TR cells were significantly higher than that in U251 cells (P<0.01), while those expressions in U251-TR siPOU3F3 cells were significantly lower than those in U251-TR cells (P<0.01).Conclusion: lncRNAPOU3F3 is the key factor to promote TMZ resistance in human high-grade gliomas cells, which may exert certain guiding significance in the clinical treatment for TMZ resistance.
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Objective@#To investigate the prognostic impact of alterations of epidermal growth factor receptor(EGFR) and MGMT in glioblastoma.@*Methods@#The retrospective study included 161 supratentorial glioblastomas diagnosed in the Department of Pathology, Xuanwu Hospital, Capital Medical University from 2009 to 2015. EGFR and EGFRvⅢ protein expression was detected by immunohistochemistry; EGFR amplification was detected by fluorescence in situ hybridization; MGMT promoter methylation was detected by pyrosequencing. The change of molecular genetics EGFR and MGMT and outcome were assessed statistically.@*Results@#There were 161 patients, including 85 (52.8%) males and 76 (47.2%) females. The mean age was 53 years, and the median overall survival was 13 months. The integrated classification of glioblastoma included 16 IDH-mutant, 134 wild type, and 11 NOS. The rate of overexpression of EGFR protein was 32.9%(53/161), and that of EGFR amplification was 37.5%(18/48). There was high concordance between immunohistochemistry and FISH(85.4%, Kappa=0.475, P<0.01) and between the level of EGFR protein and EGFR amplification (P<0.01). Twelve cases showed EGFRvⅢ expression, and all also showed EGFR protein overexpression; 149 cases were EGFRv Ⅲ wild type, and EGFR protein overexpression was seen in 27.5%(41/149) of cases. There was no correlation between EGFR and EGFRv Ⅲ expression. Of all cases, 70.2%(106/151) showed MGMT promoter methylation by pyrosequencing. The changes of molecular genetics of EGFR and MGMT were not related. EGFR amplification and protein overexpression had no significant relationship with prognosis. Patients with EGFRv Ⅲ-mutant had shorter survival time than the EGFRv Ⅲ-wild type(P=0.014); patients with MGMT promoter methylation had better prognosis than without (PFS:P=0.002,OS:P=0.006),and MGMT promoter methylation was an independent predictor for overall survival (HR=0.269, 95%CI 0.124-0.583, P=0.001).@*Conclusions@#EGFR protein expression by immunohistochemistry correlates with the status of EGFR amplification. Patients with EGFRv Ⅲ-mutant tumors have poorer prognosis than that with EGFRv Ⅲ-wild type tumors. MGMT promoter methylation is closely associated with prognosis and an independent predictor for overall survival.
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OBJECTIVE To explore whether different concentrations of potassium bisperoxo (1,10-phenanthroline) oxovanadate [BPV (phen)) affect cell cycle by regulating the expression of DNA meth-yltranferases (DNMT) and cell cycle-related genes. METHODS HT22 cells were treated with BPV (phen) 0.3 and 3 μ mol • L-1 for 24 h. The cell viability was detected by MTS assay, cell cycle was detected by flow cytometry, the activity of DNMT was detected by ELISA, the mRNA expressions of p21, DNMT1, DNMT3A and DNMT3B were measured with real-time quantitative PCR, while the levels of corresponding proteins were measured by Western blotting. RESULTS Compared with the DMSO control group, BPV(phen) 0.3 μ mol• L-1 did not affect cell viability, but the cell viability of BPV(phen) 3 pmol-L-1 increased signifi-cantly (P<0.05). There was no significant difference in cell cycle between DMSO control and BPV(phen) 0.3 nmol-L-1 group, but the proportion of cells in S phase increased((76.0±1.6)%)(P<0.05) and in G2 phase decreased ((2.1 ±1.5)%) (P<0.05) of BPV(phen) 3.0 μ mol • L-1 group. The intracellular DNMT activity of BPV(phen) 3.0 Mmol • L-1 group was significantly increased compared with the DMSO control group (F<0.05). There was no significant difference in the mRNA expressions of p21, DNMT1, DNMT3A and DNMT3B between DMSO control and BPV(phen) 0.3 μ mol • L-1 group, but all increased in BPV(phen) 3.0 μ mol • L-1 group (P<0.05, P<0.01). Western blotting results showed that there was no significant difference in the protein expressions of P21, DNMT1, DNMT3A and DNMT3B between DMSO control and BPV (phen) 0.3 μ mol • L-1 group, but only the protein expressions of DNMT3B and P21 of BPV (phen) 3.0 μ mol • L-1 group increased significantly (F<0.05). CONCLUSION BPV(phen) can regulate the expression of downstream and cell cycle-related genes by altering the expression of DNMTs, which in turn affects the growth and proliferation of HT22 cells.
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O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation is an important molecular biomarker which plays a key role in tumor development and determine the molecular subtypes and prognosis of gliomas. Radiomics and imaging examinations like MR and PET can obtain information of morphology, function, metabolism and molecular alterations of gliomas, which may help comprehensively and non-invasively predict MGMT promoter methylation status in gliomas. The relationships between imaging features and MGMT promoter methylation status of gliomas were reviewed in this article.
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Objective To investigate the expressions of histone lysine-specific demethylase 1 (LSD1),O6-methylguanine DNA methyltransferase (MGMT) and cell proliferation-associated antigen Ki-67 in high-grade glioma and their influences on prognosis.Methods Sixty-five cases of grade Ⅲ and Ⅳ glioma confirmed by pathology from January 2011 to June 2017 in the First Affiliated Hospital of Xinjiang Medical University were selected.Immunohistochemistry (SP method) was used to detect the expressions of LSD1,MGMT and Ki-67 in pathological specimens.The therapeutic effect was evaluated by long-term follow-up.The relationships between the three markers and pathological grade,progression-free survival (PFS) and overall survival (OS) were analyzed.Results The overall positive rates of LSD1,MGMT and Ki-67 in the 65 high-grade glioma specimens were 70.8％ (46/65),60.0％ (39/65) and 100.0％ (65/65),respectively.There were no significant differences in the expressions of LSD1 and MGMT in grade Ⅲ and Ⅳ glioma (x2 =1.588,P =0.208,x2 =0.013,P=0.908).Ki-67 expression (+),(++),(+++) in grade Ⅳ glioma were observed in 18,19 and 11 cases,respectively.Ki-67 expression (+),(++) in grade Ⅲ glioma were observed in 11,5 cases,and 1 case was (+++),and the difference in expression intensity between the two groups was statistically significant (Z =-2.083,P =0.037).Log-rank test showed that the positive expressions of LSD1,MGMT and Ki-67 were negatively correlated with the PFS of patients with high-grade glioma (x2 =12.217,P =0.007;x2=4.446,P =0.035;x2=12.536,P =0.002),also were negatively correlated with OS (x2 =11.708,P =O.008;x2 =6.637,P =0.010;x2 =11.807,P =0.003).Grade Ⅳ patients were more likely to have relapse progression than grade Ⅲ patients (x2 =6.573,P =0.010),and OS was shorter (x2 =3.974,P=0.046).Cox proportional hazards model analysis showed that the expressions of LSD1 (HR =1.361,95％CI:1.094-1.694,P=0.006;HR=1.406,95％CI:1.117-1.771,P =0.004) and Ki-67 (HR=1.703,95％ CI:1.175-2.468,P =0.005;HR =1.778,95％ CI:1.209-2.616,P =0.003) were the independent prognostic risk factors for PFS and OS of patients with high-grade glioma.Correlation analysis results showed that the expression of MGMT was positively correlated with the expression of LSD1 (r =0.406,P =0.001).Conclusion LSD1,MGMT and Ki-67 have higher positive expression rates in high-grade glioma.MGMT is a prognostic factor for high-grade glioma,and LSD1 and Ki-67 can be used as independent predictors of prognosis for high-grade gliomas.
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Background: Patients with Glioblastoma multiforme (GBM) have a five years survival of less than 5%, but the response to chemotherapy with alkylating agents can vary depending on the methylation status of O6-methylguanine-DNA-methyltransferase (MGMT). Genetic testing has limitations for routine use, while immunohistochemistry (IHC) offers a fast and affordable technique but with heterogeneous results in the literature. Aim: To evaluate MGMT expression by IHC in tumor tissue of Chilean patients with GBM. Material and Methods: Tumor samples of 29 patients with a pathological diagnosis of GBM were studied. We performed IHC staining and manual analysis of positive and negative cells for MGMT expression. A cut-off of at least 10% of cells expressing MGMT was used. Demographic and clinical features of patients were obtained from clinical records. Results: The median number of cells counted per case was 692 (interquartile range [IQR] 492-928). Fifteen cases (52%) were positive for MGMT expression. Median overall survival was 5.3 months (IQR 3.4-12-8). The effect of MGMT expression on the therapeutic response was not studied since only 3 patients received chemotherapy. Conclusions: Our results are similar to international reports, but we were not able to determine the association between MGMT expression and therapeutic response.
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Humans , Male , Female , Adult , Middle Aged , Aged , Brain Neoplasms/enzymology , Biomarkers, Tumor/metabolism , Glioblastoma/enzymology , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Prognosis , Brain Neoplasms/genetics , Immunohistochemistry , Biomarkers, Tumor , Gene Expression Regulation, Neoplastic , Chile , Survival Rate , Retrospective Studies , Glioblastoma/genetics , O(6)-Methylguanine-DNA Methyltransferase/geneticsABSTRACT
Objective:To explore the role ofDNA methyltransferase 1 (DNMT1) in mouse skin aging.Methods:Epidermal conditional K14 Cre-mediated DNA methyltransferase 1 (DNMT1) knockout mice (Mut group,n=4) and the littermate normal mice with the same age (WT group) n=4) were used in this study.HE staining was used to detect the pathological changes of skin;the changes of number in the dermal elastic fibers were detected by Gomori aldehyde fuchsin staining,the number of 5-bromo-2-deoxyuridine (BrdU)-labeled transit amplifying cells (TAC) in epidermis were detected by immunohistochemical staining;the number of chlorodeoxyuridine (CldU)-labelretaining cells (LRC) in epidermis were detected by immunofluorescent staining.Results:Compared with the WT group,the skin showed premature aging symptoms in the Mut group concomitant with the decreased epidermal thickness as well as the number of dermal collagen fibers,while the increased dermal elastic fiber fracture.Compared with the WT group,the number of TAC in the epidermis was significantly increased (P＜0.05),and the number of LRC was significantly decreased (P＜0.05) in the Mut group.Conclusion:The phenotype of skin premature aging in epidermal stem cell conditional DNMT1-knockout mice suggests an important role of DNMT 1 in skin aging.
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Objective To establish a mouse model for short-term exposure to ambient PM and to investigate the impact on the Cytochrome P450 1A1(CYP1A1)and O6-methylguanine-DNA methyltransferase(MGMT)mRNA expression. Methods Twenty 6-week-old BALB/c mice were randomly assigned to one of two groups, each consisting of 5 male and 5 female animals. These mice were then housed in situ concurrently for 2 weeks in our lab located in urban area of Hangzhou. The first group was kept inside an individual ventilated caging(IVC)system equipped with a high-efficiency particulate-air(HEPA)filter, whereas the second was housed inside a IVC with HEPA filter removed. Then it's allowed flow-through of ambient air freely via a pipeline outside. Mice inside the HEPA filtration chamber were therefore protected from exposure to all airborne particulate. The other was in fact exposed to ambient air directly. After the exposure, the bronchoalveolar lavage(BAL)fiuid was collected for each animal and the differentials and percentages of BAL cells were determined. Paraffin sections of lungs of the mice were made and were examined for any inflammation changes. CYP1A1 and MGMT mRNA levels in the lungs were then detected by RT-qPCR. Results The mean concentration of PM2.5was(99.7±51.6)μg/m3in the exposure group. Weight increases were similar between the two groups(P>0.05). The number of total cells and macrophages in BALF from exposure mice was significantly greater than control.A mild inflammation was observed from light photomicrographs of the lung after PM exposure. CYP1A1 and MGMT mRNA levels were significantly up-regulated in the lung from the exposure group. Conclusion A mouse model for short-term exposure to ambient PM was established. CYP1A1 and MGMT may mediate the toxic effect of PM exposure.
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High grade gliomas are always the research focus in the field of neurosurgery due to their poor prognosis despite the current standard therapeutic regimen of surgical resection followed by radiation therapy and chemotherapy. Alkylating agent temozolomide has been established as the standard chemotherapy while its resistance inevitable during treatment. This phenomenon seriously influences the prognosis of patients suffering from high grade gliomas. This review aims to elucidate temozolomide chemoresistance mechanisms through three chapters including O6-methylguanine-DNA methyltransferase (MGMT) methylation, mismatch repair mutation and epigenetic regulation consisting of p21, chromatin and histone, Y-box binding protein-1 and microRNAs.
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Objective • To investigate the effect of mammalian target of rapamycin (mTOR) inhibitor rapamycin on acute myeloid leukemia (AML) with DNA methyltransferase 3A (DNMT3A) R882 mutation in mouse model. Methods • AML cell line OCI-AML3 cells with DNMT3A R882 mutation were cultured with the treatment of rapamycin or DMSO, and then these cells were injected into the tail vein of sublethally irradiated NOD/SCID mice, respectively. The disease progression was monitored by blood routine examination and flow cytometry analysis of CD45+, OCI-AML3 cells, in peripheral blood. Survival time was recorded. Samples from bone marrow, spleen and liver were harvested for flow cytometry analysis and pathological examination. Results • The increasing trend of peripheral leukocytes in the rapamycin treated group was obviously slower than that in the DMSO treated group. The proportion of peripheral blood CD45+ cells in the rapamycin treated group was (4.44±2.58)% (1 week after transplantation) and (34.42±13.64)% (2 weeks after transplantation), which were lower than (16.71±8.96)% and (51.55±5.36)% in the DMSO treated group in the same period, respectively. The median survival time of the rapamycin treated group (27 d) was significantly longer than that of the DMSO group (23 d). The ratios of CD45+ cell infiltration in bone marrow, spleen and liver of the rapamycin treated group were all less than 5%, which were significantly lower than those [(51.32±27.81)% in spleen and (50.03±28.74)% in liver] of the DMSO treated group. Compared with the DMSO treated group, the spleen size of the mice was significantly smaller, and the spleen infiltration and structural damage were significantly alleviated in the rapamycin treated group. Conclusion • Rapamycin shows effective inhibition on the progression of AML with DNMT3A R882 mutation in NOD/SCID mouse model.
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High grade gliomas are always the research focus in the field of neurosurgery due to their poor prognosis despite the current standard therapeutic regimen of surgical resection followed by radiation therapy and chemotherapy.Alkylating agent temozolomide has been established as the standard chemotherapy while its resistance inevitable during treatment.This phenomenon seriously influences the prognosis of patients suffering from high grade gliomas.This review aims to elucidate temozolomide chemoresistance mechanisms through three chapters including O6-methylguanine-DNA methyltransferase (MGMT) methylation,mismatch repair mutation and epigenetic regulation consisting of p21,chromatin and histone,Y-box binding protein-1 and microRNAs.
ABSTRACT
Objective To observe the influences of NaAsO2 on H3K36me3 modifications,mRNA transcription of O6-methylguanine-DNA methyltransferase gene (MGMT) in HaCaT cells,and to explore the relationship between the transcription of MGMT gene regulated by H3K36me3 and DNA damage induced by arsenic,in order to provide new ideas and scientific basis for prevention and intervention of arsenism.Methods HaCaT cells were treated with 1.25,2.50,5.00 and 10.00 μmol/L NaAsO2 for 24 h,and were also treated with 10.00 μmol/L NaAsO2 for 6,12 and 24 h.HaCaT cells that treated with 0.00 pmol/L NaAsO2 and 0 h were used as blank control group.The degree of DNA damage in peripheral blood cells was detected by single cell gel electrophoresis.The level of H3K36me3 modifications was detected using Western blotting.Quantitative real-time polymerase chain reaction was used to detect the mRNA levels of MGMT gene.Quantitative chromatin immuno-precipitation was used to detect the level of H3K36me3 modifications in the coding regions (ChIP1 and ChIP2) of MGMT gene.Results ①Among the groups of HaCaT cells treated with 2.50,5.00 and 10.00 μmol/L NaAsO2,the levels of tail DNA％ (11.83 ± 1.15,16.85 ± 2.52,24.23 ± 2.75) and olive tail moment (OTM,10.90 ± 1.13,16.19 ± 2.26,23.83 ± 2.79)were significantly increased compared with those of the control group (0.00 μmol/L,2.40 ± 0.51,2.26 ± 0.40,all P ＜ 0.05).After treated with 10.00 μmoFL NaAsO2 for 12 and 24 h,compared with the control group (0 h,3.66 ± 1.02,3.38 ± 1.00),the degrees of tail DNA％ (15.51 ± 1.92,24.18 ± 2.42) and OTM (13.58 ± 2.04,23.14 ± 2.11)were significantly increased (all P ＜ 0.05).②Compared with the control group (0.00 μmol/L,100.00 ± 0.00),the levels of H3K36me3 modifications (60.59 ± 9.75,57.82 ± 11.28,39.45 ± 7.09) were lower at the dosages of 2.50,5.00 and 10.00 μmol/L NaAsO2 (all P ＜ 0.05).Compared with the control group (0 h,100.00 ± 0.00),the levels of H3K36me3 modifications (48.47 ± 9.67,47.75 ± 6.98) were lower after treated with 10.00 μ mol/L NaAsO2 for 12 and 24 h (all P ＜ 0.05).③The levels of H3K36me3 modifications in HaCaT cells exposed to different doses of NaAsO2 were negatively associated with the tail DNA％ and OTM (r =-0.897,-0.903,all P ＜ 0.05).④Compared with the control group (0.00 μmol/L,100.00 ± 0.00),the mRNA levels of MGMT gene were lower at the dosages of 2.50,5.00 and 10.00 pmol/L NaAsO2 (78.20 ± 3.50,61.40 ± 2.60,49.15 ± 4.70,all P ＜ 0.05).⑤There was no observed H3K36me3 enrichmem regularity in the gene encoding ChIP1 and ChIP2 regions of MGMT gene in all doses of NaAsO2 groups (all P ＞ 0.05).Conclusions H3K36me3 may be involved in the regulation of arsenicinduced DNA damage in HaCaT cell.Amenic could inhibit the mRNA transcription of MGMT gene in HaCaT cells,but the transcription of MGMT gene regulate by H3K36me3 is not closely related to DNA damage induced by arsenic.