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Abstract Recent studies have found that lncRNA-MEG3(MEG3) plays an important role in the development of EMs (Endometriosis), but the specific mechanism needs to be further explored. This study aimed to investigate the effect of MEG3 on the proliferation, invasion of EMs cells. The authors used RT-qPCR to detect the expression of MEG3 and miR-21-5p in EMs tissues and hESCs cells, MTT and Transwell to detect cell proliferation and invasion, western blotting assay to detect the expression of DNMT3B and Twist, MSP to detect the methylation of Twist. The present study's detection results showed that MEG3 was lowly expressed in EMs tissues and hESCs cells, and overexpression of MEG3 could down-regulate miR-21-5p and inhibit endometrial cell proliferation and invasion. In addition, overexpression of MEG3 upregulated the expression of DNMT3B and promoted the methylation of TWIST. In conclusion, the present findings suggest that MEG3 is downregulated in EMs tissues, and overexpression of MEG3 can promote the activity of DNA methyltransferase DNMT3B by downregulating miR-21-5p, thereby promoting the methylation of Twist, downregulating Twist level to inhibits hESCs cells proliferation and invasion.
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The immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome is the only known human genetic disease involving DNA methylation defects.About 50% of the cases are caused by the compound heterozygous mutation of DNMT3B gene.About a hundred cases were reported in the world, but only a few cases came from China.There may be a misdiagnosis and missed diagnosis.To the best of our knowledge, no Chinese article systematically discusses the ICF syndrome.This paper aims to review the possible mechanisms, clinical manifestations, genetic characteristics, treatment, and prognosis of the ICF syndrome, and to improve Chinese doctors′ knowledge about this disease.
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OBJECTIVE@#To investigate the expression of DNMT3b in human bladder cancer tissues and its correlation with postoperative survival of patients with bladder cancer.@*METHODS@#Thirty-eight pairs of surgically resected human bladder cancer tissues and adjacent bladder tissues were detected by immunohistochemistry for DNMT3b expression, and the correlations of DNMT3b expression level were analyzed with the patients'age, gender, pathological grade, tumor size, T stage, lymph node metastasis and TNM stages. Kaplan-Meier survival analysis was performed to assess the effect of DNMT3b expression on survival outcomes of the patients.@*RESULTS@#High DNMT3b protein expression was detected in 63.16% of the bladder cancer tissues and in 13.16% of the adjacent tissues ( < 0.05). The expression level of DNMT3b was associated with the pathological grade (=0.002), tumor size ( < 0.001), T stage ( < 0.001), lymphatic metastasis (=0.039) and TNM stage ( < 0.001), but not with gender or age of the patients. Multivariate logistic regression analysis showed that the protein expression level of DNMT3b was correlated with tumor size (=0.008) and TNM grades of the tumor (=0.042). Kaplan-Meier analysis showed that the patients with a high DNMT3b expression had a significantly shorter overall survival than those with a low DNMT3b expression (=0.021).@*CONCLUSIONS@#DNMT3b overexpression in bladder cancer is closely related to such clinicopathological factors as pathological grade, tumor size, T stage, lymphatic metastasis, and TNM stage and a shorter overall survival of the patients, suggesting the potential value of DNMT3b as a prognostic marker and a new therapeutic target for bladder cancer.
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OBJECTIVE@#To explore the role of DNMT3B in regulating the proliferation and invasion of hepatocellular carcinoma (HCC) cells.@*METHODS@#We collected the tumor tissues and adjacent tissues from a total of 175 patients with HCC diagnosed in the Second Affiliated Hospital of Chongqing Medical University between May, 2008 and May, 2013 to prepare the tissue microarrays. The association of the expression of DNMT3B with the prognosis and the tumor-free survival and tumor-specific survival rates of the patients was analyzed. Univariate and multivariate Cox regression analyses were used to analyze the effect of DNMT3B expression on the prognosis of HCC. We used RNA interference technique to knock down the expression of DNMT3B in Huh-7 hepatoma cells and observed the changes in cell proliferation using CCK-8 assay and EDU staining and in cell migration and invasion ability using Transwell assay.@*RESULTS@#The positive rates of DNMT3B was significantly higher in HCC tissues than in paired adjacent tissues (67.4% 41.1%, =0.015). A high DNMT3B expression in HCC was significantly associated with the tumor size (=0.001), vascular invasion (=0.004), and intrahepatic metastasis (=0.018). The patients with high DNMT3B expressions had significantly lower tumor-free and tumor-specific survival rates than those with low DNMT3B expressions ( < 0.005). In Huh-7 cells, silencing DNMT3B significantly inhibited the cell proliferation and inhibited cell migration and invasion. Western blotting showed that silencing DNMT3B obviously increased LATS1 expression, decreased the expression of YAP1, and activated Hippo signaling pathway. Methylation-specific PCR showed that the methylation level of LATS1 was decreased in the cells with DNMT3B silencing.@*CONCLUSIONS@#The expression level of DNMT3B is significantly higher HCC tissues than in the adjacent tissues, and the high expression of DNMT3B is closely related to the low survival rate of the patients. Silencing DNMT3B inhibits the proliferation, migration and invasion of HCC cells. DNMT3B promotes the progression of HCC primarily by enhancing the expression of YAP1 through methylation of LATS1 and inhibition of its expression, which inhibits the anti-cancer effect of Hippo signaling pathway.
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Humans , Carcinoma, Hepatocellular , Cell Line, Tumor , Cell Proliferation , Liver Neoplasms , Protein Serine-Threonine Kinases , Signal TransductionABSTRACT
Objective To investigate the association of DNA methyltransfcrase 3B (DNMT3B) promoter region single nucleo tide polymorphism (SNP)-149C＞T and-579G＞T with genetic susceptibility of esophageal cancer (EC) of Han population in Jiangsu Suqian.Methods Gcnotypes of the-149C＞T and 579G＞T locus in DNMT3B promoter region were examinedby polymerase chain reaction with restriction fragment length polymorphism (PCR RFLP) in 246 esophageal cancer patients and 240 healthy controls.Results In-149C＞T site,the distributions of genotypes TT compared with CT in the EC group and the controls were no significant difference (x2 =0.089,P＞0.05).In-579G＞T site,the distributions of genotypes TT compared with GT+GG in the EC group and the controls were no significant difference (x2 =0.649,P＞0.05).When strat ified by age and gender,there was no significant difference in the EC group and the controls in both two sites (x2 =0.044～0.876,all P＞0.05).Conclusion The DNMT3B promoter region-149C＞T and-579G＞T were not associated with the genetic susceptibility of esophageal cancer.
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Objective To investigate the expression of DNA methyltransferase 3b (DNMT3B) in hepatocellular carcinoma (HCC) and its effect and mechanism on the proliferation,invasion and migration of HCC cells.Methods The expression of DNMT3B gene was detected by qRT-PCR in 46 cases of HCC tissues and corresponding adjacent tissues;the results and clinical pathological parameters were analyzed.SiRNA targeting DNMT3B was transfected into MHCC97-H cells by RNA interference (RNAi) technique.The mRNA and protein expression levels of related genes were detected by qRT-PCR and Western blot.The cell proliferation was measured by MTT assay,and the invasion and migration abilities were measured by Transwell assay.Results In 46 HCC patients,the expression of DNMT3B (73.91％) was significantly higher in HCC than in adjacent normal tissue.The high expression of DNMT3B gene was associated with histological type and tumor size of HCC (all P＜0.05).Inhibition of DNMT3B gene expression decreased proliferation,invasion and migration of MHCC97-H cells.Interference with DNMT3B gene increased the expressions of tumor suppressor genes RASSFA1,APC and MTSS1 at mRNA and protein levels.Conclusion DNMT3B is associated with the progression of HCC.It may inhibit the proliferation,invasion and migration of HCC cells by regulating the methylation of downstream tumor suppressor gene.
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Objective To investigate the correlationship between DNMT3a, DNMT3b protein expressions and the state of promoter methylation of ERα gene and ERα protein expression in the development of sporadic breast cancer. Methods A total of 180 patients with sporadic breast cancer and 30 patients with breast fibroadenoma were included in this study. The expressions of DNMT3a and DNMT3b protein were detected by immunohistochemical method. The state of promoter methylation of ERα gene was detected by methylation specific PCR in 97 patients with sporadic breast cancer. Results There were no significant differences in positive expression rates of DNMT3a and DNMT3b protein between breast fibroadenoma and breast cancer. There were higher expression levels of DNMT3a and DNMT3b in breast cancer patient of Ⅲ～Ⅳstages than those of Ⅰ～Ⅱstages. The expression of DNMT3a was significantly higher in patients with lymph node metastasis than that of patients without lymph node metastasis (P＜0.05). Of 97 cases of breast cancer patients, ERα gene promoter methylation occurred in 39 cases (40.2%). The positive expression of DNMT3a protein was positively correlated with the ERα gene methylation (rS=0.250). The DNMT3a protein expression showed a significant influence to the overall survival (OS) in patients of breast cancer (P=0.035), no significant influence to the disease-free survival (DFS) (P=0.064). DNMT3b protein expression showed no significant influence to OS and DFS of patients with breast cancer (P=0.914 and 0.961). Conclusion The positive expressions of DNMT3a and DNMT3b are correlated with the invasion, metastasis and poor prognosis of sporadic breast cancer. DNMT3a was positively correlated with the state of ERα gene promoter methylation. The inhibition of DNMT3a and DNMT3b may have advantages in the prevention and treatment of sporadic breast cancer.
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<p><b>OBJECTIVE</b>To investigate the effect of stable knockdown of DNA methyltransferase 3b (DNMT3b) on the proliferation and apoptosis of bladder cancer cells.</p><p><b>METHODS</b>Lentivirus expressing DNMT3b siRNA or the negative control siRNA was infected in human bladder cancer BIU-87 cells. MTT assay and flow cytometry were used to detect cell proliferation and apoptosis, respectively. The inhibitory effect of DNMT3b knockdown on xenograft tumors in nude mice was observed. Real-time PCR and Western blotting were carried out to investigate the expression level of cell apoptosis related genes. Methylation specific PCR was used to examine the methylation in the promoter region of the cell apoptosis related genes.</p><p><b>RESULTS</b>The results of real-time PCR and Western blotting showed that DNMT3b mRNA and protein level were stably knocked down in BIU-87 cells. Stable DNMT3b knockdown suppressed BIU-87 cell growth and the tumor formation ability of the cells in nude mice. DNMT3b knockdown promoted the apoptosis of BIU-87 cells, increased the mRNA and protein expression of the cell growth and apoptosis related genes including DAPK, Bax and RASSF1A, and significantly decreased the methylation of these genes.</p><p><b>CONCLUSION</b>Stable DNMT3b knockdown can affect the methylation of the cell growth and apoptosis related genes to regulate their expression, which might be a possible mechanism for suppressed cell growth and enhanced apoptosis of BIU-87 cells.</p>
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Animals , Humans , Mice , Apoptosis , Cell Cycle , Cell Line, Tumor , Cell Proliferation , DNA (Cytosine-5-)-Methyltransferases , Genetics , Gene Knockdown Techniques , Mice, Nude , Neoplasm Transplantation , RNA, Small Interfering , Real-Time Polymerase Chain Reaction , Urinary Bladder Neoplasms , Genetics , PathologyABSTRACT
In this study,RNA interference technique was employed to silence the expression of DNMT1 and/or DNMT3b in human bladder cancer T24 cells.The expression levels of their mRNA and protein were greatly decreased by up to 75% and 65% respectively after T24 cells were transfected with lipofectamine2000 for 72 h,indicating RNA interference is an effective tool in gene knockdown.Proliferation and apoptosis of T24 cells were detected by MTT,and annexin-V-FITC and propidium iodide staining flow cytometry,respectively.It was found that loss of the DNMT1 or DNMT3b expression could inhibit the cell growth and promote the cell apoptosis to some extent.However,combined treatment with shRNA targeting both DNMT1 and DNMT3b mRNA could ob-viously enhance the above effects.It was concluded that simultaneously silencing both genes could result in strong suppressing effect on tumor proliferation and promoting ceil apoptosis than separate use,suggesting combined use of DNMT1 and DNMT3b can achieve a synergistic effect in the CpG island methylation in human bladder tumorigenesis.
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Objective To explore the effects of DNMT3b on the expression and methylation sta-tus of the promoter region of DLC-1 in human hepatocellular carcinoma cell line. Methods The SMMC-7721 cell line was divided into 2 groups. The cell line in the experimental group was transfect-ed with DNMT3b siRNA, while that in the control group was transfected with control siRNA. West-ern blot was used to detect the expression of DNMT3b and DLC-1 and MSP was employed to examine the methylation status of the promoter region of DLC-1. Results The expression of DNMT3b was significantly higher in the experimental group than in the control group, while the expression of DLC-1 was just opposite. There was no significant difference in the methylation status of the promoter re-gion of DLC-1 between the 2 groups and both were methylated. Conelnsion The inhibition of expression of DNMT3b by siRNA method can enhance the expression level of DLC-1, and the methylation status of the promoter region of DLC-1 does not change at the same time. When affecting the expression of DLC-1, DN-MT3b might not play the role of methyhransferase, but can act as a transcriptional regulatory factor.
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PURPOSE: DNA methylation is an important epigenetic factor in tumorigenesis. We hypothesized that polymorphism of the promoter of the DNA methyltransferase 3b (DNMT3b) genes, which are responsible for regulating the methylation status of tumor suppressor genes, are associated with increased risk of gastric cancer. MATERIALS AND METHODS: In this hospital-based case-control study, to determine the role of this polymorphism of the promoter of DNA methyltransferase 3b (DNMT3b) genes in gastric cancer, we genotyped 176 cases and 70 control subjects. To determine the genotype, we used a polymerase chain reaction restriction fragment length polymorphism assay. We compared alleles and genotypes between the two groups and revealed an association of DNMT3b promoter polymorphism with increased risk of gastric cancer in the Korean population. RESULTS: Genotype frequencies were 14.8% (Cytosine-Cytosine), 71.6% (Cytosine-Thymine), and 13.6% (Thymine- Thymine) in the case patients and 40.0% (Cytosine-Cytosine), 42.9% (Cytosine-Thymine), and 17.1% (Thymine-Thymine) in the control subjects, respectively. Compared with CC homozygotes, CT heterozygotes had a 4.523-fold increased risk (OR, 2.13; 95% CI, 2.324~8.803), and the TT homozygotes had a 2.154-fold elevated risk (OR, 1.42; 95% CI, 0.899~5.165). For the T variant genotype (CT+TT), there was a 3.846-fold increased risk (OR, 1.88; 95% CI, 2.040~7.251). However, no significance was observed in the genotype distributions of both polymorphisms according to histopathology, stage of stomach cancer. The Ssame results were observed with Helicobacter infection. CONCLUSION: DNMT3b promoter polymorphism, especially the T variant genotype, is associated significantly with thean increased risk of gastric cancer.
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Humans , Alleles , Carcinogenesis , Case-Control Studies , DNA , DNA Methylation , Epigenomics , Genes, Tumor Suppressor , Genotype , Helicobacter Infections , Heterozygote , Homozygote , Methylation , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Stomach NeoplasmsABSTRACT
BACKGROUND: DNA methylation is the main mechanism of epigenetic modification of genes and plays an important role in carcinogenesis. The methylation of promoter can inactivate the tumor suppressor gene by repression of transcription. We investigated the relationship between the 39179G>T (-579bp from exon 1B) polymorphism in DNMT3b gene, which is involved in de novo methylation, and the risk of primary lung cancer in Koreans. METHODS: The DNMT3b 39179G>T genotypes were determined using PCR-RFLP method in 392 primary lung cancer patients and 391 healthy controls who were frequency (1:1) matched based on age and sex. RESULTS: Although the frequencies of GG, GT, TT genotypes among cases (0.8%, 19.9%, 79.3%, respectively) were not significantly different from those among controls (1.5%, 25.1%, 73.4%, respectively), the frequency of wild-type G allele among cases was significantly different from that of controls (14.1% vs 10.7%, p=0.05). The combined GT and GG genotype was associated with a significantly decreased risk of lung cancer [adjusted odds ratio (OR)=0.71, 95% confidence interval (CI)=0.51~1.00, p=0.05] when TT genotype was used as reference. When the lung cancers were categorized by tumor histology, the combined GT and GG genotype was associated with a significantly decreased risk of adenocarcinoma (adjusted OR=0.46, 95% CI=0.26~0.80, p=0.006). In adenocarcinoma, the decreased risk of the combined GT and GG genotype was statistically significant in older patients (>or=61 years, adjusted OR=0.23, 95% CI=0.09~0.58, p=0.002) and in heavier smokers (>or=35 pack years, adjusted OR=0.34, 95% CI=0.13~0.88, p=0.028) in stratification analyses. CONCLUSION: DNMT3b 39179G>T polymorphism may be a genetic determinant of lung cancer, especially adenocarcinoma in Koreans.