UHRF1/DNMT1-MZF1 axis-modulated intragenic site-specific CpGI methylation confers divergent expression and opposing functions of PRSS3 isoforms in lung cancer

As confusion mounts over RNA isoforms involved in phenotypic plasticity, aberrant CpG methylation-mediated disruption of alternative splicing is increasingly recognized as a driver of intratumor heterogeneity (ITH). Protease serine 3 (PRSS3), possessing four splice variants (PRSS3-SVs; PRSS3-V1-V4), is an indispensable trypsin that shows paradoxical effects on cancer development. Here, we found that PRSS3 transcripts and their isoforms were divergently expressed in lung cancer, exhibiting opposing functions and clinical outcomes, namely, oncogenic PRSS3-V1 and PRSS3-V2 versus tumor-suppressive PRSS3-V3, by targeting different downstream genes. We identified an intragenic CpG island (iCpGI) in PRSS3. Hypermethylation of iCpGI was mediated by UHRF1/DNMT1 complex interference with the binding of myeloid zinc finger 1 (MZF1) to regulate PRSS3 transcription. The garlic-derived compound diallyl trisulfide cooperated with 5-aza-2'-deoxycytidine to exert antitumor effects in lung adenocarcinoma cells through site-specific iCpGI demethylation specifically allowing MZF1 to upregulate PRSS3-V3 expression. Epigenetic silencing of PRSS3-V3 via iCpGI methylation (iCpGIm) in BALF and tumor tissues was associated with early clinical progression in patients with lung cancer but not in those with squamous cell carcinoma or inflammatory disease. Thus, UHRF1/DNMT1-MZF1 axis-modulated site-specific iCpGIm regulates divergent expression of PRSS3-SVs, conferring nongenetic functional ITH, with implications for early detection of lung cancer and targeted therapies.

DNMT1-mediated PPARα methylation aggravates damage of retinal tissues in diabetic retinopathy mice

BACKGROUND: Peroxisome proliferator-activated receptor alpha (PPARα) is associated with diabetic retinopathy (DR), and the underlying mechanism is still unclear. Aim of this work was to investigate the mechanism of PPARα in DR. METHODS: Human retinal capillary pericytes (HRCPs) were treated with high glucose (HG) to induce DR cell model. DR mouse model was established by streptozotocin injection, and then received 5-Aza-2-deoxycytidine (DAC; DNA methyltransferase inhibitor) treatment. Hematoxylin-eosin staining was performed to assess retinal tissue damage. PPARα methylation was examined by Methylation-Specific PCR. Flow cytometry and DCFH-DA fluorescent probe was used to estimate apoptosis and reactive oxygen species (ROS). The interaction between DNA methyltransferase-1 (DNMT1) and PPARα promoter was examined by Chromatin Immunoprecipitation. Quantitative real-time PCR and western blot were performed to assess gene and protein expression. RESULTS: HG treatment enhanced the methylation levels of PPARα, and repressed PPARα expression in HRCPs. The levels of apoptotic cells and ROS were significantly increased in HRCPs in the presence of HG. Moreover, DNMT1 was highly expressed in HG-treated HRCPs, and DNMT1 interacted with PPARα promoter. PPARα overexpression suppressed apoptosis and ROS levels of HRCPs, which was rescued by DNMT1 up-regulation. In DR mice, DAC treatment inhibited PPARα methylation and reduced damage of retinal tissues. CONCLUSION: DNMT1-mediated PPARα methylation promotes apoptosis and ROS levels of HRCPs and aggravates damage of retinal tissues in DR mice. Thus, this study may highlight novel insights into DR pathogenesis.

DNMT1 protein promotes retinoblastoma proliferation by silencing MEG3 gene / 南方医科大学学报

OBJECTIVE@#To investigate whether DNMT1 protein induces retinoblastoma proliferation by silencing MEG3 gene.@*METHODS@#Two retinoblastoma cell lines (HXO-RB44 and SO-RB50) and a normal human retinal pigment epithelial (RPE) cell line were transfected with the plasmid pcDNA-DNMT1 or si-DNMT1 for up-regulating or interference of DNMT1 expression, and with pcDNA-MEG3 or si-MEG3 for up-regulating or interference of MEG3 expression. Western blotting was used to detect the changes in the expression of DNMT1 protein in the transfected cells, and CCK-8 and EdU assays were used to detect the changes in cell proliferation. Real-time quantitative PCR (qRT-PCR) was performed to detect MEG3 expression in SO-RB50 and HXO-RB44 cells after transfection, and the methylation level of MEG3 gene promoter after interference of DNMT1 expression was detected using methylation-specific PCR.@*RESULTS@#SO-RB50 and HXO-RB44 cells showed significantly increased expression of DNMT1 protein as compared with normal RPE cells ( < 0.05). In HXO-RB44 cells, transfection with pcDNADNMT1 resulted in significantly increased expression of DNMT1 protein, enhanced cell proliferation ability, and significantly reduced expression of MEG3 ( < 0.05). In SO-RB50 cells, transfection with si-DNMT1 significantly reduced the expression of DNMT1 protein, suppressed the cell proliferation, and increased MEG3 expression ( < 0.05). Interference of DNMT1 significantly reduced the methylation level of MEG3 gene promoter. After reversing the regulatory effect of DNMT1 on MEG3 gene, DNMT1 protein showed significantly weakened ability to regulate retinoblastoma cell proliferation ( < 0.05).@*CONCLUSIONS@#In retinoblastoma cells, the up-regulation of DNMT1 protein induces promoter methylation and inactivation of MEG3 gene and eventually leads to abnormal cell proliferation.

Phenotypic diversity of hereditary sensory and autonomic neuropathy type IE: a case series and review of the literature

@#Objective: DNA methyltransferase 1 (DNMT1) is crucial to maintaining methylation during DNA replication and DNA repair. DNMT1 mutations have been identified in two neurological syndromes, including hereditary sensory and autonomic neuropathy type IE (HSAN IE) with dementia and hearing loss and autosomal dominant cerebellar ataxia, deafness and narcolepsy. It is likely that DNMT1 mutations lead to various symptoms of the central and peripheral nervous system. The aim of this study was to examine the clinical characteristics, especially the initial symptoms, in the cases of DNMT1 mutations. Methods: We investigated the clinical manifestation and examination findings of four cases of HSAN IE from one family with the DNMT1 mutation c.1531Y>C (p.Try511His). Results: All four cases exhibited sensory neuropathy, cerebellar ataxia, and hearing loss, all of which were demonstrated by the audiograms. The initial symptoms of the four cases included hearing loss (n=1), gait disturbance (n=1), and depressive mood (n=2). Depressive symptoms are reported in some cases with HSAN IE, however, there are currently no published reports that describe them as primary symptoms. The CSF orexin level was measured in three cases, revealing normal values in two cases and intermediate values in one case, in which the patient exhibited rapid eye movement (REM) sleep behavior disorder. Conclusion: Our findings suggest that in cases with HSAN IE or the DNMT1 mutation, psychiatric symptoms should be taken into account as one of the initial manifestations of the disease.

Inhibitory effect of polyphyllin Ⅰ on the proliferation of prostate cancer PC3 cells via ERK1/2/P65/DNMT1 and its molecular mechanism / 中华男科学杂志

<p><b>Objective</b>To explore the inhibitory effect of polyphyllin Ⅰ (PPⅠ) on the proliferation of castration-resistant prostate cancer PC3 cells and its molecular mechanism.</p><p><b>METHODS</b>We cultured human prostate cancer PC3 cells in vitro and treated them with PPⅠ at the concentrations of 0 (blank group), 0.4, 0.8, 1.2, 1.6, 2.0, and 2.4 μmol/L for 24, 48, and 72 hours, respectively. Then we detected the proliferation of the cells by MTT assay, measured their apoptosis by flow cytometry, and determined the expressions of p-ERK1/2, ERK1/2, NF-κB/p65 and DNMT1 proteins as well as the level of NF-κB/p65 in the cells additionally treated with the ERK1/2 inhibitor SP600125 by Western blot.</p><p><b>RESULTS</b>Compared with the blank control group, the PPⅠ-treated PC3 cells showed a concentration- and time-dependent reduction of the survival rate (1.00 ± 0.00 vs 0.85 ± 0.05, P < 0.01) at 0.4 μmol/L after 48 hours of intervention, concentration-dependent early apoptosis at 0.8 μmol/L (4.83 ± 0.95 vs 13.83 ± 2.97, P < 0.01), time-dependent increase of the expressions of p-ERK1/2 (1.00 ± 0.00 vs 1.73 ± 0.17, P < 0.01) and ERK1/2 (1.00 ± 0.00 vs 1.36 ± 0.12, P < 0.01) at 2 hours, and concentration-dependent decrease of the expressions of NF-κB/p65 and DNMT1 at 1.2 μmol/L (1.00 ± 0.00 vs 0.78 ± 0.10 and 0.63 ± 0.06, P < 0.01) and 1.6 μmol/L (1.00 ± 0.00 vs 0.67 ± 0.11 and 0.52 ± 0.09, P<0.01). Inhibition of ERK1/2 phosphorylation with PD98059 markedly reversed PPⅠ-induced decrease of the NF-κB/p65 expression as compared with that in the PPⅠ group (0.86 ± 0.18 vs 0.43 ± 0.09, P < 0.05).</p><p><b>CONCLUSIONS</b>PPⅠ induces the early apoptosis and suppresses the proliferation of PC3 cells, probably by activating the ERK1/2 pathway and inhibiting the expressions of the NF-κB/p65 and DNMT1 proteins.</p>

A novel mutation in the DNMT1 gene in a patient presenting with pure cerebellar ataxia

Mutations in the DNA methyltransferase 1 gene (DNMT1) were reported to cause two phenotypes: OMIM 604121 and OMIM 614116. The first phenotype includes autosomal dominant cerebellar ataxia, deafness, and narcolepsy, which were reported to be caused by mutations in exon 21. The second phenotype includes hereditary sensory and autonomic neuropathy type 1E, which was suggested to be caused by mutations in exon 20 and 21. In this article, we report a novel heterozygous missense variant c.898A>C, p.(Lys300Gln) in exon 12 of DNMT1 in a young woman who presented with pure cerebellar ataxia. This report indicates that a mutation in exon 12 may lead to pure cerebellar ataxia. Another possibility is that the patient is currently in an early stage of the disease, and as the disease progresses, she will have more manifestations. To confirm or exclude this possibility, a subsequent follow-up study reporting the disease progression in this patient may be needed. Further reports of cases with the same mutation are needed to confirm the phenotype of this mutation.

Study on miR-148a regulation function of cardiac differentiation on MSCs by targeting DNMT1 / 中国免疫学杂志

Objective:To verify DNA methyltransferase 1(DNMT1) is the direct target of miR-148a and explore the internal mechanism by which miR-148a regulates the cardiac differentiation of mesenchymal stem cells(MSCs)by directly targeting DNMT1.Methods:miRNA microarray was used to screen out the abnormally expressed miRNAs in MSCs before and after 5′-azacytidine(5′-aza) treatment.Target scan was uesd to predict the target of miR-148a.miR-148a mimics and DNMT1-wt,scramble and DNMT1-wt,miR-148a and DNMT1-mut,scramble and DNMT1-mut were co-transfected in MSCs cells and luciferase activity were detected by single photon.MSCs cells were transfected with miR-148a lentivirus plasmid.Respectively extract DNA,RNA and protein 1,7,14 and 28 days after transfection.The CpG methylation level on DNA regulatory sequences of Gata-4 upstream gene was detected by methylation detection,the mRNA and protein expressions of myosin heavy chain(MHC),cardiac troponin T(cTnT),CD90,CD29,Nkx2.5 and Gata-4 were detected by qRT-PCR and Western blot.Results:miRNA Microarray screened out the abnormally expressed miRNAs in MSCs before and after 5′-aza-induced,including miR-146a-5p,miR-148a,miR-539,etc.Among which miR-148a was remarkably upregulated.Targetscan,Luciferase Reporter Gene and qRT-PCR verified that DNMT1 was the direct target of miR-148a.By infected MSCs cells with miR-148a lentivirus plasmid,we confirmed that the methylation level of Gata-4 gene upstream was changed,and with prolong of transfection,the methylation level of Gata-4 was decreased,the expression of MHC and cTnT were increased,while CD90 and CD2 were continually decreased,the mRNA expression of Nkx2.5 and Gata-4 were increased MSCs cells started myocardial differentiation.Conclusion:miR-148a can regulate the cardiac differentiation of MSCs by directly targeting DNMT1.

Relationship between Methylation of MGMT Gene and Protein Expression of DNMT1 in Gastric Cancer / 胃肠病学

Background:Studies have shown that promoter methylation of MGMT gene is closely related to many malignant tumor including gastric cancer.DNA methyltransferase 1 (DNMT1) is highly expressed in many malignant tumor tissues.However, studies on relationship between methylation of MGMT gene and DNMT1 expression in gastric cancer are rare.Aims:To investigate the relationship between methylation of MGMT gene, protein expression of DNMT1 and gastric cancer.Methods:Promoter methylation status of MGMT gene in 60 gastric adenocarcinoma tissues and corresponding paracancerous tissues were detected by methylation-specific PCR (MSP).RT-PCR and immunohistochemistry were used to measure mRNA and protein expressions of MGMT and DNMT1, respectively.Results:Methylation rate of MGMT gene promoter in gastric cancer was significantly higher than that in paracancerous tissue (45.0% vs.13.3%, P<0.001).The positivity rate of MGMT mRNA in gastric cancer was significantly lower than that in paracancerous tissue (41.7% vs.93.3%, P<0.001), while the positivity rate of DNMT1 mRNA expression in gastric cancer was significantly higher than that in paracancerous tissue (76.7% vs.18.3%, P<0.001).Methylation rate of MGMT promoter in MGMT mRNA-negative expressed gastric cancer tissue was significantly higher than that in MGMT mRNA-positive expressed gastric cancer tissue (57.1% vs.28.0%, P<0.05).It showed a negative correlation between MGMT protein expression and DNMT1 protein expression in gastric cancer tissue (r=-0.795, P<0.01).Conclusions:Promoter methylation of MGMT gene and high expression of DNMT1 may be associated with the development and progression of gastric cancer.

Characterization of the Relationship of CDKL5 with MeCP2 and Dnmt1 in PrimaryRat Cortical Neurons

ABSTRACT Cyclin-dependent kinase-like 5 (CDKL5) is a protein kinase that is homologous to mitogen-activated protein kinases (MAPKs) and cyclin-dependent kinases (CDKs). Mutations in the CDKL5 gene cause X-linked infantile spasms and phenotypes that overlap with that of Rett syndrome, which is a neurodevelopmental disorder caused primarily by mutations in the methyl CpG binding protein 2 gene (MECP2). Previous studies in transfected cell lines showed that CDKL5 interacts with MeCP2 and DNA (cytosine-5)-methyltransferase 1 (Dnmt1). However, little is known about the relationships of CDKL5 with interacting proteins in primary neuronal cultures. In this study, we investigated the expression patterns of CDKL5, MeCP2 and Dnmt1, and their interaction in cultured rat cortical neurons. Using real-time PCR analysis, we found that CDKL5, MeCP2 and Dnmt1 have similar expression patterns at the mRNA level. In contrast, the expression patterns of those proteins at the protein level are different and could be inversely correlated, as shown by western blotting. Using co-immunoprecipitation, we further demonstrated that CDKL5 interacts with MeCP2 and Dnmt1 in primary rat cortical neurons. These data suggest that a functional link exists among CDKL5, MeCP2 and Dnmt1 during neuronal development and may provide further insight into the pathogenesis of Rett syndrome.

Effect of Silencing DNMT1 gene on histone methylation modulation, cell proliferation and apoptosis in Molt-4 cell line / 中国药理学通报

Aim To investigate the effect of small in-terfering RNA(siRNA) targeting DNMT1 gene on cell proliferation, apoptosis and histone modulation in acute lymphoid leukemia cell line, Molt-4. Methods The small interfering RNA targeting DNMT1 gene was transfected into Molt-4 cells by LipofectamineTM 2000. The DNMT1 mRNA and protein level were detected by RT-PCR and Western blot. Cell proliferation was de-termined by MTT. Cell apoptosis was measured by Flow Cytometry. The expression of Bcl-2, procaspase-3, P15, histone methylation and histone acetylation was detected by Western blot. Results DNMT1 was suppressed by siRNA targeting DNMT1 in a concentra-tion-dependent manner. DNMT1 siRNA suppressed cells proliferation and induced apoptosis in Molt-4 cells. Apoptotic rate was (4. 27 ± 1. 42)% , (15. 25 ± 1. 54)% , (35. 63 ± 2. 54)% , (66. 27 ± 3. 02)%after transfecting with DNMT1 siRNA at 0, 30, 60, 120 nmol·L - 1 for 24 hours, P < 0. 05. The expres-sion of Bcl-2, procaspase-3 was suppressed and P15 was promoted after transfecting of DNMT1 siRNA. DN-MT1 siRNA downregulated histone methylated H3K9 and upregulated histone methylated H3K4. The altera-tion of histone acetylation of H3 was not seen. Conclu-sion DNMT1 siRNA suppresses DNMT1 efficiently in Molt-4 cells. The depletion of DNMT1 downregulates histone methylation of H3K9, and upregulates histone methylation of H3K4. It inhibits cell growth and in-duces cell apoptosis in Molt-4 cell line.

Effects of DNMT1-siRNA on the pain behavior and the expression of SOCS1,p-ERK,p-CERB in CCI rats / 临床麻醉学杂志

Objective To investigate the effects of DNMT1 on neuropathic pain behavior and neuropathic pain modulation.Methods Thirty-two male SD rats, weighing 200-250 g, were randomly assigned into 4 groups (n =8 each):sham operation group (group S),chronic constrictive injury group (group CCI),CCI+ DNMT1-siRNA group (group CDS),CCI+ control-siRNA group (group CCS).Group CDS were intrathcally injected of DNMT1-siRNA (2 μg/10 μl),and group CCS were intrathcally injected of control-siRNA 7,8,9 days after operation.Mechanical withdrawal threshold (MWT)and thermal withdrawal latency (TWL)were measured before operation and on day 3,5,7,9,12,14 after operation.The rats were then sacrificed and L4-L6 segments of the spinal cord were removed for determination of SOCS1,p-ERK,p-CREB expression using Western blot on day 14.Results Compared with group S,MWT and TWL in group CCI and CCS were significantly decreased on day 3,5,7,9,12,14 after operation (P <0.05).Compared with group CCS,MWT and TWL in group CDS were significantly increased on day 9,12,14 after operation (P < 0.05 ). Compared with group S and CDS,SOCS1 was significantly downregulated,p-ERK and p-CREB were significantly upregulated in group CCI and CCS (P <0.05 ).Conclusion Intrathcal injection of DN-MT1-siRNA significantly relieves neuropathic pain by upregulating SOCS1,downregulating p-ERK and p-CREB in rats spinal cords.

Research progress and prognostic significance of EZH2 in acute myeloid leukemia / 中国肿瘤临床

Objective:To investigate the relationship between DNA methyltransferase (DNMT1) and EZH2 gene expression levels and their clinical significance in patients with acute myeloid leukemia (AML). Methods:The mRNA expression levels of DNMT1 and EZH2 in 50 AML cases and 30 healthy controls were quantified through real-time PCR. The relationship among DNA methyltransferase (DNMT1) and EZH2, clinicopathological factors, and prognosis was analyzed. Results:The mRNA expression of DNMT1 in the AML cases (2.72 ± 0.73) was significantly higher than that in the healthy controls (0.89 ± 0.27) (P<0.01). The mRNA expression of EZH2 in the AML cases (4.39±1.06) was also significantly higher than that in the healthy controls (1.87±0.33) (P<0.01). The expression of DNMT1 was positive-ly associated with that of EZH2 (r=0.51, P=0.002). The expression of DNMT1 was also associated with PB%and WBC≥50 × 109/L (P<0.05). The overall survival of the group with a high-mRNA-expressing DNMT1 was 15 months (95%CI=9-19 months). This period was significantly shorter than that of the group with low-mRNA-expressing DNMT1 (32 months, 95%CI=27-40 months;P=0.006). Conclu-sion:DNMT1 and EZH2 expression levels were downregulated. These levels were associated with poor AML prognosis. The expression of DNMT1 was also positively associated with that of EZH2.

Epstein - Barr virus-associated lymphoepithelioma-like gastric carcinoma located on gastric high body: Two case reports.

Lymphoepithelioma-like gastric carcinoma (LELGC) is a rare neoplasm of the stomach with an incidence of 1-4% of all gastric cancers. It is characterized by the presence of a lymphoid stroma with cells arranged primarily in micro alveolar, thin trabecular, and primitive tubular patterns or isolated cells. It is one of the histological patterns observed in patients with Epstein — Barr virus (EBV)-associated gastric carcinoma (EBVaGC). In situ hybridization was usually used to confirm the presence of EBV. There are two types of EBVaGC, LELGC, and ordinary type. Approximately, 15-25% of EBVaGC exhibit the LELGC pattern. Here, we described two cases of LELGC and the related literatures were reviewed as well. The two cases were submucosal mass from a 59- or 63-year-old man. We found LELGC has special clinicopathologic features and protein expression profile. This should promote us to make a true diagnosis.

Effect of folate in modulating the expression of DNA methyltransferase 1 and methyl-CpG-bingding protein 2 in cervical cancer cell lines / 中华流行病学杂志

Objective To explore the effects of folate on the expression of DNA methyltransferase 1 (DNMT1) and methyl-CpG-bingding protein 2 (MeCP2) in cervical cancer cell lines.Methods Experimental study was carried out in vitro.Human cervical cancer cell lines,including C33A cell with HPV negative and Caski cell with HPV16 positive,were treated with different concentration of folate.The expression of DNMT1 and MeCP2 protein (by Western blot)and mRNA (by real-time PCR) were then detected in the two cell lines.Results It was found that supplement of folate was able to reduce the cell proliferation in C33A cell (r=0.984,P＜0.001) and Caski cell (r=0.978,P=0.002),as well as induced the cell apoptosis (C33A:r=0.989,P＜0.001 ;Caski:r=0.994,P＜0.001).Results showed that the expression levels of DNMT1 protein (C33A:r=-0.914,P＜ 0.001 ; Caski:r=-0.859,P=0.003) and MeCP2 protein (C33A:r=-0.830,P=0.005 ;Caski:r=-0.981,P＜0.001) decreased gradually with the increase of folate concentrations,but the expression of DNMT1 and MeCP2 mRNA was not observed in Caski or C33A cell.When at the same levels of folate,the expression of DNMT1 protein or mRNA was higher in Caski cell than in C33A cell.However,the expression of MeCP2 protein or mRNA was higher in C33A cell than in Caski cell.Conclusion Our fimding indicated that adequate foleta could effectively inhibit the proliferation of cervical cancer cells and facilitate their apoptosis in vitro,thus would reverse the aberration protein expression of DNMTl and MeCP2.That there might be a synergistic action between HPV16 infection and parafunction of DNMT l in cervical cancer,being noticed.

Genome-wide DNA methylation changes induced by low dose radiation in mouse / 中华放射医学与防护杂志

Objective To study the whole genome DNA methylation changes induced by low dose radiation (LDR) in mouse,and mRNA expression profiles of DNMT1 and MBD2 in peripheral blood mononuclear cell (PBMC) and tissues.Methods Thirty male BALB/c mice were randomly divided into 3 groups:control,single exposure (0.5 Gy),and fractionated exposure of 6 MV X-rays for 10 d (0.05 Gy/d × 10 d).Control mice were sham-treated.To determine the immediate (early) effect of irradiation,15 mice (5/group) were sacrificed 2 h after the last irradiation.The other 15 mice were sacrificed 1 month after the last irradiation (delayed effect).Before sacrifice,blood was sampled immediately.Kidney,liver,spleen,brain and lung tissues were collected.A global DNA methylation quantification Kit and highperformance liquid chromatography (HPLC) were used to investigate the methylation level in blood DNA.The expressions of DNMT1 and MBD2 were determined by RT-PCR.Results For the early effects of irradiation,as compared with controls,fractionated exposure to X-ray irradiation led to the significant depression of global DNA methylation level in blood (t =10.19 and 8.93,P ＜ 0.05).DNMT1 and MBD2 mRNA were down-regulated in PBMC,kidney and liver (t =5.06,3.01,3.97,12.25,3.50 and 3.73,P ＜0.05),and MBD2 was also down-regulated in spleen (t =3.03,P ＜ 0.05).However,no changes were observed in single exposed group.As for the delayed effects,the methylation levels of blood were not changed in the single or fractionated exposed groups,and only MBD2 mRNA was down-regulated in PBMC and brain of fractionated exposed group (t =3.52 and 2.85,P ＜ 0.05).Conclusions Fractionated LDR exposure can induce genome DNA hypomethylation,which is tissue-specific,and may be related with down-regulation of DNMT1 and MBD2.

Effects of DNA methyhransferase 1 gene silencing on DNA methylation of pancreatic cancer / 中华胰腺病杂志

ObjectiveTo assess the effects of DNA methyhransferase 1 ( DNMT1 ) gene silencing on DNMTs activity and methylated CpG sites of hMLH-1 in pancreatic cancer cell line PaTu8988.Methods DNMT1 siRNA and negative control siRNA was constructed by Ambion Company of United States.Then they were transfected into pancreatic cancer cell line PaTu8988 at the concentrations of 15,30 nmol/L,and the cells without transfection was used as the control group.Real-time PCR and Western blotting were applied to detect the DNMT1 mRNA and protein expression,and DNMTs activity was detected by using DNMTs activity assay kit.Change of methylation of CpG island of hMLH-1 was detected by bisulfite sequencing PCR (BSP).The expression of hMLH-1 mRNA was detected by Real-time PCR.ResultsAt 48 h after transfection,Realtime RT-PCR analysis showed that the levels of DNMT1 mRNA in DNMT1 siRNA group ( 15 nmol/L) and DNMT 1 siRNA group (30 nmol/L) were 0.573 ± 0.026 and 0.143 ± 0.044,which were significantly lower than those in control group 1.020 ±0.217 and negative siRNA 15 nmol/L group 0.900 ±0.475,and negative siRNA 30 nmol/L group 0.938 ± 0.327 (P ＜0.05 ).Western blotting analysis showed that the level of DNMT1 protein of DNMT1 siRNA group was also lower than those of negative siRNA and control groups.DNMT activity in DNMT1 siRNA15,30 nmol/L groups was 0.364 ± 0.124and 0.250 ± 0.072,which were significantly lower than those in control group 0.931 ± 0.065and negative siRNA group 0.665 ± 0.055 and 0.472 ± 0.040.DNMT activity was positively correlated with DNMT1 mRNA expression ( r =0.69,P ＜ 0.01 ).DNMT1 RNA interference decreased 8 methylated CpG sites of hMLH-1 to 1 site.Concluslons DNMT1siRNA can specifically inhibit the expression of DNMT1 gene of PaTu8988 and DNMT activity,and can decrease methylated CpG sites of hMLH-1 gene.

Expressions and clinical significance of DNA methyltransferase 1 in pancreatic carcinoma / 中华胰腺病杂志

Objective To investigate the expressions of DNA methyltransferase 1 (DNMT1) in pancreatic carcinoma and its clinical significance.Methods 30 samples of pancreatic cancer tissues and paired para-cancerous tissues were collected from patients who underwent curative pancreatectomy.The levels of DNMT1 mRNA were detected by real-time RT-PCR.Expressions of DNMT1 protein were detected by streptavidin peroxidase immunohistochemistry.The relationships between expression of DNMT1 and clinicopathological findings were analyzed.Results The value of relative quantification (RQ) of DNMT1 mRNA in human pancreatic cancer tissues was 2.32 (1.17 ～ 5.17 ), which was significant higher than 0.78 (0.07 ～3.14) in para-cancerous tissues(P ＜0.05).The index of expression of DNMT1 protein in human pancreatic cancer tissues was (54.5 ±21.2)% ,which was significant higher than( 10.9 ± 15.0)% in paracancerous tissues (P ＜ 0.01 ).Patients were divided into the high DNMT1 group (n = 19) with above 54.5% of the DNMT1 positively cancer cells and the low DNMT1 group ( n = 11 ) with less than 54.5% of the DNMT1 positively cancer cells.The high expression of DNMT1 was correlated significantly with clinical staging ( x2 =6.897, P = 0.029), lymph node metastasis ( x2 = 4.739, P = 0.029) and neural invasion ( x2 = 5.44, P =0.020).On the other hand, no association between DNMT1 expression and age, gender, tumor location,tumor size, tumor differentiation, the serum CEA and CA19-9 levels could be found.Conclusions DNMT1 mRNA and protein was highly expressed in pancreatic cancer tissues.High expression of DNMT1 might be related to the aggressiveness of pancreatic cancer, lymph node metastasis and neural invasion.

Effects on Biological Behavior of Bladder Carcinoma T24 Cells via Silencing DNMT1 and/or DNMT3b with shRNA In Vitro / 华中科技大学学报（医学）（英德文版）

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.

RNA interfering on the cell cycle of gastric cancer by the recombinantplasmid affecting DNMT1 gene expression / 重庆医科大学学报

Objective:To clone the recombinant plasmid affecting gene DNMT1 expression by RNA interfering and transfect it into AGS in order to further search its effects on the cell cycle of gastric cancer AGS.Methods:Two DNA sequences containing short hairpin structure was designed and synthesized.The complement form was obtained by annealing and inserted into vector pTZU6+1.The recombinant plasmid was transfected into AGS.Finally the cell cycle was evaluated through flow cytometer.Results:The S cycle cells of the transfected AGS dropped dramatically.And the G2-M cycle cells increased significantly.Conclusion:Successful cloning and transfecting the recombinant make it possible to search new gene therapy for the tumors.

Demethylation of progesterone receptor in leukemia cell line induced by DNMT1 antisense oligonucleotide wrapped in liposome / 第三军医大学学报

Objective To investigate the effects of the transfection of DNMT1 antisense oligonucleotide wrapped in liposome on the promotor methylation and expression of progesterone receptor in leukemia cell lines so as to find a new pathway of methylation for gene therapy. Methods Artificially synthesized DNMT1 antisense oligonucleotide MG88 wrapped in liposome was transfected into the leukemia cell line. Changes of DNMT1 & PRB mRNA were detected by RT PCR, and the changes of the methylation status of PRA & PRB were analyzed by MSP. Results After transfection of leukemia cell line with MG88, DNMT1 mRNA decreased significantly. PRA & PRB promotors were demethylated, and the expressions of PRB mRNA increased significantly. Conclusion MG88 can induce the demethylation of PR promoters in leukemia cell line, resulting in the increase of PRB mRNA expression. So MG88 can be used in the experimental study of DNA methylation for gene therapy.