Expression and Significance of DNA Methyltransferases in Gastric Cancer / 胃肠病学

Background: DNA methylation plays an important role in the development of gastric cancer, but it needs the modification with DNA methyltransferases (DNMTs). Aims: To investigate the expression and clinical significance of DNMTs in the occurrence and development of gastric cancer. Methods: A total of 80 cases of gastric cancer tissues and corresponding adjacent normal tissues were collected. Immunohistochemistry was used to detect the expressions of DNMT1, DNMT3a, DNMT3b, and their correlations with clinicopathological features of gastric cancer were analyzed. mRNA and protein expressions of DNMT1, DNMT3a, DNMT3b in 4 gastric cancer cell lines and human gastric epithelial cell line were determined by qRT-PCR and Western blotting, respectively. Results: Compared with adjacent normal tissue, the positive expression rate of DNMT1 was significantly increased in gastric cancer (68.8% vs. 10.0%, P<0.01). The positive expression of DNMT1 in gastric cancer tissue was significantly higher than that of DNMT3a and DNMT3b (68.8% vs. 38.8%, 40.0%, P<0.05), while the positive expression of DNMT1 in adjacent tissue was significantly lower than that of DNMT3a and DNMT3b (10.0% vs. 60.0%, 52.5%, P<0.05). The positive expression of DNMT1 was correlated with depth of invasion, lymph node metastasis and TNM stage in patients with gastric cancer (P<0.05), while the positive expression of DNMT3a, DNMT3b were not correlated with clinicopathological features of gastric cancer. The expression of DNMT1 in gastric cancer cells was significantly higher than that in normal gastric epithelial cells, while the expressions of DNMT3a and DNMT3b were significantly decreased. Moreover, the expression of DNMT1 was related to the degree of differentiation of gastric cancer cells (P<0.05). Conclusions: DNMT1 maybe play an important role in the occurrence and development of gastric cancer.

Analysis of the correlation of MGMT and XRCC1 gene expression with the pathogenesis of glioma and its mechanism analysis / 中国医师杂志

Objective To investigate the relationship between the expression of 06-methyl guanine DNA methyltransferase (MGMT),X-ray repair cross complementation gene 1 (XRCC1) and the incidence of glioma.Methods From February 2015 to September 2017,53 glioma patients (glioma group) in our hospital were enrolled in the study.50 patients with hypertensive intracerebral hemorrhage were selected as control A group,and 106 healthy volunteers as control B group.Immunohistochemical staining was used to detect the expression of MGMT and XRCC1 in brain tissue of glioma group and control A group,and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) were used to detect the polymorphism of MGMT and XRCC1 gene in glioma group and control B group.Results The positive expression rates of MGMT and XRCC1 in the tissues of brain glioma were 47.17％ and 39.62％,respectively,which were significantly higher than those in the control A group (P ＜ 0.05).There was no significant difference in the positive expression rate of MGMT and XRCC1 in patients with grade Ⅰ,,Ⅲ and Ⅳ (P ＞ 0.05);There was no correlation between the expression of MGMT and XRCC1 in glioma tissues (rs =0.162,P ＞ 0.05);The proportion of XRCC1 genotype AG + GG in brain glioma group was 58.49％,which was significantly higher than that of control B group (P ＜ 0.05);The proportion of MGMT genotype LP + PP in brain glioma group was 28.30％,which was significantly higher than that of control B group (P ＜ 0.05).Conclusions MGMT and XRCC1 are increased significantly in glioma brain tissues,but not correlatedwith pathological grades;The polymorphism of MGMT and XRCC1 genes may be related to the susceptibilityof gliomas.

mRNA expression of DNA methyltransferase 2 and 3a in the epidermis of patients with psoriasis vulgaris / 中华皮肤科杂志

Objective To investigate the expression of DNA methyltransferase 2 (DNMT2) and 3a (DNMT3a) in the epidermis of patients with psoriasis vulgaris.Methods Between March 2009 and December 2010,46 patients with psoriasis vulgaris were enrolled from the Department of Dermatology,Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College,and the Department of Dermatology of Yixing People's Hospital,and 28 healthy controls were enrolled from the Department of Dermatologic Surgery,Hospital for Skin Diseases,Chinese Academy of Medical Sciences and Peking Union Medical College.Real-time quantitative PCR was performed to determine the mRNA expression of DNMT2 and DNMT3a in the epidermis of the lesional and nonlesional skin of patients with psoriasis vulgaris,as well as in the epidermis of normal skin of the healthy controls.Results The mRNA expression of DNMT2 (expressed as 2-△△Ct) in the lesional skin,non-lesional skin of the patients and normal skin of the healthy controls was 0.62 ± 0.02,0.36-± 0.05 and 0.15 ± 0.11,respectively.The mRNA expression of DNMT2 was significantly higher in the lesional skin than in the non-lesional skin of the patients (t =6.23,P ＜ 0.01),and higher in the non-lesional skin of the patients than in the normal skin of the healthy controls (t =7.33,P ＜ 0.01).Additionally,the mRNA expression of DNMT3a was significantly higher in the lesional skin (0.85 ± 0.03) than in the non-lesional skin (0.43 ± 0.04) of the patients (t =5.66,P ＜ 0.01),and higher in the non-lesiona] skin of the patients than in the normal skin of healthy controls (0.18 ± 0.09,t =8.62,P ＜ 0.01).Conclusion Both DNMT2 and DNMT3a mRNA were abnormally expressed in the epidermis of patients with psoriasis vulgaris.

Aminoglycoside resistance genes in carbapenem-resistant Klebsiella pneumoniae / 中华临床感染病杂志

Objective To analyze the aminoglycoside ( AG ) antibiotics resistance rate of carbapenem-resistant Klebsiella pneumoniae ( CRKP ) and its molecular mechanisms.Methods One hundred and four strains of CRKP isolated from 4 hospitals in Zhejiang Province from January 2013 to June 2014 were collected, including 56 strains from Sir Run Run Shaw Hospital , Zhejiang University School of Medicine ( S hospital ), 22 from the First Affiliated Hospital, Zhejiang University School of Medicine ( Z hospital), 13 from Yiwu TCM Hospitals (Y Hospital) and 13 from Fuyang First People's Hospital (F Hospital).VITEK 2 Compact method and K-B disk method were used to detect the susceptibility of commonly used antibiotics including three kinds of AGs (kanamycin, gentamycin and amikacin ).PCR and sequencing techniques were used to screen the aminoglycoside resistance -related 16S rRNA methylation genes (rmtA, rmtB and armA) and the aminoglycoside modified enzyme resistance gene [aac(6′)Ⅰb].The relationship between drug resistance and carrier status of drug resistance genes was analyzed .Homologous analysis of rmtB-positive strains was performed using PFGE to examine the epidemic spread of strains in each hospital.Results All 104 CRKP strains were multi-drug resistant and had high resistance to cephalosporins, fluoroquinolones ( ciprofloxacin, levofloxacin ) and nitrofurantoin.The resistance rates to gentamicin, kanamycin and amikacin were 73.1%(76/104), 64.4%(67/104) and 56.7%(59/104), respectively.The carrying rates of aminoglycoside-resistance genes were: rmtB 56.7%( 59/104 ), aac (6′)Ⅰb 17.3%(18/104), armA 1.9%(2/104); while no rmtA was detected.Thirty-seven strains did not carry the screened genes.Amikacin-resistant strains were resistant to both kanamycin and gentamicin, and both were rmtB-positive strains.The PFGE classification results showed that 104 strains were divided into 11 clonal populations, and there were scattered non-population clones in each hospital. There were seven major clonal populations (Ⅰ-Ⅶ) carrying rmtB genes, of which typeⅠ, typeⅢand typeⅤwere prevalent in S hospital ; typeⅡ, typeⅥand TypeⅦwere popular in Z hospital ; the distribution of strains in Y hospital was scattered ; F hospital had one independent clone type Ⅳ(3 strains).Conclusion AGs still have certain sensitivity to CRKP strains.The main mechanism of strain resistance to AGs is the rmtB gene-mediated 16S rRNA methylase.

A novel approach for treating hepatocellular carcinoma: epigenetic therapy / 临床肝胆病杂志

Recent studies have shown that the pathophysiology of hepatocellular carcinoma (HCC) involves epigenetic alterations in DNA methylation, histone modifications, and microRNAs. In this review, we describe the general characteristics of epigenetic machinery in HCC as well as the current advances in epigenetic therapy for HCC. It is pointed out that epigenetic therapy has good prospects in the treatment of HCC, but there is still a long way before clinical application.

Expression of DNA methyltransferases 1,3a,and 3b in retinoblastoma / 中华眼底病杂志

Objective To observe the expressions of DNA methyltransferases (DNMTs) 1,3a and 3b in retinoblastoma (RB).Methods Sixty-two RB samples and six normal retinas were studied,including 17 poorly differentiated and 45 well differentiated samples; 16 invasive and 46 non-invasive samples.The expressions of DNMT1,3a,and 3b,and Ki-67 were detected using immunohistochemical analysis.Brown staining of nuclei was considered to represent the positive stain for DNMT1,3a and 3b,and ki-67,blue staining as negative.The level of high expression of nuclear staining was,positive cells in DNMT1≥65 ％,in DNMT3a≥60％ and in DNMT3b≥40％.The correlations of DNMT1,3a and 3b expression in RB samples,and MIB-1 labeling index were analyzed.Results Viewed under the light microscope,negative expressions of DNMT1,3a and 3b were demonstrated in normal retinas,however,positive expression was observed in RB samples,with 100％ in DNMT1,98％ in DNMT3a and 92％ in DNMT3b.Comparing well differentiated RB samples with poorly differentiated samples,significant differences were found in high expression of DNMT1 (x2 =12.57,P＜0.05) and DNMT3a (x2 =10.54,P＜0.05) ; also in the positive cells of DNMT1 (U=179,P＜0.05) and DNMT3a (U=198,P＜0.05).No significant difference was found comparing high expression (x2=1.5,P＞0.05) and positive cells (U=307,P＞0.05) of DNMT3b.When comparing invasive tumor tissues with non-invasive tumors,significant differences were shown between high expression (x2 =4.72,P＜0.05) and positive cells comparing DNMT1 (U=236,P＜0.05).No significant difference was shown in high expression (x2=3.53,0.84; P＞0.05) in DNMT3a and DNMT3b,or in comparison with positive cells (U=338,257; P＞0.05).The expression of DNMTs was positively correlated with the MIB-1 labeling index in RB tissues (R2=0.554,0.376,0.219; P＜0.05).Conclusion There are high expressions of DNMT1,3a,and 3b in RB.

Pseudomonas aeruginosa multirresistente: um problema endêmico no Brasil / Multidrug-resistant Pseudomonas aeruginosa: an endemic problem in Brazil

Relatos mundiais têm documentado a problemática da endemicidade de isolados clínicos de Pseudomonas aeruginosa multirresistente (MDR) aliada a elevados índices de morbidade/mortalidade. No Brasil, surtos de infecção ocasionados por P. aeruginosa têm sido relacionados com uma disseminação clonal da espécie. Atualmente, as opções terapêuticas para o tratamento das infecções causadas por esse microrganismo são limitadas, muitas vezes restringindo-se ao uso de carbapenêmicos (p. ex., imipenem [IPM]). Assim, a resistência ao IPM é uma questão de saúde pública, uma vez que esse antibiótico é empregado como último recurso no tratamento de infecções de origem hospitalar, causadas por bactérias Gram-negativas multirresistentes. No Brasil, os principais mecanismos relacionados com fenótipos multirresistentes de P. aeruginosa são produção de metalobetalactamase (MBL) do tipo SPM-1, presença de metilase 16S rRNA RmtD, perda de porina OprD e superexpressão de bombas de efluxo, o que pode explicar os altos índices de resistência a carbapenêmicos e aminoglicosídeos. A emergência de cepas com essas características é preocupante, tendo em vista a escassez de terapias efetivas no tratamento de infecções por esse patógeno. Finalmente, com base em relatos nacionais, publicados por diferentes grupos de pesquisa, podemos deduzir que a convergência de múltiplos mecanismos de resistência em P. aeruginosa tem sido um evento favorável para a seleção de diferentes clones endêmicos multirresistentes disseminados no Brasil.

Global reports have documented the endemicity of multidrug-resistant (MDR) Pseudomonas aeruginosa associated with high levels of morbidity/mortality. In Brazil, outbreaks of MDR P. aeruginosa have been related to clonal dissemination. Currently, therapeutic options for the treatment of these infections are restricted to carbapenemic antibiotics (i.e., imipenem [IPM]). Thus, carbapenem resistance is a public health issue, since carbapenems are considered the last resort to nosocomial infections caused by MDR Gram-negative bacteria. In Brazil, the main mechanisms associated with MDR P. aeruginosa phenotypes are metallo-betalactamase (MBL) production (SPM-1 enzyme), presence of 16S rRNA methylase RmtD, loss of OprD porin, and overexpression of efflux pumps, which may explain the high level of carbapenem and aminoglycoside resistance. Accordingly, the emergence and dissemination of MDR strains is worrisome. Finally, based on national reports published by different groups of investigators, it is deduced that the convergence of multiple mechanisms of P. aeruginosa resistance has played a major role in the selection of endemic MDR clones widespread in Brazil.

The expression and role of DNA methyltrsansferase 3a in colonic mucosa of patients with ulcerative colitis / 中华消化杂志

Objective To explore the association between DNA methyltrsansferase 3a (DNMT3a) expression and interleukin-4 (IL-4),interferon-gamma (IFN-γ) in mononuclear cells of colonic laminapropriamucosae in ulcerative colitis (UC) patients.Methods From November 2009 to March 2010,sixty colonic mucosa biopsy specimens through colon scopy were collected,specimens of active UC or normal controls were 30 in each group.The expression of DNMT3a,IL-4,and IFN-γ was detected with immunohistochemical method (SABC) in mononuclear cells of colonic laminapropriamucosae,and their relation with different degrees of colonic inflammatory response was analyzed.Results The positive expression rate of DNMT3a,IL-4,IFN-γ in mononuclear cells of colonic laminapropriamucosae in active UC group was significant higher than that in control group and there were statistically significant differences (x2 =16.596,P=0.000;x2 =42.857,P=0.000;x2 =6.667,P=0.024;).The expression of them was higher in sever inflammatory response than that in mild inflammatory response,and its expression intensity increased as inflammatory activity became more severe.There was positive correlation between IL-4,IFN-γ and DNMT3 expression in UC group (r=0.46,P=0.01 ;r=0.559,P=0.001).Conclusions The expression of DNMT3a,IL-4 and IFN-γ is associated with the degree of colonic inflammatory response.DNA methylation maybe involved in Th1/Th2 immune balance regulation in UC pathogenesis.

Effects of TSA on promoter methylation and expression of E-cadherin gene in hepatocellular carcinoma cell lines / 中华普通外科杂志

Objective To study the effects of histone deacertylase inhibitor (TSA) on promoter methylation and expression of E-cadherin gene in a hepatocellular carcinoma cell line SMMC7721. Methods Hepatocellular carcinoma cell line SMMC-7721 was treated with TSA (300 nm/L), MTT method was used to investigate the growth inhibition ratio, TUNNL was conducted to measure the apoptosis ratio, methylation-specific PCR (MSP) was employed to detect changes in the CpG island methylation of E-cad promoter region, Western blot technique was used to detect the expression of E-cad gene and DNMT3b before and after TSA treatment, respectively. Results TSA decreases the SMMC-7721 cell viability and induces apoptosis, the growth inhibition ratio was 21.85% compared with control group. The apoptosis ratio of control group was (4.69±0.56)% ,the apoptosis ratio of TSA treatment group was (14.94±0.91)%. The apoptosis ratio of TSA treatment group was significantly higher than that of control group(P = 0.000). Before treated with TSA, the CpG island of E-cad promoter region was methylated, and the expression of E-cad was negative. TSA treatment induces demethylation of the CpG island in E-cad promoter region, causes the re-expression of E-cad. TSA reduces the expression of DNMT3b. Conclusions TSA decreases the SMMC-7721 cell viability and induces apoptosis, reverses the methylation status of E-cad promoter region, and resumes E-cad gene expression. TSA may induce demethylation through down-regulating the expression of DNMT3b.

Coordinated change of a ratio of methylated H3-Iysine 4 or acetylated H3 to acetylated H4 and DNA methylation is associated with tissue-specific gene expression in cloned pig

Various cell types in higher multicellular organisms are genetically homogenous, but are functionally and morphologically heterogeneous due to the differential expression of genes during development, which appears to be controlled by epigenetic mechanisms. However, the exact molecular mechanisms that govern the tissue-specific gene expression are poorly understood. Here, we show that dynamic changes in histone modifications and DNA methylation in the upstream coding region of a gene containing the transcription initiation site determine the tissue-specific gene expression pattern. The tissue-specific expression of the transgene correlated with DNA demethylation at specific CpG sites as well as significant changes in histone modifications from a low ratio of methylated H3- lysine 4 or acetylated H3-lysine 9, 14 to acetylated H4 to higher ratios. Based on the programmed status of transgene silenced in cloned mammalian ear-derived fibroblasts, the transgene could be reprogrammed by change of histone modification and DNA methylation by inhibiting both histone deacetylase and DNA methylation, resulting in high expression of the transgene. These findings indicate that dynamic change of histone modification and DNA methylation is potentially important in the establishment and maintenance of tissue-specific gene expression.

Enzyme Production and Drug Resistance of Pseudomonas aeruginosa / 中华医院感染学杂志

OBJECTIVE To investigate the 16S rRNA methylases gene and AMEs of 30 strains multi-resistant Pseudomonas aeruginosa isolated from clinic work in our hospital.METHODS Collected 50 P.aeruginosa strains from two hospitals(30 strains of multi-resistant P.aeruginosa from Yantai,Shandong,20 strains of pan-drug resistant P.aeruginosa from Jiangsu),and analyzed 6 kinds of 16S rRNA methylases(armA,rmtA,rmtB,rmtC,rmtD and npmA) and 6 kinds of AMEs gene aac(3)-Ⅰ,aac(3)-Ⅱ,aac(6′)-Ⅰb,aac(6′)-Ⅱ,aac(3″)-Ⅰ,and aac(2″)-Ⅰ by PCR and sequence analysis.RESULTS Among 30 strains of multi-resistant P.aeruginosa from Yantai,the positive rate of 4 kinds of genes aac(3)-Ⅱ,aac(6′)-Ⅱ,aac(3″)-Ⅰ,and aac(2″)-Ⅰ was 20%(6/30),46.7%(14/30),36.7%(11/30) and 3.3%(1/30).The other 8 kinds genes were all negative.The total positive rate of AMEs gene was 46.7%(14/30).In 20 strains of pan-drug resistant P.aeruginosa,the positive rate of 6 kinds of genes rmtB,aac(3)-Ⅱ,aac(6′)-Ⅰb,aac(6′)-Ⅱ,aac(3″)-Ⅰ,and aac(2″)-Ⅰ was 55%(11/20),60%(12/20),35%(7/20),60%(12/20),50%(10/20) and 45%(9/20).The other 6 kinds genes were all negative.The total positive rate of AMEs gene was 95%(19/20).CONCLUSIONS It is the first report that 16S rRNA methylases gene is existed in P.aeruginosa;there is very high positive rate of AMEs genotypes in P.aeruginosa;there are differences in gene existing among two hospitals.

Genes of 16S rRNA Methylases and Aminoglycoside Modifying Enzymes in Strains Continuously Isolated from PAE in two Hospitals / 中华医院感染学杂志

OBJECTIVE To investigate the drug-resistance and the existence of genes in 16S rRNA methylases and aminoglycoside modifying enzymes in strains continuously isolated from Pseudomonas aeruginosa(PAE) in two hospitals of Jiangsu and Zhejiang Provinces.METHODS The drug-resistance of the strains continuously isolated from PAE was detected with K-B test,five kinds of genes in 16S rRNA methylases(rmtA,rmtB,rmtC,rmtD and armA) and six kinds of aminoglycoside modifying enzymes [aac(3)-Ⅰ,aac(3)-Ⅱ,aac(6′)-Ⅰb,aac(6′)-Ⅱ,ant(3″)-Ⅰ and ant(2″)-Ⅰ] were detected by PCR.RESULTS The strains were just sensitive to cefoperazone/sulbactam,imipenem,meropenem and amikacin(hospital A:74.2%,80.0%,82.9% and 68.5%;hospital B:90.0%,50.0%,50.0% and 95.0%,respectively).There was a high rate in the drug-resistance to ?-lactamase medicines,ciprofloxacin and sulfamethoxazole co.Genes in 16S rRNA methylases were not detected from PAE strains in the two hospitals.CONCLUSIONS The rates of genes in aminoglycoside modifying enzymes detected from strains in continuously isolated from PAE are different in different hospitals.

Transfer RNA methylases and carcinogenesis.

An increased tRNA methylase activity (100 %) accompanied by a 40 % decrease in the regulatory glycine methyltransferase activity was demonstrated in livers of mice fed on the carcinogenic (thioacetamide) diet, long before the onset of malignant transformation. Shortterm treatment with thioacetamide and phenobarbital independently, also brought about a significant increase in the rat liver tRNA methylase activity. A significant increase in the tRNA methylase activity was observed in the mammary glands of pregnant as well as lactating mice as against the negligible enzyme activity in the normal mammary glands of C3H and CBA mice, whereas a large increase in the tRNA methylase activity was evident in the spontaneously induced mammary tumours in these strains. Hepatic tRNA methylase activity was shown to remain unaffected in rats during various physiological stress conditions. It is suggested that elevation in the tRNA methylase activity may be one of the prerequisites during malignant transformation. A considerable increase in the tRNA methylase activity in host tissues of the tumour-bearing mice was also demonstrated.