ZO-1 gene methylation status and its clinical significance in children with non-Hodgkin lymphoma / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the methylation status of zonula occludens-1 (ZO-1) gene promoter and its clinical significance in children with stage IV non-Hodgkin lymphoma (NHL) and to provide a basis for further etiological study and early diagnosis of this disease.</p><p><b>METHODS</b>Fifty-five children with a confirmed diagnosis of stage IV NHL (40 cases of T-NHL and 15 cases of B-NHL) were selected as the case group, and 20 children with diseases other than hematologic malignancies were selected as the control group. Bone marrow samples were collected from these subjects. Methylation-specific PCR (MS-PCR) was applied to evaluate the methylation status of ZO-1 gene promoter, and the integrated optical density (IOD) was determined. RT-PCR was used to measure the mRNA expression of ZO-1.</p><p><b>RESULTS</b>MS-PCR showed that the methylated bands of ZO-1 gene promoter were found in 39 (70.9%) of 55 patients in the case group before treatment, while no ZO-1 gene promoter methylation was detected in the control group. With close tracking of 47 cases in the study group, consisting of 32 cases of T-NHL and 15 cases of B-NHL, the rates of ZO-1 gene promoter methylation prior to treatment were 72% and 67%, respectively, (P>0.572). The cases of T-NHL and B-NHL showed no significant changes in methylation rate in the early and middle phases of chemotherapy (P>0.05), but they showed significant changes in methylation rate in the late phase of chemotherapy (P<0.05). RT-PCR showed that the NHL cases carrying methylated ZO-1 gene had no mRNA expression of ZO-1, while all children in the control group had mRNA expression of ZO-1. There was no linear relationship between the total number of peripheral blood leukocytes and ZO-1 gene IOD (r=0.093, P=0.575); a positive correlation was found between the number of malignant cells in bone marrow and ZO-1 gene IOD (r=0.669, P<0.001).</p><p><b>CONCLUSIONS</b>ZO-1 gene shows a hypermethylation status in children with NHL, and the methylation level is positively correlated with the number of malignant cells in bone marrow. ZO-1 may be used as a novel molecular marker in early diagnosis, outcome assessment, prognostic evaluation, and detection of minimal residual disease.</p>

Detection of malignant lymphoma in children by using Id4 gene methylation indicator / 中华实用儿科临床杂志

Objective To study and discuss what part does methylated Id4 gene participate in malignant lymphoma stage Ⅳ by detecting the extent of how much Id4 gene has been methylated in afflicted children who suffer from malignant lymphoma.Methods Forty-two patients who had diagnosed with malignant lymphoma [Hodgkin's disease (HD),Non-Hodgkin's lymphoma(NHL)] were selected as study group.Their chemotherapy stages of pre-treatment,early-treatment,mid-treatment,post-treatment and clinical remissions or relapse throughout the entire treatment had been traced.At each stage the expression of methylated Id4 gene mRNA was detected by methylation-specific polymerase chain reaction (MS-PCR) and compared with the control group.The control group consisted of 20 non-neoplastic hematologic disorder affected children as sample donors.Results MS-PCR detection:in pre-treatment stage,there were 27 patients who were found methylated or partially methylated Id4 genes.Methylated ratio was thus at 64.3％ (27 patients out of a total of 42 patients).Those 27 patients were actively traced down along with different stages of treatment (21 NHL patients,6 HD patients).Before NHL there was 55.6％ methylated Id4 gene (15 cases out of 27 NHL patients).During chemotherapy treatment,there was 51.9％ of methylated Id4 gene positive (14 cases out of 27 patients).In post chemotherapy treatment,there was 48.1％ of methylated Id4 gene positive (13 cases out of 27 patients).Totally there were 9 patients showed clinical recovery after chemotherapy.There was 44.4％ of traceable methylated Id4 gene after recovered chemotherapy patients (4 recovered patients still carrying positive reading of methylated Id4 gene out of totally 9 recovered patients).There were 2 patients relapsed,with traceable methylated Id4 gene re-appeared in them afterwards.Throughout different treatment stages,there was no significant correlation in the treatment result and the appearance of methylated Id4 gene in early treatment stages (all P ＞ 0.05).But in latter treatment of chemotherapy,the correlation started to emerge (P ＜ 0.05) ; The overall statistics on NHL and HD share the same statistical pattern on different stages (all P ＞ 0.05).The control group had 20 patients,none of them had methylated Id4 gene.The study group showed noticeable difference in methylated Id4 gene before and after the experiment (P ＜ 0.001).RT-PCR result showed that before chemotherapy treatment,all of those who carried methylated Id4 gene had no expression of mRNA,by comparison to the control1 group which all had expression of mRNA.Conclusions Methylated Id4 gene is closely related in affected children who suffer from malignant lymphoma and its complications.The expression of Id4 gene is depressed when it has been methylated.The state of methylated Id4 gene is changed as patient's condition changed,so the methylated Id4 gene is thus a possible indicator of early diagnostic tool for children lymphoma.

Methylation of Id4 gene and inhibitive effect of arsenic trioxide on it in Raji cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study methylation of Id4 gene and demethylation effect of arsenic trioxide (ATO) in Raji cells.</p><p><b>METHODS</b>Human Burkitt's Raji lymphoma cells were cultared and treated with ATO at different concentrations and different time points. Methylated degree of Id4 gene was detected by methylation specificity polymerase chain reaction (MS-PCR), Id4 mRNA expression in Raji cell by reverse transcription polymerase chain reaction (RT-PCR), the growth of cell by MTT assay, and cell apoptosis and cycle distribution by Flow Cytometry (FCM).</p><p><b>RESULTS</b>(1) The Id4 gene exhaustive methylation in control group, and hypermethylation in experimental group were reversed by ATO in a dose-dependent manner. (2) Id4 mRNA expression in Raji cells treated with ATO for 48 h increased gradually with ATO concentration increasing in experimental group. (3) Raji cell growth inhibited rates after different concentrations of ATO treatment for 24, 48, 72 h were 12.15% ∼ 92.17% in the experimental group (P < 0.05). (4) Apoptosis peak emerged after ATO treatment for 48 hours in experimental group, while a much lower apoptosis in control group. (5) After ATO treatment for 48 h in experiment group, the cells were arrested at G(0)/G(1) phase in a dose-dependent manner.</p><p><b>CONCLUSION</b>Id4 gene presents exhaustive methylation in Raji cells. ATO can reverse the hypermethylation of Id4 gene and recover the expression of Id4 mRNA. Hypermethylation of Id4 gene is one of the reasons of Raji cells malignant proliferations.</p>

Construction of eukaryotic expression vector of short hairpin RNA for transforming growth factor-beta1 / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To construct the plasmid containing short hairpin RNA (shRNA) of TGF-beta1 expression vector.</p><p><b>METHODS</b>Short chain oligonucleotide was designed according to the TGF-beta1 mRNA sequence provided by Genebank, then DNA segment was gained through annealing after chemosynthesis, and then was cloned to pWH1 vector. The recombinant TGF-beta1 shRNA expression vector was evaluated by using enzyme cutting. At last, the constructed TGF-beta1 expression vector was transfected into salivary gland mucoepidermoid carcinoma (Ms) cells by Lipofectomine TM 2000, and its effect on TGF-beta1 expression was observed by RT-PCR and immunohistochemistry.</p><p><b>RESULTS</b>Successful construction was identified by enzyme cutting and the constructed plasmid was called pWH1-TGF-beta1. The shRNA and it inhibited the TGF-beta1 mRNA and protein expression effectively.</p><p><b>CONCLUSION</b>The constructed TGF-beta1 shRNA expression vector can block the TGF-beta1 expression in salivary gland mucoepidermoid carcinoma cells.</p>

Screening and identification of metastasis-related gene expression in tongue carcinoma with cDNA microarray assay / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To identify metastasis-associated genes in tongue carcinoma and to better understand the mechanism underlying tongue carcinoma metastasis. To compared mRNA expression profiles of two tongue carcinoma cell strains with high and low metastatic potentials using microarray technology.</p><p><b>METHODS</b>Tca8113 and Tb cells were used as model systems to study the molecular mechanism of tumor metastasis. Two fluorescent cDNA probes labeled with Cy3 and Cy5 dyes were prepared from the mRNA samples of Tca8113 and Tb cells by reverse transcription method. The two color probes were then mixed and hybridized to the cDNA chip constructed by double dots of 1 152 human genes, and scanned at two wave lengths. Differential expression genes from the above two cell lines were analyzed using computer. Then six of the different expression genes were further validated by RT-PCR technique.</p><p><b>RESULTS</b>In the 1 152 clones of known genes and expressed sequence tags that were analyzed, 37 showed significantly different (minimum 2 folds) expression levels in two cell lines. Among the 37 genes, 15 were up regulated (with ratio more than 2) and 22 down regulated (with ratio less than 1/2). The results of RT-PCR analysis were coincident with those of microarray assay.</p><p><b>CONCLUSION</b>Some of these genes are known to be involved in human tumor antigen, immune surveillance, adhesion, cell signaling pathway and growth control. It is suggested that the microarray in combination with a relevant analysis facilitates rapid and simultaneous identification of multiple genes of interests and in this study it provided a profound clue to screen candidate targets for early diagnosis and intervention.</p>