Objective To investigate the effects of DNA methyltransferase inhibitor (DNMTi) and histone deacetylase inhibitor (HDCAi) on expression of E-cadherin gene and invasiveness of cholangiocarcinoma cell. Methods According to different treatment, the QBC939 cells were divided into four groups: blank control group, hydralazine group, valproic acid group and hydralazine and valproic acid combined group. After 48 h, the expression of E-cadherin was evaluated by reverse transcription-PCR (RT-PCR), mehtylation specific PCR (MSP) and Western blot, the invasiveness of QBC939 cells was evaluated by Transwell method. Results There was no expression of E-cadherin mRNA and protien in blank control group and valproic acid group. The expressions of E-cadherin mRNA and protien in hydralazine and valproic acid combined group were higher than those in hydralazine group ( P ＜ 0.01), while the invasiveness of QBC939 cells of hydralazine and valproic acid combined group was much lower than that of blank control group, hydralazine group and valproic acid group ( P ＜ 0.01). Conclusion DNMTi and HDACi can synergistically re-express E-cadherin gene and weaken the invasiveness of QBC939 cell, which plays an important part in treatment of cholangiocarcinoma.
Objective To investigate the expression of the histone deacetylases 1( HDAC1) and the level of whole histone acetylation and methylation in lung T cells of asthmatic rats, and investigate their role in the pathogenesis of asthma.Methods Sixteen wistar rats were randomly divided into a control group and an asthma group( n =8 in each group) . The rats was sensitized with ovalbumin( OVA) and challenged with aerosol OVA to establish asthma model. The asthmatic ratmodel was confirmed by measurement of pulmonary function, histochemical staining, HE staining, and the levels of interleukin-4 ( IL-4 ) , interferon-gamma ( IFN-γ) and immunoglobulin E( IgE) in serum and bronchoalveolar lavage fluid ( BALF) . T cells were isolated fromrat lungs and the purity was identified. The expression of the HDAC1, the level of whole histone H3 and H4 acetylation, and whole H3K9 dimethylation were analyzed by Western blot in lung T cells. Results Compared with the control group, the protein expression of HDAC1 was significantly lower( 0. 465±0. 087 vs 0. 790 ±0. 076, P lt;0. 05) in lung T cells of the asthma group. No significant differences werefound in regard to the level of whole histone H3 and H4 acetylation and whole H3K9 dimethylation betweenthe two groups. Conclusions HDAC1 in lung T cells may be involved in the pathogenesis of asthma.Histone modification by HDAC1 may be a specific eventwith gene transcription which may not be associated with asthma.
Objective To examine the expression of promoter CpG island methylation of Notch1 gene and explore the variable sites for DNA methylation in lung CD4 + T cells of asthmatic rat models.Methods An ovalbumin ( OVA) sensitized- challenged asthmatic rat model was established. Total T cells were isolated and CD4 + T lymphocytes were purified using magnetic beads. Twenty Wistar rats were randomly divided into a control group and an asthma group ( n = 10 in each group) . CD4 + T cells were isolated by immunomagnetic beads and identified by flow cytometry ( FCM) . Realtime PCR was employed to examine the mRNA expression of Notch1 gene in lung CD4 + T cells and the methylation level of Notch1 gene was examined by methylation-specific PCR. Results The mRNA expression of Notch1 in lung CD4 + T cells of the asthma group was 2. 254 ±0. 403 times as much as that of the control group. The total methylation level of asthma group was lower than that of the control group ( 0. 150 ±0. 108 vs. 0. 300 ±0. 667, P lt;0. 01) . Conclusion Promoter demethylation is one of the major mechanisms of Notch1 gene up-regulation in pathogenesis of asthma.
Objective To investigate the possible mechanism of arsenic trioxide (As2O3) inducing P16 gene demethylation and transcription regulation in the retinoblastoma (RB) Cell Line Y79． Methods The induced growth inhibition of Y79 cell was assayed by MTT; The DNA content of Y79 cell was analyzed by flow cytometry after being exposed to As2O3; the methylation status of the P16 gene in Y79 cell line before and after treatment with As2O3 was detected by the nestedmethylation specific PCR and DNA sequencing; the mRNA of P16,DNA methyltransferases (DNMT3A and 3B)gene were determined by RT-PCR． Results As2O3 was able to inhibit the growth of Y79 cell and increase the cell number in G0-G1 phase；P16 gene was not expressed in Y79 cell line and As2O3 can induce itrsquo;s mRNA expression；after 48 hour disposal of As2O3，the methylation levelof P16 gene was apparently attenuated in Y79 cell line,the expression of DNMT3A and DNMT3B was obviously down-regulated． Conclusions P16 gene is the hypermethylation in the retinoblastoma cell line Y79, and As2O3 can inhibite the methylation of P16 gene and upregulate the expression of p16 gene mRNA which inhibits the proliferation of Y79 cell by inducing the G0-G1 arrest, by inhibiting the expression of DNA methyltransferases.
Objective To investigate the role of DNA methylation on regulation of cell apoptosis and proliferation in ischemia-reperfusion of small intestine. Methods Thirty-five male Wistar rats were randomly divided into normal group， sham operation group， and ischemia-reperfusion group. The apoptotic cell was assessed by TUNEL and electron microscopy and the expression of Ki-67 was examined by immunohistochemistry in the small intestinal parts （villi epithe-lium， crypt epithelium， and lamina propria mucosa of small intestine）. The DNA methylation was detected by DNA histo-endonuclease-linked detection of methylated DNA sites. Results ①The apoptotic positive cells increased at 3 h， 6 h，and 12 h after ischemia-reperfusion in the villi epithelium， crypt epithelium， and lamina propria mucosa of small intestine as compared with the normal group and sham operation group （P＜0.01）；Moreover， the apoptotic cells in the lamina propria mucosa of small intestine were identified as T cells by electron microscopy. ②The expressions of Ki-67 markedly increased at 3 h， 6 h， 12 h， and 24 h after ischemia-reperfusion in the villi epithelium cells as compared with the normal group and sham operation group （P＜0.01）. ③The weak expression of DNA methylation was found in the villi epith-elium and crypt epithelium in the normal group and sham operation group， the b expression was examined in the crypt epithelium cells nearby stem cell site in the ischemia-reperfusion of small intestine， the change of expression was gradually weak from crypt epithelium to villi epithelium. Conclusion This initial results indicate that the DNA methyl-ation in the ischemia-reperfusion of small intestine might regulate cell apoptosis and proliferation.
ObjectiveTo explore the relationship between aberrant promoter CpG islands methylation status of E-cadherin gene and hepatocarcinogenesis, and to assess its significance in clinical early diagnosis of hepatocellular carcinoma (HCC). MethodsSurgically resected specimens, among which cancerous and corresponding noncancerous liver tissues from 34 HCC patients, 10 liver cirrhosis from patients without HCC and normal liver tissues from 4 accidental deaths, were collected in West China Hospital. Breast cancer cell line MDA-MB-435 with promoter CpG islands hypermethylation of E-cadherin as positive control was gained from the Cell Bank of Chinese Academy of Sciences in Shanghai. The methylation status of promoter CpG island of E-cadherin gene was detected by nested methylationspecific polymerase chain reaction (nested-MSP). ResultsE-cadherin gene promoter CpG islands hypermethylation was found in 61.76% (21/34) of cancerous tissues, in 29.41% (10/34) of noncancereous tissues from the 34 HCC patients and in 50.00% (5/10) liver cirrhosis from patients without HCC. None of the 4 normal liver samples were detected E-cadherin mehylation positive. Moreover, the methylation of E-cadherin gene was significantly more frequent in 34 cancerous than that in corresponding noncancerous liver tissues (Plt;0.05), which had no significant difference between the 10 cirrhotic samples and cancerous or non-cancerous liver tissues (Pgt;0.05). In 34 cancerous samples, with the combination of both biomarkers of E-cadherin methylation and AFP400 (serum AFP level at a cutoff of 400 μg/L), the diagnostic sensitivity of HCC increased to 82.35%. ConclusionsThe aberrant promoter methylation of E-cadherin gene may play a vital role in the development and progression of HCC. Moreover, it might be an early event in hepatocarcinogensis. It is of high value to make further study to confirm the significance of E-cadherin gene methylation in clinical diagnosis and therapy.
ObjectiveRecent advancements in the researches on cholangiocarcinoma (CC) related genes methylation in CC were reviewed and the clinical significances of aberrant DNA methylation for the diagnosis and treatment of CC were discussed. MethodsRelevant literatures about the relation between CC-related genes methylation and CC published recently were collected and reviewed. ResultsThe genesis of CC resulted from abnormal expressions of many genes. Many researches had shown that the abnormal methylation of CC-related genes had a close relation with CC. Epigenetic alteration had been acknowledged as an important mechanism contributing to early CC carcinogenesis. ConclusionsAbnormal methylation of CC-related genes is related with CC. The detection of CC-related genes methylation might provide new specific biomarkers for early noninvasive diagnosis of this disease. Using epigenetic agents such as azacytidine to modulate the activities of DNA methyltransferase and reverse the methylation status of CC-related gene might be an attractive strategy for future treatment of CC, which could be combined with conventional therapies.
ObjectiveTo explore the clinical significance of promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) in cholangiocarcinoma. MethodsPromoter methylation status of MGMT gene and expression of MGMT protein were detected in cholangiocarcinoma by methylationspecific PCR and immunohistochemical staining, respectively. ResultsAberrant methylation of MGMT gene was detected in 17 patients (47.2%). Twentyone cases showed negative immunoreactivities. Of 21 patients with negative MGMT expression, 14 patients had aberrant methylation of MGMT gene. In 15 patients with positive MGMT expression, aberrant methylation of MGMT gene was only found in three cases. There was a negative correlation between promoter methylation status of MGMT gene and the expression of MGMT protein (rs=-0.816, Plt;0.05). Promoter methylation status of MGMT gene was related to depth of invasion, degree of differentiation, and TNM stage (Plt;0.05), but not to age of patient, gender, pathological type, and lymph node metastasis (Pgt;0.05). ConclusionsHypermethylation of MGMT promoter is a frequency molecular event in cholangiocarcinoma and may be involved in carcinogenesis. Methylation status of MGMT gene may be used to evaluate malignant degree of cholangiocarcinoma.
Objective To review the advance of gene diagnosis and gene therapy on gastric cancer. Methods Literatures about the advance of gene diagnosis and therapy on gastric cancer were reviewed. Results Detection of tumor marker by gene technique is important for early diagnosis, follow-up and therapy evaluation of gastric cancer in clinic. But there are still many problems in gene therapy of gastric cancer. Conclusion Gene detection and gene therapy will become important supplementary means for diagnosis and treatment of gastric cancer.
ObjectiveTo determine the level of CDH1 gene promoter hypermethylation in human gastric carcinoma by establishing MS-PCR method, and analyze retrospectively the possible statistical relationship between CDH1 gene promoter hypermethylation in human gastric carcinoma and HP infection, tumor differentiation, invasion, lymph nodal and distant metastasis, respectively. MethodsThe bisulfite conversion MS-PCR method was adopted to examine the level of CDH1 gene promoter hypermethylation in 40 cases of human gastric carcinoma tissue collected between January 2008 and December 2009. The statistical relationship between CDH1 gene promoter hypermethylation in human gastric carcinoma and HP infection, tumor differentiation, invasion, lymph nodal and distant metastasis were examined respectively with SPSS statistical tools. ResultsThe positive rate of CDH1 gene promoter hypermethylation in gastric carcinomas (67.5%) was higher than that in paired normal gastric mucosae (12.5%), and the difference was significant (P<0.05). In gastric carcinomas, the positive rate of CDH1 gene promoter hypermethylation in well differentiated or moderately differentiated groups (22.2%) was lower than that in poorly differentiated groups (80.6%), and the difference was significant (P<0.05). The positive rate of CDH1 gene promoter hypermethylation in HP positive groups (78.1%) was higher than that in HP negative groups (25.0%), and the difference was significant (P<0.05). ConclusionCDH1 gene promoter hypermethylation may play an important role in the process of tumor carcinogenesis in gastric carcinomas. Meanwhile, the CDH1 gene promoter hypermethylation may lead to poor differentiation in gastric carcinomas. CDH1 gene promoter hypermethylation is related to HP infection in the original gastric carcinomas, which shows that HP may get involved in the process of tumor suppressor gene methylation/inactivation and tumor development process.