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Objective To explore the mechanism of transcription factor EB (TFEB) in autophagy of aging cardiomyocytes. Methods Animal experiment: Twenty aged Wistar rats were randomly divided into the sham operation group (Sham group) and the ischemia-reperfusion injury group (I/R group). Cell experiments: (1) Aging cardiomyocytes were cultured in vitro, incubated with 8 g/L D-galactose for 8 days, and then divided into the normoxia group and the hypoxia/ reoxygenation group (H/R group). (2) Aging cardiomyocytes transfected by adenovirus overexpressing and interfering with TFEB were divided into the Ad-GFP group, the Ad-GFP+H/R group, the Ad-TFEB group, the Ad-TFEB+H/R group, the sh-NC group, the sh-NC+H/R group, the sh-TFEB group and the sh-TFEB+H/R group. (3) Aging cardiomyocytes treated with specific inhibitors of DNMT1, DNMT3a and DNMT3b after hypoxia/reoxygenation were divided into the H/R group, the DNMT1 specific inhibitor (DC-05) group, the DNMT3a specific inhibitor (TFD) group and the DNMT3b specific inhibitor (NA) group. (4) Aging cardiomyocytes transfected by adenovirus interfering with DNMT3b were divided into the sh-NC group, the sh-NC+H/R group, the sh-DNMT3b group and the sh-DNMT3b+H/R group. Quantitative real-time PCR (qPCR) was used to detect the mRNA level of TFEB, and Western blot assay was used to detect the autophagy related proteins TFEB, LC3B and p62 in aging cardiomyocytes. The DNA methylation levels of TFEB promoter in aging myocardium and cardiomyocytes were detected by nested methylation specific PCR (nMS-PCR). Results Compared with the Sham group or the normoxia group, the mRNA and protein expression of TFEB were decreased in the I/R group and the H/R group (P＜ 0.01). The protein expression of LC3B-Ⅱ/Ⅰ was decreased in aging cardiomyocytes after overexpression of TFEB in the Ad-TFEB group compared with the Ad-GFP group, while the protein expression of p62 was increased (P＜0.01). The opposite results were obtained after interfering with TFEB (P＜0.01). Compared with the Sham group or the normoxia group, the DNA methylation level of the TFEB promoter was increased in the I/R group and the H/R group (P＜0.05). Compared with the H/R group, it was found that the mRNA and protein expression level of TFEB were increased in the NA group (P＜ 0.01). And the TFEB mRNA and protein expression were increased in aging cardiomyocytes after overexpressed interference with DNMT3b (P＜0.01). Conclusion DNMT3b inhibits the TFEB expression by regulating DNA methylation of TFEB promoter, thus to promote autophagy of aging cardiomyocytes. [ABSTRACT FROM AUTHOR]

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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~Ⅳ stages than those ofⅠ~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. [ABSTRACT FROM AUTHOR]

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 ( P < 0.05). The expression level of DNMT3b was associated with the pathological grade ( P =0.002), tumor size ( P < 0.001), T stage ( P < 0.001), lymphatic metastasis ( P =0.039) and TNM stage ( P < 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 ( P =0.008) and TNM grades of the tumor ( P =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 ( P =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.

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% vs 41.1%, P =0.015). A high DNMT3B expression in HCC was significantly associated with the tumor size ( P =0.001), vascular invasion ( P =0.004), and intrahepatic metastasis ( P =0.018). The patients with high DNMT3B expressions had significantly lower tumor-free and tumor-specific survival rates than those with low DNMT3B expressions ( P < 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.

Objective: To investigate the effects of apple polyphenol on cell proliferation and migration of breast cancer cell line MDA-MB-231, and explore the potential mechanism., Methods: The breast cancer cells in the logarithmic phase were treated with 0, 50, 100, 200, 400 and 800 μg/m L of apple polyphenol for 24, 48 and 72 h, then Trypan blue staining was employed to detect cell vitality; CCK8 kit was used to determine cell growth and proliferation; cell migration ability was observed by scratch experiment, and the change of proteins expression level was assessed by Western blot., Results: Apple polyphenol could content-dependently inhibit breast cancer cell's vitality and proliferation between 0 and 800 μg/m L, decrease the proteins level of ubiquitinlike with PHD and ring finger domain 1( UHRF1) and matrix metalloproteinases-2( MMP2), and the protein levels of DNA methyltransferases 3 a( DNMT3 a) and DNMT3 b, themolecular targets of UHRF1, also decreased. Apple polyphenol inhibition of cell 's migration was more significant between 400 and 800 μg/mL., Conclusion: Apple polyphenol inhibited the vitality, cell growth and proliferation, and migration of breast cancer cells MDA-MB-231 by decreasing the proteins expression of UHRF1 and MMP2, and the downstream targets of UHRF1, DNMT3 a and DNMT3 b, also decreased.