Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells / 癌症

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the -486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The -486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Preventive effects of emodin on cerebral ischemia injury and expression of the inflammatory factors in rats with cerebral ischemia / 中国中药杂志

<p><b>OBJECTIVE</b>To assess emodin antagonism to cerebral ischemia injury, and to discuss the mechanism of emodin inhibiting the inflammatory cascade reaction from the levels and expressions of cytokines.</p><p><b>METHOD</b>Rats were divided into sham-operated group, model group, Ligustrazine group and emodin groups (low, middle, high dosage). After focal cerebral ischemic model of cerebral middle artery occlusion was duplicated with nylon thread, we took the speciments after ischemia 6 hours, observed the changes of the evaluating score of neural symptoms, brain water ratio and cerebral infarction area, determined the levels of TNF-alpha, IL-beta and TGF-beta in rats brain tissue by radioimmunoassay, detected the expressions of TNF-alpha and VCAM-1 by immunohistochemistry, and measured VCAM-1-mRNA expression by in-situ hybridization.</p><p><b>RESULT</b>Compared with sham-operated group, the evaluating score of neural symptoms, brain water ratio and cerebral infarction area of rats in model group were higher (P < 0.01) , the levels of TNF-alpha and IL-1beta of rats brain tissue in model group increased, while the level of TGF-beta was lower, and the expressions of TNF-alpha and VCAM-1 increased (P < 0.01). The evaluating score of neural symptoms, brain water ratio and cerebral infarction area improved obviously in every emodin group, especially in emodin low dosage group. Levels of TNF-alpha, IL-1beta and the expressions of TNF-alpha and ICAM-1 in emodin low dosage group and Ligustrazine group were lower, while the level of TGF-beta was higher. Compared with Ligustrazine group, the changes aboved are more significant in emodin low dosage group (P < 0.01).</p><p><b>CONCLUSION</b>The increase of inflammatory cascade reaction mediated by various cytokines such as TNF, IL-1beta, ICAM-1 and the decrease of TGF protection are the important mechanism of cerebral ischemia injury. The mechanism of emodin antagonism to cerebral ischemia injury may be implemented by inhibiting inflammatory cascade reaction and increasing the brain protective factors, such as TGF.</p>