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Effects Of Phosphatidylinositol 3 Kinase/Akt Pathway On The Regulation Of DNA Methyltransferase 3B Gene Expression And Its Mechanism

Posted on:2011-01-16Degree:DoctorType:Dissertation
Country:ChinaCandidate:C Z MeiFull Text:PDF
GTID:1100360305997254Subject:Biochemistry and Molecular Biology
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Cancer cells show widespread and distinguished epigenetic changes when compared with their normal parental tissue, including DNA methylation and chromatin modification. DNA methylation is the best-known epigenetic marker and controlled by DNA methyltransferases (DNMTs). To date, three catalytically active DNMTs have been identified in human, DNMT1, DNMT3A, and DNMT3B, their encoding gene respectively located in 19p13.2.,2p23 and 20q11.2.. DNMT1 prefers hemimethylated DNA substrates and is therefore primarily responsible for maintenance methylation, which must occur during DNA replication. Dnmt3A and Dnmt3B are necessary for de novo methylation and for the establishment of new methylation patterns in mammalian cells. Aberrant DNA methylation in cancer cells includes global DNA hypomethylation and specific promoter hypermethylation of genes. Hypermethylation of the CpG-island promoter can affect genes involved in the development of cancer cells, and the levels of DNMTs are elevated in various malignancies and transition from a precancerous to a malignant state. There are reports about that de novo methyltransferase DNMT3B has more important role in the development of cancers. Within the cells, diverse processes are regulated by phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway including growth and survival and cell-cell communication. Abnormal PI3K/Akt activity has been linked to the formation of tumors, metastasis. In our previous study we found that phosphatidylinositol 3 kinase (PI3K)/Akt pathway stabilizes DNMT1 protein and advance the carcinogenesis. However, the relationship between PI3K/Akt pathway and DNMT3B gene expression is unknown. In first section of our research, we found that Akts (Aktl or Akt2) transcripts are associated with the total DNMT3B transcripts in 6 human hepatocellular carcinoma (HCC) cell lines. We further found that there are various in the ratio of DNMT3B3 mRNA to DNMT3B4 mRNA of 6 HCC cell lines. In addition, we confirmed that DNMT3B protein was strongly higher expression in HCC tissues than each matched adjacent noncancerous tissues.To explore the mechanisms of Akt on the regulation of DNMT3B gene expression, in second section of our work, we firstly assessed that PI3K, a upstream molecule of Akt, how to modulate DNMT3B gene expression, and found that LY294002, a small molecule specific inhibitor of PI3K pathway, down-regulated DNMT3B gene expression at the RNA and protein level in hepatocarcinoma cell line BEL-7404 in a dose-and time-dependent manner. We investigated that LY294002 down-regulated DNMT3B mRNA is prior to the down-regulation of DNMT3B protein after LY294002 treatment, which suggested that there was translational regulation. Moreover, we observed that down-regulation of DNMT3B gene expression by LY294002 was not accompanyed with the sub-cellular location of DNMT3B. Furthermore, down-regulation by LY294002 was also found in other two hepatocarcinoma cell lines SMCC7721 and L02. In addition, we performed a CHX chase protein decay assay between LY294002 treatment and without treatment to assesse the relationship between down-regulation of DNMT3B with LY294002 treatment and de novo synthesis of protein machinery. The result showed that LY294002 did not alter the stability of DNMT3B protein. In second section of our work, we found higher DNMT3B mRNA and protein levels in Myr-Akt2-MEFs constructed by our lab and expressed constitute activation of Akt2 than in MSCV-MEFs. Meanwhile, we found that the knockdown of Aktl and Akt2 caused the decrease of DNMT3B mRNA and protein levels in BEL-7404 stably transfected with Aktl/2 shRNA human lentiviral particles (Aktl/2 siRNA). To further confirm Akt regulate the DNMT3B gene expression, we observed Lithium chloride, an inhibitor of GSK3βwhich is a downstreamed molecule of Akt, moderately increased the DNMT3B protein in the BEL-7404 cells. All these findings suggest that PI3K/Akt modulates the DNMT3B gene expression.In order to deeply demonstrate the mechanism of regulating DNMT3B gene expression by PI3K/Akt pathway, in the third section of our study, we assessed that how the promoter of DNMT3B was affected by LY294002. Firstly, we constructed four specific DNMT3B promoter fragments which included core promoter region and base promoter sequences according to Yanagisawa's study, and were inserted upstream of the firefly luciferase gene in the pGL3-Basic vector. Next these constructs were sequenced and aligned with GeneBank. Furthermore we assayed luciferase activities of these plasmids with transiently transfection experiments and Dual-Luciferase Reporter Assay, we found the core promoter fragment revealed the highest activity in BEL-7404 cells, the data were similar to that reported in document. Moreover we investigated that both the luciferase activities of firefly and renilla were decreased in BEL-7404 cells transiently transfected with these pGL3 plasmids under with LY294002 treatment, which were consistent with the data of cell cycle assay that represented cycle arrest of the G1 phase in BEL-7404 cell treated with LY294002. Whereas the above investigation, we took the ratio of relative luciferase activity of the pGL3-Basic (occupying base transcription activity) with LY294002 treatment or untreatment to be the criterion, and found the ratio of the DNMT3B promoter constructs were lower than the criterion. These results indicated that PI3K/Akt pathway regulates the expression of DNMT3B gene at transcription control. Furthermore, we observed that LY294002 stimulation induced the increase of DNMT3B degradation using mRNA decay analysis, which suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Moreover we evaluated that the HuR expression was also modulated by LY294002 in BEL-7404 cells in a time-dependent manner. Whereas DNMT3B mRNA stabilized by HuR was reported, we hypothesized that DNMT3B was down-regulated by LY294002 via HuR decrease. Taken together, these findings in the fourth sections of our study indicated that PI3K/Akt pathway regulates the expression of DNMT3B gene at transcription and post-transcription level.In summary, we have, for the first time, demonstrated that PI3K/Akt pathway regulates the expression of DNMT3B gene at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt pathway and DNMT3B gene on hepatocarcinogenesis.
Keywords/Search Tags:DNA methyltransferase, PI3K/Akt pathway, human hepatocellular carcinoma
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