| Hepatocellular carcinoma (HCC) is one of the malignant tumors that having high death rate, in China the incidence of which is55%of the global number. Compared with normal cells HCC has many changes, one of which is DNA methylation. DNA methylation is majorly catalyzed by DNA (cytosine-5) methyltransferase. There are three catalytic DNMTs currently being accepted: DNMT1, DNMT3A and DNMT3B, whose encoding genes are located in chromosome19p13.2.、2p23and20q11.2separately. DNMT1catalyzes hemi-methylated DNA double-strand, thus having a maintenance function. Dnmt3A and Dnmt3B are necessary for de novo methylation and for the establishment of new methylation patterns in mammalian cells. There is abnormal methylation in tumor cells, which is hypomethylation of global DNA and hypermethylation of specific genes’promoters. This epigenetic change is closely associated with the processing of tumor cells. Staurosporine (STS) can inhibit various protein kinases as well as inhibiting proliferating and inducing apoptosis, the mechanism of apoptosis induced by it is mainly through mitochondrial pathway. Whether DNMTs was involved in the STS induced apoptosis and what the role they might play was seldom studied, on which we are based for this thesis.In the first section of research, we studied the relevance between STS induced HCC apoptosis and the alteration of DNMT3B. First, we selected a series of different doses of STS to observe its impact on cell viability of HCC. We found that the decreasing of cell viability induced by STS was dose-dependent. At the same time, we observed the cleavage of PARP and caspase-3in protein level to testify that STS could induce apoptosis and this effect was also dose-dependent. On this basis, we selected a dose1μM STS that inhibited cell viability by about50%and induced apparent apoptosis in the both cell lines for the following experiments. Cells were treated with1μM STS for0h、4h and12h, then apoptotic bodies were stained by DAPI and apoptosis was examined by flow cytometry respectively. We found that the number of apoptotic bodies was increasing along the time, and the total apoptotic rate was increasing too. This result suggested that HCC apoptosis induces by STS was time-dependent. After that we wanted to know how DNMT1and DNMT3B changed during the process. We set time gradient of STS treatment, finding that DNMT1protein did not have apparent change but DNMT3B protein decreased along the time. So we focused on DNMT3B examining mRNA level、protein decay/mRNA decay to discuss in which level STS down regulated the experiment of DNMT3B. The results showed that the level of mRNA decreased according to the time, the protein stability was also influenced, but mRNA stability did not show statistically difference. These results indicated that STS could regulate the expression of DNMT3B on protein stability and mRNA levels.In the second section we futher explored the possible molecular mechanism of STS regulating DNMT3B in transcriptional level and whether DNMT3B was involved in STS induced cell apoptosis and its possible mechanism. Using DNMT3B core promoter plasmid to transfecte cell and then treat cell with STS we found that STS decreased the activity of DNMT3B core promoter plasmid. The results showed that p-JNK was activated and the other MAPK pathways were diminished gradually, so we supposed that JNK pathway involved in DNMT3B regulation by STS. After treating cells with p-JNK inhibitor the down-regulation of DNMT3B protein by STS was reduced by it. This indicated that STS might regulate DNMT3B protein expression through JNK pathway. Then we assessed the function of DNMT3B reduction in STS induced apoptosis. We designed small interfering RNA sequence (siRNA) and transfected it into BEL-7404cell. First, we proved that siRNA could interfere DNMT3B efficiently. We tested DNMT3B expression in the mRNA level, finding that it could down regulate DNMT3B expression. Next we wanted to explore whether the decrease of DNMT3B promoted STS induced apoptosis in HCC. After interfering DNMT3B for48h we treated cell with STS for another4h, finding that compared with using STS alone to treat cell the splicing of PARP and caspase-3were more significantly enhanced, that is to say it could notably facilitate cell sensitivity to STS and promote STS induced apoptosis. Then we further discussed the mechanism of DNMT3B promoting STS induced apoptosis. After knocking down DNMT3B we found up-regulation of a tumor suppressor gene p16. We consider this may be the key of down-regulation of DNMT3B promoting apoptosis.In summary, we found that the down regulation of DNMT3B protein and mRNA level was due to protein stability and its transcriptional activity. JNK pathway involved in the STS induced DNMT3B protein and transcription activity alteration. Down expression of DNMT3B promoted STS induced HCC apoptosis. Our research elucidated the function and molecular mechanism of DNMT3B gene expression in STS induced apoptosis, which should have theoretical significance and clinical significance in deep explore the function of epigenetic regulation in tumor cells. |
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