| Survivin is an inhibitor of apoptosis protein (IAP) family member, it is the apoptosis inhibitor as well as the mitotic regulator. Survivin is absolutely undetectable in normal tissues, but over-expressed in all the most common human cancers. Also, it is required for maintaining cancer-cell viability. Thus, Survivin is considered as a therapeutic target in cancer.According to previous research, in mammalian cells, a short methylated sense oligonucleotide that is complementary to the sequence of the promoter region of a given gene can induce hypermethylation of the promoter region of that gene and thus inhibit that gene transcription. This mechanism might be used to cure diseases by inhibiting the expression of a certain gene harmful to health. By virtue of this, we designed a methylated oligodeoxynucleotide (SurKex, 22 bases) complementary to a region of part promoter of survivin. Our early experiments showed that SurKex can inhibit not only survivin mRNA and protein expression, but also tumor growth of tumor-bearing mice, and prolong survival of animal model of nude mice by pharmacodynamics in vivo and in vitro. At the same time, we had a preliminary study on the mechanism of SurKex. The results showed that SurKex could lead survivin gene silencing by inducing DNA methylation of survivin promoter region.In this article, at the basis of our previous research, we first had a further study on methylation mode of survivin promoter inducing by SurKex through bisulfite-sequencing and confirmed that SurKex can lead to methylate survivin promoter in CpG-site specific way, that is targeting induction.When gene silencing, some changes of covalent modifications of histone combined with a certain gene will accordingly happen. Next, we used chromatin immunoprecipitation (ChIP) to investigate the modifications of acetylation/deacetylation or methylation on the specific lysine residue of histone H3, H4 combined with survivin promoter sequence after survivin promoter site-specific methylation was induced by SurKex. The results showed that SurKex increased histone H3-K9 dimethylation,H3-K27 trimethylation and decreased H4,H4-K16 acetylation levels. These changes of histone modifications positively related with gene silencing. Although DNA methylation and histone covalent modifications are both important biochemical events of epigenetics in regulating gene expression, there is a hot debate in epigenetics about the order and the causal relationship between them when they regulate gene expression. In our experiment, SurKex not only induced methylation of survivin promoter but also changes of histone tail modifications. Now that how did they collaborate with each other on survivin expression and control survivin"on or off"precisely? Whether DNA methylation guides histone covalent modifications or histone covalent modifications guide DNA methylation would be a key issue to be solved in our next study.Then we studied the effects on the efficacy of SurKex in blocking DNA methylation, histone deacetylation or histone methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR), histone deacetylase inhibitor trichostatin A (TSA) and histone methyltransferase inhibibor BIX-01294 respectively. We determined the expression of survivin mRNA by RT-PCR, analyzed the site-specific CpG methylation of survivin promoter region by bisulfite sequencing and detected the covalent modifications of histone tails combined with survivin promoter by chromatin immunoprecipitation (ChIP). These results showed that DNA methylation was the crucial step in survivin silencing, but histone deacetylation and methylation guided DNA methylation, which could play a role through DNMT1.Finally, we would be in an attempt to verify whether our inference was correct or not. We investigated the expression of DNMT1 mRNA by RT-PCR in blocking histone deacetylation and methylation by TSA and BIX-01294 respectively. The results showed that the expression of DNMT1 mRNA was decreased greatly after histone deacetylation or histone methylation being blocked, which revealed that both of them could affect DNA methylation by upregulating the expression of DNMT1 mRNA. Of course, this is only a possible means of their roles.In summary, we first confirmed that SurKex is a new type of targeted, site-directed CpG DNA methylation drug candidate, and it could result in survivin silencing by inducing site-specific CpG methylation of survivin promoter region; Next we revealed that SurKex could increase histone H3-K9 dimethylation and H3-K27 trimethylation levels and decrease histone H4 and H4-K16 acetylation levels besides DNA methylation; Then we verified that DNA methylation is a vital step in survivn silencing, but the change of histone covalent modifications (including histone deacetylation and methylation) is more advantage event, and it guide DNA methylation; Finally, we initially proved that histone deacetylation and methylation guide DNA methylation by affecting the expression of DNMT1 mRNA.This research provides not only new ideas for the design of antineoplastic drugs and a new method for cancer targeted therapy from cancer epigenetics, but also the experimental evidence for solving the major theoretical problem in the field of Epigenetics.
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