Meth-Ylation Profiling Of Tumor Supressor Genes In Hepatocellular Carcinoma And Epigenetic Regulation In Gene Expression

Background and abstractTwo mechanisms are involved in carcinogenesis: genetic alterations with changes in DNA sequence and epigenetic alterations without changes in DNA sequence. Genetic alterations include mutation, chromatin deletion and rearrangement, etc. Epigenetic alterations mainly refer to the methylation of DNA 5'-flanking region, but recently it has been reported that variable covalent histone modifications which can induce chromatin remodeling are a major step in the epigenetic regulation of genes. Acetylation and methylation are especially important among these histone modifications.DNA methylation is the unique common modification in human DNA and the final stage in all the epigenetic alterations, which mainly regulates gene expression in transcription level, and is closely correlated with tumor suppressor genes (TSGs) silencing. Many studies have demonstrated that tumors of almost any type involved a group of tumor-related genes methylation but nor did normal cells, so methylation can be regarded as tumor specific biomarkers. While in different tissues or different growth periods of the same type cells, the different methylation status in different DNA CpG islands make up of the DNA methylation profiling, the tissue-specific DNA methylation profiling is a remarkable character inmammalian. So it is very important to make a tissue and tumor specific gene methylation profiling especially the TSGs methylation profiling to diagnose a specific tumor. Previous studies have shown that tumors of any type had relatively instable chromosomes. Restriction fragment length polymorphism studies of paired HCC and non-tumorous liver samples has revealed relatively frequent chromosomal allelic losses (loss of heterozygosity, LOH) on lp,3p,4, 5q, 8p, lOq, lip, 13q, 16, 17p, and 22q, which suggests that these sites may harbor TSGs involved in the pathogenesis of HCC. The "two hits" hypothesis about inactivation of TSGs believed that the gene occurred one hit may be apt to be hit by the second one, and many researches revealed that methylation could be as the other hit to inactive one gene together with LOH, so we conferred that the TSGs located on the chromosomes where occurred LOH frequently might be apt to occur methylation. Based on above, we detected the methylation status of ten TSGs located on instable chromosomes, six TSGs located on stable chromosomes and three DNA mismatch repair (MMR) genes, and made a TSGs methylation profiling in HCC and selected the clinical biomarkers of diagnosis.Until now no one gene therapy has acquired satisfactory effects, however, the emergence of epigenetic mechanism brought us new hope, and it has been clarified as a crucial significant mechanism to find out new targets of drugs and therapy approaches. Two major steps are involved in epigenetic mechanism: one is DNA methylation, the other is histone modifications. In 2000, David Allis provided the "histone code" hypothesis: there were many covalent modifications in histone N-terminal tails (including methylation, acetylation, phosphorylation, ubiquitination, and ribosylation), the modifications are amino acid-specific and specify unique downstream function in a combination or sequential fashion. In 2001, the study in Neurospora showed that a histone H3 Iys9 (H3K9) specific methyltransferase mutation could induce the lost of DNA methylation, and later some complexes of DNA methyl-binding proteins and histone deacetylases were identified, this suggested that there was crosstalk between histone modifications and DNA methylation. H3K9 is a crucial site in H3 N-terminal tail, which canoccur both acetylation and methyl ation and play an important role in forming of heterochromatin and in gene transcriptional regulation.On the basis of identification of biomarkers that can be used to detect and diagnose cancer in its earliest stages, we cultured six HCC cell lines and further detected the mRNA levels of the biomarkers, we treated the cells with 5-Aza-2'-deoxycytidine (5-AZA-CdR) and Trichostatin A (TSA") respectively and in combination, and observed their reversal of DNA methylation, transcriptional regulation and their effects on H3K9 acetylation and trimethylation, then we discussed the interactions of two histone modifications and DNA methylation.Latest studies have shown that some. leukemic cells can be induced to differentiate to mature granulocytes and/or macrophages without correcting its genetic abnormailities, both 5-AZA-CdR and TSA played differentiation role in. tumor cells of many types. In our study, we observed the differentiation-induction role of 5-AZA-CdR and TSA respectively and in combination and provided the experimental data for clinical improvement. Method 1. Make TSGs methylation profiling in HCCExtract the genomic DNA from 48 pairs of HCC tissues and corresponding noncancerous tissues, and modify the DNA by sodium bisulfite treatment. Sodium bisulfite treatment of unmethylated DNA causes conversion of the cytosine bases in DNA to uracils. Methylated (Me) cytosines are resistant to bisulfite treatment and remain as cytosines. Therefore, bisulfite treatment results in a sequence difference between methylated and unmethylated DNA. Subsequent PCR amplification, using primers specific for methylated or unmethylated DNA desighed by MethPrimer software, enables the methylation status of a given DNA sequence to be determined. We selected ten TSGs located on instable chromosomes in HCC, six TSGs located on stable chromosomes in HCC and three DNA mismatch repair genes, to detect the methylation status by Methylation-specific PCR (MSP), and make the profiling of TSGs in HCC and identify the biomarkers that can be used to diagnose HCC in its earliest stages.2. Observe the reversal role of 5-AZA-CdR on DNA methylation and its transcriptional regulation on MMR gene.We cultured 6 HCC cell lines and treated the cells with l|j.mol/L 5-AZA-CdR, the methlation status of above genes before and after lu.mol/L 5-AZA-CdR treatment were detected by MSP, and the mRNA changes of MMR genes were evaluated by RT-PCR, then we discussed the relationships between DNA methylation and gene transcription.3. Observe the effects of 5-AZA-CdR and TSA on DNA methylation, H3K9 acetylation and trimethylation and gene expression respectively and in combination, and discuss their potential interactions.The gene selected as the biomarkers were our study objects. Extract the DNA, RNA and nuclear proteins before and after 5-AZA-CdR and TSA treatment respectively and in combination , the methylation status of the genes were detected by MSP; the mRNA changes were evaluated by RT-PCR and H3K9 acetylation and trimethylation levels were determined by western blot; we'also detected the effects of 5-AZA-CdR and TSA on expression of HDAC1 and HDAC3 proteins by western blot; at last, we used chromatin imunoprecipitation (CHIP) assay to detect the interactions between gene promoter and H3K9 acetylation/methylation and discuss their roles in gene transcriptional regulation.4. Discuss the role of differentiation-indution of 5-AZA-CdR and TSA respectively and in combination on HCC cells.HepG2 cells were treated with 5-AZA-CdR and TSA respectively and in combination, cell growth inhibition was assayed by MTT method and the cell growth curve was made; the changes on cell morphology were observed using optical microscope and mitosis indexes were accounted; Colony formation rate was determined by Colony formation experiment in soft agar; assessment of cell cycle and apoptosis were performed by Flow Cytometry; the activities of TAT (tyrosine-a-ketoglutarate transainase) were detected by Diamondstone's method and the levels of caspase-3 and bcl-2 proteins were investigated by western blot.Result1. Nine often genes located on instable chromosomes occurred hypermethylation in more than 50% HCC tissues, while only one of six genes located on stable chromosomes occurred hypermethylation in more than 50% HCC tissues. Among all the genes, RIZl and TMS1 gene occurred hypermethylation in HCC tissues, and their methylation rate was significantly difference between HCC and corresponding concancerous tissues (62.5% versus 22.9%, 66.7% versus 6.3%, respectively;p<0.0\), so, they can be regarded as effective biomarkers to diagnose HCC in its earliest stages. In the three DNA mismatch genes, hMSH2 was the unique gene whose hypermethylation rate was more than 50%.2. Nine of ten genes located on instable chromosomes occurred hypermethylation in more than 50% HCC cell lines, while three of six genes located on stable chromosomes occurred hypermethylation in more than 50% HCC cell lines. There were more than one gene occurred hypermethylation in per cell line. 5-AZA-CdR could reverse methylation well, moreover, some genes methylation could be completely reversed. hMSH2 gene occurred hypermethylation in more than 50% HCC cell lines, treated by lumol/L 5-AZA-CdR, the methylation status in some cell lines could be partly or completely reversed, and subsequently their mRNA levels were increased.3. Our RT-PCR results showed that RIZl expression was low in HepG2 cell line and TMS1 expression was lost in SNU449 cell line. RIZl methylation could not be reversed by 5-AZA-CdR and TSA respectively, but could partly reversed by the combination. TMS1 methylation could be partly reversed by 5-AZA-CdR and TSA respectively, and completely reversed by the combination. The expression of RIZl was increased and the expression of TMS 1 was restored by 5-AZA-CdR and TSA treatment respectively, there was a cooperative interaction between them. Western blot results showed that 5-AZA-CdR and TSA could increase H3K9 acetylation while decrease H3K9 trim ethyl ati on respectively and in combination, and there was a cooperative interaction between them; 5-AZA-CdR and TSA could inhibit the expression of HDAC1 respectively and in combination, HDAC3 proteinexpression could also be inhibit by the combination but not by them respectively. CHIP result showed that 5-AZA-CdR and TSA could increase the H3K9 acetylation and decrease the H3K9 trimethylation in the promoter of RIZ1 and TMS1 respectively and in combination, and there was a cooperative interaction between them.4. Both 5-AZA-CdR and TSA could inhibit the growth of HepG2 cell line, and decrease the clone formation rate, observed through microscope, the morphology of cells treated with 5-AZA-CdR had slight change, the maximum of mitosis index decreased but not advance and the activity of TAT increased moderately; while the morphology of cells treated with TSA changed distinctly, cells extended like slim branches, the maximum of mitosis index decreased and advanced one day and the activity of TAT increased prominently. All these demonstrated that the effect of TSA on differentiation was much better than 5-AZA-CdR. Flow Cytometry showed that 5-AZA-CdR could decrease S-stage cells, but no distinct effect in Go/G] and G2/M stages cells. TSA caused block in G2/M and decrease S-stage cells, â–  the combination mainly caused cell block in Go/G], while G2/M-stage cells were also increased and S-stage cells were decreased than control. 5-AZA-CdR and TSA increased the apoptosis rates respectively and in combination, and there was a cooperative interaction between them. Western Blot results demonstrated that the protein level of caspase-3 was increased and bcl-2 protein level was decreased prominently by 5-AZA-CdR and TSA respectively and in combination, and there was a cooperative interaction between them. Conclusion1. The TSGs located on instable chromosomes are apt to occur methylation. RIZ1 and TMS1 methylation can be regarded as biomarkers to diagnose HCC in clinic. ,2. DNA methylation, histone H3K9 acetylation and trimethylation all take part in the regulation of RIZ1 and TMS1 expression in HCC, and there are strong interactions among of them.3. Both 5-AZA-CdR and TSA can restore or increase gene expression throughDNA methylation dependent and DNA methyl ation-independent way. There isa cooperative interaction between them. "-'?â– .4. Both 5-AZA-CdR and TSA can enhance H3K9 acetylation, and HDAC1 playan important role in the process. HDA'C3 may be the compound of the proteincomplex which regulates gene expression and the complex is relatively stable. "5. Both 5-AZA-CdR and TSA can decrease H3K9 trimethylation, but the processis not accomplished by effecting on DNA methylatiom 6. TSA is an. effective differentiation inductive drug; 5-AZA-CdR has somedifferentiation-induction role by itself, and can be regarded as an enhancer ofTSA.