| 1. BackgroundEpigenetic modification, including DNA methylation and histone modification, plays critical roles in gene transcription and carcinogenesis. It is one of the hot spots in life science researches which focus on DNA methylation. DNA methylation is catalyzed by DNA methyltransferase (DNMT) to conjunct a methyl group onto cytosine in CpG island. The maintenance of normal DNA methylation pattern is critical for precise control of gene transcription, which makes sure the functions of cells. DNA methylation is one of the epigenetic modifications playing key role during gene transcription. Increasing evidences have demonstrated that histone modification, following by DNA methylation, may play greater roles than DNA methylation.MeCP2, potential therapeutic target in clinical treatment, is the founder member of methyl-CpG binding protein (MBP) family. Numerous researches indicated that MeCP2 is a multiple function protein playing key roles in gene transcription. MeCP2 has been regarded as a gene transcription repressor since its discovery. But recent evidences revealed that MeCP2 also can activate gene transcription. The activating genes by MeCP2 are related with Rett syndrome, till now, no evidences on MeCP2 research in environmental pollutant have appeared.Exposure to benzene, an environmental pollutant and important industrial solvent, is associated with increased acute myelogenous leukemia (AML) risk. Among the metabolites derived from benzene, hydroquinone (HQ) is one of the most important, which is also widely used in various industries as antioxidant or stabilizer for certain materials, production of antioxidants, antiozonants, agrochemicals and polymer industry. HQ could induce several types of changes, including genetic changes and epigenetic changes. Aberrant expression of MeCP2 is very common in tumor tissues and cells, but rare researches on MeCP2 expression induced by environmental pollutant has been reported. Based on previous finding that MeCP2 expression was associated with Rb expression at different time, we got a hypothesis that Rb may be probably regulated positively by MeCP2.Rb, one of the critical targets of clinical treatment, whose functions refers to cell apoptosis and senescence, cell proliferation, cell cycle, DNA response and maintenance of DNA stability. Rb dysregulation has been associated with carcinogenesis, but the mechanism remains elusive. The function of Rb and MeCP2 overlap with each other, enhancing the possibility that MeCP2 activates Rb expression, and bioinfomatic analysis demonstrated that within the promoter of Rb there have 25 binding sites for MeCP2.Together with previous results, bioinfomatic analysis and finding from other researchers, we hypothesized that a positive regulatory loop composed by MeCP2 and Rb may playes important roles during carcinogenesis induced by HQ. To explore whether a positive regulatory loop composed by MeCP2 and Rb be existed and uncover its bilogical fucntion and the epigenentics mechanism, this researche was conducted.2. Methods2.1. Cell line and HQ concentration selectionTK6 lymphoblastiod cell line was use in this research. The HQ concentration was selected according to the cytotoxicity assay results and the actual concentration of benzene exposure among Chinese occupational population. The cell viability of the cell line treated with the selected HQ should maintain above 75%.2.2. Detection of cell biological characteristicsCell growth was tested by cell counting and CCK-8 cell counting assay kit. Cell cycle was detected by PI staining on flow cytometer. Cell apoptosis was assayed by PI/Anexin-V staining on flow cytometer.2.3. Chemical treatmentCells was treated with DNA methyltransferase (DNMT) inhibitor (5-AZA), histone deacteylation (HDAC) inhibitor (TSA) or DNA damage inducer (DOX) and harvested at indicated time.2.4. Gene expression assayTotal cellular RNA was extracted using TRIzol reagent, cDNA was generated and quantitated by real-time PCR using SYBR green mastermix. Total cellular protein was extracted using cell lysis buffer and protein concentration was detected with BCA protein assay kit, related protein expressions were tested by western blotting. Cells were fixed methanol, Rb and MeCP2 protein expression were detected by immunofluorescence on flow cytometer.2.5. DNA methylation detection using methylation specific PCR (MSP)PCR primers were design with methyl primer express software according to the promoter DNA sequence got from UCSC database. Genomic DNA was extracted with the protocols from EZNA-DNA kit. Then,1 μg of the purified DNA was subjected to bisulfite modification using a CpGenome DNA Modification Kit.2.6. Bioinformatic analysisRelated documents were collected and bioinformatics analysis was conducted using consite software according to the DNA sequence of Rb gene promoter got from UCSC database.2.7. MeCP2 protein binding rate in Rb promoter assay using Chromatin immunoprecipitation (ChIP)Cells were crosslinked with 1% formaldehyde at room temperature, and then nuclear was extracted for sonication and digestion with nuclear emzyme. Chromatin, purified after immuoprecipiptation using ChIP-grade antibody, was quantitated with PCR.2.8. Construction of MeCP2 lentivirus vector and stable expression cell lineMeCP2 coding region was cloned into PLVX eukaryotic expression vector, termed as PLVX-MECP2, which was packaged with Lenti-XTM Lentiviral Expression Systems and then cells were infected, screened with puromycin to generate stable expression cell line. Western blotting was used to verified the protein expression of MeCP2 protein.2.9. Protein complex assay using co-immunoprecipitationProteins samples were adjusted to equal concentrations after extraction, and then antibodies were added according the protocol from manufacturer. Shaking overnight at 4 degree, then protein G/A agarose beads suspension was added, shaking at 4 degree for 1 h. Supernatant was collected and the beads was rinsed triplicate.3. Results3.1. HQ concentration selectionWe selected the HQ concentration based on the following two princinples, a) the cell viability should be maintained above 75% and b) the actual concentration of benzene exposure among occupational population currently. So 2.5～20.0μM HQ were selected for our further studies.3.2. Cell growth assay of HQ induced malignant cellsResults from cell counting assay demonstrated that the number of HQ induced malignant cells was 2.88 folds comparing with the control. And results from assay using CCK-8 cell counting kit verified the results from cell counting assay. These evidences indicated that cells treated with HQ have malignant characteristics of tumor.3.3. Apoptosis and cell cycle assay of HQ induced malignant cellsThe fraction of S-phase cells in HQ induced malignant cells was 48.2%, which was greater than that of control (41.1%) (P<0.05). Results from PI/FITC staining using flow cytometer indicated the apoptosis rates had no significance, but after serum free starvation, the alternation of apoptosis showed significane (P=0.001).3.4. Expressions of Rb, PTEN, p53 and TPOR in HQ induced malignant cellsExpressions of Rb, PTEN, p53 and TPOR mRNA were tested using RT-PCR, comparing with the control, mRNA expressions of Rb and PTEN were decreased to 65%(P<0.05) and 51%(P<0.05), respectively. TPOR mRNA was increased by 189% (PO.05), and no significance was observed in p53 mRNA. Rb and TPOR protein expression tested by western blotting assay got the same results from RT-PCR, but p53 protein was decreased.3.5. Rb and TPOR expressions influenced by DNMT inhibitor 5-AZA and HDAC inhibitor TSA5-AZA treatment could restore the mRNA expression of Rb, PTEN and p53 did not affected, but the TPOR expression increased comparing with the HQ induced malignant cells. TSA treatment got the same results from 5-AZA treatment. Protein expression tested using western blotting demonstratd that Rb expression was also restored, but TPOR expression was decreased.3.6. Biological characteristics change induced by 5-AZA and TSA in HQ induced malignant cellsBoth 5-AZA and TSA treatment could decreased the cell viability to 49.1% and 43.1%, respectively (P<0.05), accompanying with Gl-phase arrest. The fractions of Gl-phase cells in 5-AZA and TSA treated cells were increased from 37.5% to 60.8% and 70.3%, respectively (P<0.05). Meanwhile, the cell apoptosis rate were also increased from 10.4% to 23.8 and 15.1%, respectively (P<0.05).3.7. Dynamic expressions of Rb, DNMTs, and MBDs in cells treated with HQqRT-PCR analysis showed that expression of Rb mRNA was upregulated (P<0.05)in cells treated with HQ for 48 h, but DNMT1, DNMT3a, DNMT3b, and MBD1-4 were downregulated (P<0.05), and the change of DNMT1, DNMT3a, DNMT3b were dose-dependent. Western blotting analysis showed that the protein expressions of Rb, DNMT1, DNMT3b, MBD2, and MeCP2 were consistent with the results from qRT-PCR, but DNMT3a, and MBD1 showed reverse expression manner. The protein expressions of Rb, DNMT1, DNMT3a, DNMT3b, and MeCP2 in cells treated with HQ for 72 h or 5 w, were consistent with results from cells treated for 48 h, but the MBD1 was decreased and MDB2 increased. Cells treated with HQ for 5 w and then cultured without HQ for ten days, the protein expressions of all genes exception MBD2 were firstly upregulated and then downregulated (MBD1 not detect) along with the concentration of HQ.3.8. Bioinformtaic analysisBioinformtaic analysis showed that Rb promoter contained 2 CpG islands, which had 25 binding sites for MeCP2 protein.3.9. Methylaiton of Rb promoter and binding rate of MeCP2 to Rb promoterMSP was performed with DNA from cells treated with HQ for 5 w plus ten days culture without HQ, revealed that degree of Rb promoter methylation was firstly increased and then decreased concomitant with the concentration of HQ. The binding rate of MeCP2 to Rb promoter in HQ treated cells for 5w was 4.54 fold, comparison to control cells (P<0.05).3.10. MeCP2 and MBD2 protein expressions influenced by 5-AZA and TSAMeCP2 protein expression was restored by 5-AZA and TSA, comparison to the HQ induced malignant cells its expression was increased. But the MBD2 protein expression was decreased in cells treated with TSA,5-AZA treatment had no significant change.3.11. Rb and MeCP2 protein formed a protein complexResults from co-IP assay showed that Rb and MeCP2 protein formed a protein complex3.12. Restoration of MeCP2 expression enhanced the expression of RbComparing with the cells with GFP empty vector, MeCP2 mRNA expression of cells with Mecp2 restoration using MeCP2 expression vector was 6.25 fold, and the protein result was in the same manner. Rb expression manner was similar with MeCP2, these results indicated that Rb was activated by MeCP2.3.13. Rb activated MeCP2 expressionRb protein expression was activated by DOX treatment, and followed by upregulation of MeCP2 protein expression.4. Conclusion4.1. Expressions of Rb, DNMT1, DNMT3a5 DNMT3b, MBD1, MBD2 and MeCP2 were aberrant in cells treated with HQ and its malignant cells, along with the alternation of cell malignant characteristics.4.2 Expressions of Rb and MeCP2 were upregulated in cells treated with HQ, but downregulated in malignant cells, and their expression trends always kept in the same manner.4.3 Silencing of regulatory loop composed by Rb and MeCP2 plays important roles in carcinogenesis induced by HQ.4.4 Mechanism basing on epigenetics plays critical role in the silence of regulatory loop composed by Rb and MeCP2. |
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