The Relationship Between CHFR DNA And Histone Abnormal Regulation And The Tumorigenesis Of Laryngeal Carcinoma

Researches suggest that the tumorigenesis and development of malignant neoplasm is a multivariate, multistage and complicated process with the functional abnormality of multiple genes. The abnormal expression of genes includes genetic and epigenetic changes. The epigenetic change is heritable, which can not alter DNA sequence. DNA methylation and histone modification of tumor suppressor genes are main epigenetic changes. In recent years, the study of epigenetic changes has been developed sufficiently and become the clinical and investigative frontier of Laryngeal squamous cell carcinoma (LSCC). Recently, it has been discovored that the epigenetic alteration and DNA mutation are the same in tumorigenesis, in which the former is earlier than the latter.It has been kowmn that aberrant methylation of DNA has been described as an alternative mechanism of gene inactivation besides mutation and deletion. As one of epigenetic mechanisms, histone methylation can alter the chromosome status and control the gene transcription, which can lead tumorigenesis. The methylation of histone H3 lysine 9 is associated with gene repression. But the methylation histone H3 lysine 4 is associated with active gene transcription. The methylation of DNA and H3 lysine 9 are both involved in genes silencing, in which the methylation of DNA and H3 lysine 9 are synergistic, but the methylation of histone H3 lysine 4 is antagonistic to them.It appears that DNA and histone methylation likely have a mutually reinforcing relationship, and play a vital role of the the tumorigenesis and development of tumors.Laryngeal carcinoma is a common head and neck malignancy and the 11th common malignancy in the world, with a tendency towards an increasingly occurrence of new cases and deaths annually. It was shown that LSCC was one of the tumor with no increase of five-year survival rates. Therefore, it is essential to investigate the molecular mechanism of LSCC in order to identify the early diagnostic and therapeutic marker genes. Some studies have shown that the detection of the specific methylation status may be helpful to the early diagnosis of tumors. Therefore the further study of the effect on DNA and histone abnormal regulation of tumor suppressor genes plays an important role on the tumorigenesis of laryngeal carcinoma.CHFR (checkpoint with forkhead and ring finger) is a new cell cycle mitotic stress checkpoint gene.Recently, it has been known that the abnormal CHFR involed in the primary carcinogens, and closely related to gastric cancer, lung cancer and esophagal cancer.Its encoding product is ubiquitin ligase of Plkl. Plkl regulates both the Wee 1 kinase and the Cdc25 phosphatase, which in turn control the Cdc2 kinase activity at the G2 to M transition. The CHFR gene can ubiquitinate and degenerate the Plkl, which prevents cells from entering prophase and metaphase. CHFR gene expresses in normal tissue while it is low expressed or inactivated in cancer, in which losses the ability to prevent abnormal cells proliferating from G2 to M phase, thus cells abnormal differentiation and proliferation occur.In this study, we investigated the expression, the methylation of the promoter CpG islands and the methylation histone histone H3 lysine 9, histone H3 lysine 4 of CHFR in LSCC, and the effect on 5-Aza-dCand/orTSA to the Hep-2 cell line by MSP, Real-time PCR Western Blotting and CHIP. We explore the relationship between the level of expression and methylation of the CHFR and the tumorigenesis and development of LSCC. This investigation revealed a new pathway for the laryngeal tumorigenesis and early diagnosis and therapy.Material and Methods1. Subjects50 speciments of LSCC.Hep-2 cell lines, derived from human LSCC. 2. MethodsThe subjects were 50 patients with LSCC diagnosed and treated between January 2007 and January 2009 at Shengjing Hospital of China Medical University. All patients were diagnosed pathologically to be LSCC preoperatively and received no radiation and chemotherapy. We collected control mucosa from 15 patients under larngectomy and 2.0 cm away from the margin of tumor was considered as safe. The mucosa samples were histologically normal. Liquid nitrogen was used to snap-frozen all the samples and then stored in-80℃freezer. The collection of all the samples was informed patients. We extracted clinopathologic data (age, gender, primary site, T stage, clinical stage, differentiation and the nodal status) of patients were extracted from the patients' files. And the staging of the tumors was carried out according to UICC 2002 TNM classification.(1) Cell cultureHep-2 cell line was cultured in RPMI 1640 supplemented with 10% fetal bovine serum(Gibco), penicillin (100 IU/mL) and streptomycin (100 mg/mL), and incubated in a humidified incubator containing 50 mL/L CO2 at 37℃.(2) Experimental teams5-Aza-dC (5mmol/L) was used for 72h in the treatment. Culture medium containing 5-Aza-dC was exchanged every 24 h. TSA (300 nmol/L) was used for only 24 h in the treatment.5-Aza-dC was used for 48 h followed by TSA for an additional 24 h in the combined treatment.(3) Primer designThe primer sequences and reaction conditions of MSP, Real-time PCR and CHIP were described in table 3. The primer synthesis were in TaKaRa company.(4) mRNA extraction, expression analysis of CHFR using Real-time PCR Total-RNA was extracted and purified from the LSCC specimens and cell line using an RNeasy mini-Kit (TAKARA) following the manufacturer's instructions. cDNA was synthesized with 1μg of total-RNA using a SuperScript II Reverse Transcriptase kit (TAKARA). The mRNA expression levels of CHFR in the laryngeal tissues were quantified by SYBR Green I real-time quantitative PCR. The relative expression was normalized to a human glyceraldehyde-3-phosphatase dehydrogenase (GAPDH) endogenous reference standard relative to a calibrator and was evaluated by 2 -△△CT(5) DNA extraction and methylation-specific PCR (MSP) analysis of CHFRDNA was extracted from 65 laryngeal specimens using liquid-based cytology with a DNA Tissue Kit (TAKARA). Genomic DNA was denatured by NaOH and modified by sodium bisulfite.then purfied using Wizad DNA clean-up. An aliquot of bisulfite-treated genomic DNA (2μl) was used as the MSP template solution.10×Taq buffer, Taq enzyme and dNTP (TaKaRa) were used in the PCR analysis. PCR products were separated by electrophoresis on 2% agarose gels and quantitated with the FluorChem 2.0 system. The primers used for MSP and additional PCR conditions are described elsewhere.(6) Chromatin immunoprecipitation assay (CHIP)Detected methylation of histone H3 lysine 9 and histone H3 lysine 4 by chromatin immunoprecipitation assay (CHIP). Collected cell and treated it with Formaldehyde. Treated by H3-K9, H3-K4 methylation antibody, Sera or Tris-EDTA buffer. Then collected antibody-protein complex. PCR amplificated precipitated chromosome. Alpha Image 2000 collected imaging and analysed the result. All the experiments repeated three times. The average value is used to be statistically analysed.(7) Western BlotDetected CHFR protein,H3 general protein, histone H3 lysine 9 methylation protein and histone H3 lysine 4 methylation protein. Extracted protein, electrophoresis, transmembrane by PVDF membrane, and then closed by BSA. Treated by monoclonal antibody of CHFR, H3, H3K9 methylation, H3K4 methylation and actin. Then hybrided with second antibody, imaging and collected result. All the experiments repeated three times. The average value is used to be statistically analysed.3. Statistical analysisThe data were processed by SPSS 17.0 statistical software. Measurement data were expressed as mean±SD. The 2-sample T test was used to examine the expression levels of mRNA and correlations between CHFR mRNA expression and clinicopathological parameters. The Fisher's exact test was used to analyze the DNA hypermethylation of CHFR as well as correlations of aberrant DNA hypermethylation of CHFR with clinicopathological parameters., respectively. P<0.05 was considered as significance.Results1. CHFR promoter methylation in LSCCs specimentsThe frequency of the aberrant hypermethylation of CHFR promoter in LSCCs was 22%(11/50), including 10 in the stage T1+T2, and 1 in the stage T3, whereas there was no aberrant hypermethylation of CHFR promoter in the control specimens. The frequency of the aberrant hypermethylation in the early stage (41.6% 10/24) was significantly higher than that in the advanced stage (3.8% 1/26) (P<0.05). There was no significant difference in the others (including ages, sexes, the size of the tumor, the pathogenic differentiation and the lymphatic metastasis.2. CHFR mRNA expression in LSCCs specimentsThe results showed that CHFR expression was down-regulated in all LSCCs compared with the normal laryngeal tissues (P<0.05). Moreover, the CHFR gene silencing was discovered in 2 LSCCs specimens (4%). The relative ratio of CHFR mRNA between the two groups was 0.50±0.12, which was 0.29±0.18 at the early stage of LSCCs and 0.69±0.15 at advanced ones. There was significant difference in the clinical stages (P<0.05) but no significant difference in the others (including ages, sexes, the size of the tumor,, the differentiation and the lymphatic metastasis. The CHFR mRNA with the aberrant methylation in LSCC specimens (0.16±0.13) was much lower than that one with un-methylation (0.65±0.17).The CHFR gene silencing was discovered in 2 LSCCs with hypermethylation (4% 2/50).3. CHFR promoter methylation in Hep-2 cell lineThe cell line showed a characteristic DNA methylation status.5-Aza-dC and combined 5-Aza-dC and TSA resulted in demethylation of CHFR. In contrast, TSA alone did not affect the DNA methylation status of CHFR.4. CHFR mRNA expression in Hep-2 cell lineCompared with the control team,5-Aza-dC alone reactivated expression of the CHFR in Hep-2 cell line (1.75±0.21). TSA had no effect on gene expression (1.05±0.13). The combined treatment with 5-Aza-dC and TSA increased gene expression (2.15±0.18).5. CHFR histone H3 Lysine 9, H3 Lysine 4 in Hep-2 cell lineHistone H3 Lysine 9 methylation directly correlated with DNA methylation, but histone H3 Lysine 4 methylation inversely correlated with DNA methylation of CHFR. Gene down-expression associated with DNA methylation was accompanied by histone H3 Lysine 9 hypermethylation, but the methylation of H3 Lysine 4 is antagonistic to them.5-Aza-dC had effects on histone H3 Lysine 9 methylation. TSA alone had no effect on histone H3 Lysine 9 methylation. The combination of 5-Aza-dC and TSA had similar effects on histone H3 Lysine 9 methylation to that of 5-Aza-dC.TSA did not affect histone H3 Lysine 4 methylation.5-Aza-dC, or the combination of 5-Aza-dC and TSA increased histone H3 Lysine 4 methylation.6. CHFR, H3, H3-K9methylation, H3-K4 methylation protein CHFR protein of 50 tumor and 15 control mucosa specimens were detected. 46.0% (23/50) LSCC specimens had not CHFR protein expression; and all of the mucosa specimens showed detectable CHFR protein.β-actin expression were similar in all LSCCs and control mucosa samples. The differences were statistically significant (χ2=13.85; P<0.001).Compared with the control team,5-Aza-dC alone reactivated protein expression of the CHFR in Hep-2 cell line. TSA had no effect on gene protein expression. The combined treatment with 5-Aza-dC and TSA increased gene protein expression. But 5-Aza-dC and TSA didn't affecd the protein level of histone H3, methylation-H3 K9, methylation-H3 K4.ConclusionThe aberrant methylation of CHFR promoter was related to the down-expression or inactivation, which closely related to the clinical stage. The detection of aberrant methylation of CHFR may be helpful to the early diagnosis of LSCC.5-Aza-dC and combined 5-Aza-dC and TSA can reverse the methylation of CHFR, simultaneously elevated the mRNA and protein expression level.Histone H3 Lysine 9 methylation positively correlated with DNA methylation, while histone H3 Lysine 4 methylation negatively correlated with DNA methylation of the tumor suppressor gene CHFR.Methyltransferase inhibitor 5-Aza-dC affected the ststus of Histone H3 Lysine 9 and Histone H3 Lysine 4.Therefore TSA had not effect on them.