| 1. Topic resource:The funded project of Shenzhen technology bureau, Number 2007020322. Aim and significance of the study:The development of carcinoma is a complicated procedure of multi-step and polygene change. In resent years, with the development of molecular biology technique more attention has been put on the apparent genetics instead of caryogenetics, including RNA interference, DNA methylation, which is one of the important molecule mechanisms in apparent genetics.The thyroid carcinoma is the most frequent malignant tumor in the endocrine glands. Due to the slow progress, the early diagnosis and active operation therapy would bring the better prognosis. Nowadays the character of thyroid tumour, difficult to be identified clinically, can be merely identified by the post-operation pathology, which has been a big puzzle in the clinical diagnosis- therapy and resulted in the delay of therapy or the unnecessary the operation. There is no good early diagnostic method because of the unclear pathogenesis.The deletion of genetic expression product induced by the anti-oncogene methylation is one of the focus in the tumorigenesis study. As concerned to its effect on the cell cycle and cell growth, it would be the target of tumor gene therapy in the future. The detection of anti-oncogene methylation level in the patients is helpful to the diagnosis of tumor and judgement of tumor relapse or residual. There have been the reports about the gene methylation and some kinds of tumors nationwide, but few on the study about the thyroid carcinoma and anti-oncogene methylation.The anti-oncogenes of thyroid carcinoma, such as TSHR,P16INK4A,RASSF1A, have been studied by the methods of RT-PCR, MSP and gene sequencing in the research, to investigate the transcription expression of diverse anti-oncogene in thyroid carcinoma nationwide and the relationship between the anti-oncogene promoter region methylation and thyroid carcinoma, to seek the related gene in thyroid carcinoma.Our study can been used clinically to make judgement in the character of thyroid tumour before operation, make early discovery and help clinicians to institute the rational therapy. The new therapeutic strategy on thyroid carcinoma could also be made in according to the deletion expression of anti-oncogene because of 5’CpG methylation, such as the introduction of wild-type anti-oncogene into tumor cells by gene engineering, which can replace or compensate the methylated anti-oncogene to produce the normal regulation function and promote the cell differentiation. Through the suppression of mutational anti-oncogene by antisense technology, design of thyroid carcinoma vaccinum by targeting monoclonal antibody technique and control of thyroid carcinoma growth by demethylation medicine, the new methods would emerge in the early prevention, diagnosis and therapy of thyroid carcinoma.3. Study objects:PTC group:the fresh operation specimen of 50 PTC patients in our surgery have been collected from 2007 to 2009,15 male patients and 35 female patients, with the mean age 40±12 yeas. Control group:32 patients of benign thyroid tumor, including 20 patients of nodular goiter and 12 of thyroid adenoma,9 male patients and 23 female patients, with the mean age 37±12 yeas. All operation specimens were affirmed by pathology and put in -80℃immediately after ablation.4.Study methods:4.1 Methylation of anti-oncogene:4.1.1 Extraction of tissue DNA:Genome DNA was extracted by the QIAamp DNA Mini Kits 51304 (Qiagen) and stored in -20℃, the integrality of which was detected by 2% agarose gel electrophoresis, the content and purity of which were detected by ultraviolet spectrometer(figure 1).4.1.2 DNA methylation:The extracted DNA was treated and purified by sodium bisulfite with CpGenome TM DNA Modification Kit S7820(CHEMICON) and stored in -20℃.4.1.3 Nest methylation specific PCR:P16 gene was amplificated by the nest methylation specific PCR, with the outer primer designed by Primer 3 software, the primer sequence of methylation and demethylation of three anti-oncogene refered to the documents (tablel), with the primer designed by Shanghai bioengineering company. PCR kits were 2×PCR master mix K0171 (Fermentas). The reaction system was 25ul in methylation or demethylation PCR, the primer in upstream and downstream was lul (10pmol/μl) respectively, DNA after methylation was 2ul. Every product was detected by 2% agarose gel electrophoresis. The first PCR amplification condition of P16 gene:95℃force-degeneration 5min,95℃degeneration 30s,60℃renaturation 30s,72℃elongation 30s, after 35 circulation, 72℃elongation 8min finally.0.3ul of the first cycle PCR product was used in MSP. TSHR gene (methylation and demethylation) amplification condition:95℃force-degeneration 5min,95℃degeneration 30s,65℃renaturation 30s,72℃elongation 30s, after 30 circulation,72℃elongation 5min finally.P16 gene (methylation and demethylation) amplification condition:95℃force-degeneration 5min,95℃degeneration 30s,65℃renaturation 30s,72℃elongation 30s, after 30 circulation,72℃elongation 5min finally.RASFF1A gene (methylation and demethylation) amplification condition:95℃force- degeneration 5min,95℃degeneration 30s,60℃renaturation 30s,72℃elongation 30s, after 30 circulation,72℃elongation 5min finally.4.1.4 Anti-oncogene cloning and sequencing:The sequencing of each anti-oncogene was carried out in 2 PTC and 2 control randomly, the PCR products of P16 gene (methylation and demethylation) were put into sequencing after purification, due to the short gene fragment TSHR gene and RASFF1A gene were put into sequencing after cloning. (ABI3700 sequenator, cloning and sequencing by Huada gene company)4.2 The mRNA transcription expression of three anti-oncogene:4.2.1 Extraction of tissue RNA:According to the TRIZOL kit (Invitrogen), the integrality of RNA was detected by 1% methanal degeneration agarose gel electrophoresis, the content and purity were detected by UV-2450 ultraviolet spectrometer. (figure2).4.2.2 Synthesis of cDNA:With the template of the overall extracted RNA, the synthesis of cDNA was carried out according to the inverse transcription kit, the kit of first synthesis of cDNA was K1622 (Fermentas), the integrality was detected by 2% agarose gel electrophoresis.4.2.3 mRNA gene expression by RT-PCR:PCR primers of three gene were designed by Primer 3 software, the primer sequence (table 1) was designed by Shanghai bioengineering company. In the PCR kits K0171(Fermentas), the reaction system was 25ul,2ul cDNA was amplificated by PCR, PCR reaction system:2×PCR Master mix 12.5μl, the primer in upstream and downstream was 1ul (10pmol/μl) respectively, with GAPDH as intra-reference. The amplification condition is as following:TSHR gene:95℃force-degeneration 5min,95℃degeneration 30s,59℃renaturation 30s,72℃elongation 30s, after 30 circulation,72℃elongation 8min finally.P16 gene:95℃force-degeneration 5min,95℃degeneration 30s,56℃renaturation 30s,72℃elongation 30s, after 30 circulation,72℃elongation 8min finally, with GAPDH as intra-reference.RASFF1A gene:95℃force-degeneration 5min,95℃degeneration 30s,59℃renaturation 30s,72℃elongation 30s, after 30 circulation,72℃elongation 8min finally, with GAPDH as intra-reference.Samples of GAPDH products and three gene were detected by 2% agarose gel electrophoresis, the results were analyzed as absorbance (A) through image analysis system, the ratios of the gene to GAPDH were calculated as the correspondent expression quantity of three gene.4.3 Statistics4.3.1 With SPSS 13.0 software to evaluate the basaline data equilibrium between the PTC and control. The data of age were showed as x±s, the statistical difference was compared by t test. The sex equilibrium was compared by Pearsonχ2 test,with the statistical difference of P<0.05.4.3.2 The difference of methylation between group PTC and group control was analyzed by Pearsonχ2 test, with the statistical difference of P<0.05.4.3.3 The data of mRNA expression were showed as x±s, different independent sampler was compared by t test. when the variance was heterogeneity, the Satterthwaite approximation t test was uesed, with the statistical difference of P<0.05.4.3.4 The correlation analysis was done by Spearman test, r value represented the correlation. when r was closer to 1, indicating that the more closely related, with the statistical difference of P<0.05.4.3.5 The relation was analyzed by Pearsonχ2 test between promotor methylation and clinical pathological parameter, with the statistical difference of P<0.05.5. Results:5.1 The analysis of promotor methylation of three anti-oncogene:Methylation was showed as straps in amplification product.5.1.1 TSHR gene:The rate of promotor methylation in group PTC was 68%(34/50). The rate in control group was 21.9%(7/32),4/20 of nodular goiter and 3/12 of thyroid adenoma (figure 3.1) with the statistical difference of x2=16.61 and P=0.000.5.1.2 P16 gene:The rate of promotor methylation in group PTC was 54%(27/50). The rate in control group was 15.6%(5/32),3/20 of nodular goiter and 2/12 of thyroid adenoma (figure 3.2) with the statistical difference of x2=12.08 and P=0.001.5.1.3 RASFF1A gene:The rate of promotor methylation in group PTC was 60% (30/50). The rate in control group was 31.25%(10/32),6/20 of nodular goiter and 4/12 of thyroid adenoma (figure 3.3) with the statistical difference of x2=6.46 and P=0.011.5.2 Sequencing result:Gene sequencing of MSP product and non-methylation product were carried out randomly in two samples of PTC and two of control. There was methylation in all CpG situs of CpG insula of three gene promoter, C remained unchanged in CpG situs of methylation primer amplification product, however C shifted to T in CpG situs of non-methylation primer amplification product (figure 4.1-4.3).5.3 mRNA expression of three anti-oncogene:5.3.1 TSHR gene:The value of mRNA semiquantitative expression in group PTC was 0.413±0.107, the value in control group was 0.629±0.078 (figure5.1), with the statistical difference of t=-10.534 and P=0.000.5.3.2 P16 gene:The value of mRNA semiquantitative expression in group PTC was 0.513±0.170, the value in control group was 0.723±0.224 (figure5.2), with the statistical difference of t=-4.542 and P=0.000.5.3.3 RASFFIA gene:The value of mRNA semiquantitative expression in group PTC was 0.562±0.114, the value in control group was 0.669±0.156 (figure5.3), with the statistical difference of t=-3.368 and P=0.001.5.4 The relationship among three anti-oncogene methylation in PTC:5.4.1 The relationship between TSHR and RASFFIA methylation in PTC:The methylation rate of both TSHR and RASFFIA was 26/50 in PTC, the methylation rate of TSHR with RASFFIA non-methylation was 8/50, the methylation rate of RASFFIA with TSHR non-methylation was 4/50, the non-methylation rate of TSHR and RASFFIA was 12/50. There was relevance between the methylation of TSHR and RASFF1A in PTC with r=0.490 (p=0.000)5.4.2 The relationship between TSHR and P16 methylation in PTC:The methylation rate of both TSHR and P16 was 21/50 in PTC, the methylation rate of TSHR with P16 non-methylation was13/50, the methylation rate of P16 with TSHR non-methylation was 6/50, the non-methylation rate of TSHR and P16 was 10/50. There was no relevance between the methylation of TSHR and P16 in PTC with r=0.227 (p=0.113)5.4.3 The relationship between P16 and RASFFIA methylation in PTC:The methylation rate of both P16 and RASFFIA was 16/50 in PTC, the methylation rate of P16 with RASFFIA non-methylation was11/50, the methylation rate of RASFFIA with P16 non-methylation was 14/50, the non-methylation rate of P16 and RASFF1A was 9/50. There was no relevance between the methylation of P16 and RASFF1A in PTC with r=-0.016 (p=0.910)5.5 The relationship between three anti-oncogene methylation and clinical pathological information in PTC:Based on the clinical pathological information, there was no relevance between the RASSF1A methylation and the age, gender, clinical stage, lymphoid node metabasis of the patients, no relevance between TSHR、P16 and the age, gender, clinical stage, but there was relevance between TSHR、P16 and the lymphoid node metabasis of the patients (P<0.05)6. Conclusions:6.1 The rate of promoter methylation of three anti-oncogene in PTC was higher than that in control, the mRNA expression rate in PTC was lower than that in control. It concluded that the PTC carcinogenesis might be related to the methylation of three anti-oncogene.6.2 The relevance analysis among three anti-oncogene methylation showed that there was relevance between TSHR and RASFF1A, but no relevance between TSHR and p16, RASFFIA and p16. It may due to the different functions of three anti-oncogene:TSHR and RASFFIA in signal transmission, p16 in cell cycle modulation.6.3 There was relevance between the promoter methylation of three anti-oncogene in PTC and the tumor clinical stage, which concluded that the methylation might take place in the early stage of thyroid neoplasm. The rate of the promoter methylation of TSHR and p16 with lymphoid node metastasis in PTC was higher than that without lymphoid node metastasis, which concluded that the promoter methylation of TSHR and p16 might play a role in the malignant progress of thyroid carcinoma.Based on our findings, we concluded that the promoter methylation of three anti-oncogene might play a role in the PTC carcinogenesis and exist in the early stage of carcinoma. |
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