Methylation Of Promoter And Expression Of RUNX3 Gene In Papillary Thyroid Carcinoma

Background and ObjectiveThyroid carcinoma is a common malignant tumor in head and neck, which represents 1%of all malignant diseases with an increasing incidence of the disease worldwide. According to the type of tumor pathology, four types of thyroid cancer comprise all thyroid mailignancies:papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), undifferentiated(anaplastic) thyroid carcinoma(UTC) and medullary thyroid carcinoma(MTC). PTC is the most common malignant thyroid neoplasm and comprises 60% to 70% of all thyroid malignancies. At present, although the mechanism of carcinogenesis of thyroid cancer makes some progress, the specific molecular mechanisms remains unclear. Tumorigenesis is a long-term complex process in which multi-factor, multi-stage and multiple genes are involved. Changes in gene sequences such as gene mutation, gene loss of heterozygosity, microsatellite instability can lead to tumors, but It has recently found that certain tumor suppressor genes are not aware of its complete gene sequence mutations, deletions and other changes.Using of classic genetic control theory is difficult to explain this phenomenon. Epigenetics is the level of gene expression whose occurs can be inherited without changing the DNA sequence.It includes DNA methylation and histone acetylation and DNA methylation is the main form of epigenetic regulation.DNA methylation is established as the third mechanism by which tumor suppressor genes are inactivated. Recent studies show that the existence of inactivation in many tumors, including lung cancer, cholangiocarcinoma, pancreatic cancer and hepatocellular carcinoma, with its mechanism of inactivation of the gene promoter region CpG island methylation. But by now, there is not any evidence to show that the relationship between status of methylation of RUNX3 (human runt-related transcription factor 3) gene promoter and expression of its protein in papillary thyroid carcinoma (PTC). In the present study, we compared the methylation status of the Runx3 gene promoter as well as the expression of its mRNA, protein by using MSP, RT-PCR, and Western blot technique, and investigated the correlation of the promoter methylation of Runx3 gene patho-clinical features of PTC. We use MSP, RT-PCR, Western-blot to discuss their role as a tumor-suppressor gene in the invasion and metastasis of the papillary thyroid carcinoma. A better understanding of Runx3 promoter methylation and phenotypic expression will provide new insights into PTC carcinogenesis, cancer treatment, and feasible chemopreventive measures for the future.Material and Method1. 56 cases of papillary thyroid carcinoma (PTC) and non-carcerous epithelium (NCE) had been collected from January 2008 to March 2010 at thyroid surgery of the first affiliated hospital of Zhengzhou University. All cases are diagnosed by experienced pathological doctor, all of these have not been accepted any treatment before operation. One part of specimens are put into -80 fridge to preserve for the extraction of DNA and methylation, the others are for RT-PCR and Western-blot.2. The methylation of promoter and expression of Runx3 gene in papillary thyroid carcinoma are detected by methylation specially polymerase chain reaction(MSP) and RT-PCR and Western-blot.3. SPSS13.0 statistical software is used for statistical analysis. The relative quantitative RT-PCR results were expressed as mean and standard deviation and tested for statistical significance with Student's two-tailed t-test.Significant differences between the expression of Runx3 and clinicopathological parameters were compared by the X2-test.All P-values were derived from two-sided tests and a probability less than 0.05 was considered statistically significant.Results1. In NCE, there was no promoter methylation of RUNX3 gene, while in PTC the rate of it was 35.7%(20/56). There was significant difference between the two groups (P<0.01). The rate of promoter methylation of RUNX3 gene was related to the TNM stage, tumor pathological grade and lymph node metastasis (P<0.05), but not to other clinicopathological features in PTC (.P>0.05).2. We found that Runx3 mRNA was expressed in all the adjacent non-carceious epithelium but was not detectable in 32 of 56 papillary thyroid carcinoma. There is significant difference between the expression of Runx3 mRNA in PTC specimens with that in NCE(P<0.05). The mRNA expression of RUNX3 gene was related to the tumor pathological grade and lymph node metastasis(P<0.05), but not to other clinicopathological features in PTC(P>0.05).3.34 cases of 56 PTC showed a reduced or absent Runx3 protein expression. There is significant difference between the expression of Runx3 protein in PTC specimens with that in NCE(P<0.05) The protein expression of RUNX3 gene was related to the tumor pathological grade, TNM stage and lymph node metastasis(P<0.05), but not to other clinicopathological features in PTC(P<0.05).4. The promoter methylation of Runx3 gene is inversely correlated with its mRNA expression in PTC (P<0.05); The mRNA expression of Runx3 gene is positive correlated with its protein expression in PTC (P<0.05)Conclusions1. In our study, in PTC, the promoter methylation is an important inactive mechanism of RUNX3 gene, which is related to the tumor pathological grade, TNM stage and lymph node metastasis, and the results indicate that RUNX3 gene may play important roles in carcingenesis and development of PTC, and may be one of candidate tumor suppressor genes.2. In PTC, the promoter methylation is an important inactive mechanism of Runx3 gene, which play a role in the invasion and metastasis of the papillary thyroid carcinoma.Runx3 expression could serve as a usefull biomarker in evaluating the biological behavior of Runx3 and have clinical utility in devising innovative treatment strategies.3. The methylation and expression of RUNX3 are related to the tumor pathological grade and lymph node metastasis. It suggests that loss of RUNX3 protein may involve in the tumor progression and be useful to estimate the prognosis of PTC.