Relationship Between Expression And Promoter Methylation Of P16～(INK4a) Gene In Ameloblastoma

IntroductionEpigenetic code is the switch in gene expression by histone modification and DNA methylation, other than alteration in the primary nucleotide sequence of a gene. DNA me thy ltransferase (DNMT) add a methyl group to the cytosine to form methyl CpG islands, blocking transcription factor and transcription re-pressors attach to the DNA double - stands, adjusting the type and number of transcription factor attach to the gene promoter regions, could decide the gene is expressed and the amount. Thus, DNA methylation is an important way to adjust the gene expression. Cyclin dependent kinase inhibitors (CDKI) bind to and inhibit CDK, arrest cell cycle in the G1 - S or G2 - M checkpoint, to inhibit cell proliferation. Most cacer cell maintain limitless replicative potential by inactivating CDKI expression. The INK4 gene family ( pl6^INK4a, pl5^INK4b , p18^INK4c, pl9^INK4c in CDKI, not noly could inhibit cyclin D — CDK4/6 compound, but also could boost CIP/KIP family (p21^CIP/WAF1, p27^KIP1, p57^KIP2) expression by ARF - MDM2 - p53 path, arrest cell go into S phase together, pl6^INK4a is the classical delegate. In many cancer cell, pl6^INK4a gene could be found loss expression, paralleling cell high reproductive activety.Ameloblastoma ( AB) is the most occurring odontogenic tumor, and it may progressively invade the jaw, recur locally and transform malignantly. It destroys jaw greatly, not only impact the function of taking food, but also damage the face of patient terrible. Its histopathological origin may be enamel organ, odontogenic epitholium, etc. How it losing the ablity to goto diffeentiation and maturation, the molecular mechanism has not been completely clarified. We detectedthe methylation level of pl6 a promoter, and the expression of its mRNA and protein, so as to explore the pathway to aberrant high reproductive activety, and to provide some theoretic basis for the gene taget treatment of AB.Materials and methodsThe specimens used by in situ hybridizztion and immunohistochemical method were surgically removed from 54 patients with AB ( malignant AB 4 cases) , normal oral mucosa 11 cases, at the First Affiliated Hospital of China Medical University between 1996 -2005. The freeze specimens include AB 12 cases (malignant AB 2cases) , and normal oral mucosa 11 cases. We detected pi6 mRNA by in situ hybridization, and its protein by S - P immunohistochemical method, the DNA methylation by MSP. the result score: ( 1 ) IHC: The numbers of positive cells was used to evaluate the intensity of immunoexpression , <25% positive ( + );25% -49% positive ( + + );2=50% positive ( + + + ) o (2) ISH: the extent of staining was , < 10% positive( - );10 40% positive ( + );40 70% positive ( + + );^ 70% positive (+ + +).(3) MSP: PCR product from unmethylated primers is unmethylated positive, product from methylated primers is methylated positive. Chi — square test and analysis of variance were used to solve the data, and Kendall correlation was determined between pl6 DNA methylation, mRNA, protein expression. All statistical analysis was performed with SPSS 11.5 software package. The probability of P <0. 05 was regarded as statistically significant.Results1 IHC: In the normal oral mucosa, the expression of pl6INK4a protein was scarce in basal layer cell, stronge positive 90. 9% ( 10/11 ) in spinosum layer cell. In the cases of ameloblastoma, the expression of pl6 protein is scarce in the peripheral cylindrical epithelial cells and central polyhedral cells, presented more weak positive in the squamous metaplasia keratinizing cells, and the positive rate is 30. 3% (12/40). There was a stastical difference of the the expres-sion pl6 protein between the normal oral mucosa and AB( P <0. 001).2 ISH;In the normal oral mucosa, the expression of pi6 a mRNA was positive in basal layer cell, and the positive rate is 81. 8% (9/11) . In the cases of ameloblastoma, the expression of pi6 mRNA is weak and moderate positive in the peripheral cylindrical epithelial cells, and the positive rate is 31. 5% (17/ 54). There was a stastical difference of the the expression pi 6 mRNA between the normal oral mucosa and AB(P < 0.001).3 MSP: all specimens exhibit unmethylated PCR product, while the positive rate of methylated PCR product is low. The ratio DNA methylation of pl6INK4a in normal oral mucosa is 9. 1% (1/11) , in AB was 25% (3/12) , respectively. There was no stastical difference between them. With the ratio DNA methylation of pl61NK4a increasing in AB, the rate of pl6INK4a mRNA and protein expression decreaseing, there was a negative correlation between DNA methylation and mRNA or protein expression (rk= —1.0, — 1.0, P <0. 001). There was a correlation between the expression of pl6 mRNA and protein (rk = 1. 0,P<0.001). There were no significant differences among age, sex, position and histological type in the DNA methylation and expressiong of pl6Conclusions1 pi6 a promoter methylation inactivates gene express, it is maybe one of reason AB cell proliferation disordering.2 Low methylation rate of pl6INK4a promoter and high expression in normal oral mucosa, illuminate its development need pl6INK4a gene regulate.