Promoter Methylation Of RASSF1A Gene In Endometrial Carcinoma And Protein Expression

Endometrial carcinoma is a common malignant tumor of female reproductive system. It is leading cause of incidence rate in female reproductive system on developed countries ,and the death rate is second . High-risk age is 50～61years. In recent years, due to the combined effect of environmental and lifestyle factors,endometrial cancer incidence trends showed younger. But the exact cause of endometrial cancer is still unclear. All the relevant factors widely recognized are: long-term sustainability of estrogen stimulation,patient's own physical factors such as obesity, diabetes, hypertension, infertility or menopause, and genetic factors delay. The incidence of endometrial cancer is a comple result with many factors such as molecular biological changes.With the human genome sequencing project successfully completed, a research for genetic changes of endometrial cancer from the molecular level to become a hotspot in recent years.DNA methylation refers to ransfering Methyl group to the cytosine ring carbon atom No. 5, under the action of the DNA methyltransferase (DNMT).It's currently the only known mammalian way of the natural chemical modification, its essence is a Chemical modification of DNA , provided that does not change DNA base sequence,Thus affecting the downstream product of gene expression, eventually leading to cancer. DNA methylation occurres in the CpG island sites. CpG island is the DNA sequence that length > 200bp, C + G content≥50%,located in the promoter region ofsequence. CpG island of normal cells is in a non- methylation status. When the tumor suppressor gene promoter CpG island methylation, causing gene silencing, tumor suppressor loss of function.More and more studies have shown, DNA promoter methylation is one mechanisms of tumor occurrence and development. Ras associated domain family 1A gene organization (RASSF1A) is a new tumo suppressor gene discovered in 2000, the cDNA encoding the protein product of a molecular weight of 38,800 peptides, 100% in the normal tissue expression, RASSF1A proteins may be involved in cell cycle regulation, through a multi-Ways to inhibit cell growth, differentiation and promote cellular senescence,apoptosis. As a result of gene promoter methylation, expression of encoded a product of RASSF1A protein may be reduced or lost, eventually leading to cancer.Objective : About RASSF1A gene methylation and study the relationship between endometrial cancer rarely reported in China, this paper aims to detect endometrial cancer MSP in the promoter region of RASSF1A gene methylation status and protein expression of RASSF1A, RASSF1A gene revealed Promoter methylation and the occurrence of endometrial cancer, the development of relations.Methods: 40 patients, tissue with endometrial cancer and 40 cases of fresh surgical specimens of normal endometrial tissue are collected from the Third and the fourth Hospital of Hebei Medical University in 2008-2010, All specimens were pathologically confirmed, patients with endometrial cancer have no preoperative radiotherapy and chemotherapy before surgical.Nomal patients with no abnormal uterine bleeding before surgery, hormone therapy within six months. fresh specimens immediately after taking out -80℃refrigerator to prepare for extraction of tissue DNA, while part of the fresh tissue samples were fixed 4% neutral formaldehyde solution, embedded in paraffin, 4μm serial sections for immunohistochemistry Used in the experiment.Methylation specific PCR (MSP) detected the methylation status of RASSF1A .MSP is the basic principle: the formation of template DNA denaturation by NAOH single chain by sodium bisulfite, hydroquinone modified, cytosine (C) can be turned into uracil (U), the DNA methylation did not occur in ( such as normal tissue) will occur C→U, DNA methylation has occurred (eg, cancer), no occurrence of C→U, cytosine remains unchanged. Sequence differences for the modification of the design before and after methylation and unmethylated primers for PCR amplification. Methylation-specific PCR (MSP), followed by extraction of tissue samples DNA, bisulfite modification, DNA purification and recovery, methylation specific PCR and other steps, detected in 40 cases of endometrial cancer (experimental group ) and 40 normal endometrial cancer (control group) in the RASSF1A gene promoter methylation status of CpG islands to compare the difference rate of methylation between the two groups, to detect endometrial cancer and RASSF1A in normal endometrium tissue protein.RASSF1A protein expression of endometrial cancer and normal endometrium are detected by immunohistochemistry: formalin fixed endometrial tissue dehydration and paraffin embedding have been washed, 4μm serial sections. RASSF1A protein expression in immunohistochemical staining in the cytoplasm, the brown-yellow granules, showed diffuse distribution, especially in the expression of glands at the most obvious. Criteria of positive expression: View of the high magnification (×400), randomly selected from five horizons, less than 100 cells per field were calculated the percentage of positive cells and cell staining intensity, and to score: Score Positive cells Dyeing depth 0 <5% Colorless 1 5～25% jasmine 2 26～50% Yellow 3 51～75% Tan 4 >75% RASSF1Aprotein expression = percentage of positive cells×staining depth: 0～2 Negative (-) 3～8 Positive (+) 9～12 Strong positive (+ +)Results:1 RASSF1A promoter methylation status: In 40 cases of endometrial carcinoma, 27 cases of cancer tissue occurred methylation of RASSF1A gene promoter methylation,methylation rate is 67.5% (27/40); 40 cases of normal endometrium, 5 Cases of organizations have RASSF1A gene promoter methylation, methylation frequency is 12.5% (5 / 40). Endometrial cancer gene promoter methylation of RASSF1A was significantly higher than the normal endometrium, the difference was statistically significant (χ~2 = 25.208, P = 0.000 <0.01).2 RASSF1A protein expression: 40 cases of endometrial carcinoma, 17 cases of RASSF1A protein expression, the expression rate of 42.5% (17/40); 40 cases of normal endometrium RASSF1A protein expression 100% (40/40). RASSF1A protein expression in endometrial cancer rates were significantly lower than in normal endometrium, the difference was statistically significant (χ~2 = 32.281, P = 0.000 <0.01).Conclusion : RASSF1A hypermethylation and lower or loss of RASSF1A protein expression were significantly occurred in endometrial carcinoma, it can be inferred:1 RASSF1A gene promoter methylation may result in inactivation of the gene cause reduced or absent expression products and the closely related gene methylation, RASSF1A gene promoter methylation of endometrial cancer pathogenesis.2 RASSF1A protein expression decrease or loss on the incidence of endometrial cancer, played an important role.3 RASSF1A is expected to as endometrial cancer molecular marker, so as to early diagnosis of endometrial cancer and provide the theoretical basis of biological targeted therapy.