Study On The Mechanism And Correlation Of CHFR And Runx3 Gene Promoter Methylation And Expression With Pathobiological Characteristics Of Gastric Carcinomas

ObjectivesGastric carcinoma is one of the most common malignancies in the world.As the other malignant tumors,gastric carcinogenisis is a multigene and multistep involved pathological course.The correlated genes are mostly oncogenes,tumor suppressor genes,and DNA mismatch repair genes.Tumor suppressor genes may loss their functions by the way of gene mutation,loss of heterozygosity and methylation of promoters.DNA methylation is the methyl transfer,which is supplied by Sademetionine (SAM),to the specific bases under the catalysis of DNA methyltransferase(DNMT).DNA methylation of eukaryote is a way of regulating gene expression.DNA aberrant methylation can change the chromosomal structure,DNA stability,cause gene expression abnormalities,and affect cellular proliferation and differentiation.The level DNA mehtylation of eukaryote is closely correlated with tumor.Hyermethylation in promoter region causes silencing of suppressor genes,which is the key factor in carcinogenesis.Recently,some studies have shown that CHFR is an important tumor suppressor gene and its encoding product is ubiquitin ligase of Plk1. Plk1 regulates both the Weel kinase and the Cdc25C phosphatase,which in turn control the Cdc2 kinase activity at the G2 to M transition.The CHFR gene can ubiquitinate and degenerate the Plk1,which prevents cells from entering prophase and metaphase.CHFR gene expresses in normal tissue while it is low expressed or inactivated in cancer tissue, in which losses the ability to prevent abnormal cells proliferating from G2 to M phase, thus cells abnormal differentiation and proliferation occur.Some studies also show that runx3 protein coded by Runx3 gene(runt-related transcription factor 3 gene) is a regulator in the downstream of TGF-βsignal pathway.TGF-βis the effective suppressor of many cells,and the abnormality of TGF-βsignal pathway can cause carcinogenesis of many cancers.In this study we employed MSP,RT-PCR,Western blotting and immunohistochemical methods to detect the methylaion status of CHFR gene and Runx3 gene and the expression of mRNA and protein in gastric cancer tissue and normal gastric mucosa.We analyzed aberrant methylaion of the CHFR and Runx3 gene and their possible correlations to the mRNA and protein expression,further discussed the correlation between promoter methylaion of CHFR gene and Runx3 gene and mRNA abnormal expression,as well as pathologic characteristics in human gastric cancer.The studies may offer experimental basis to elucidate the effect of CHFR gene and Run3 gene in occurrence and development of human gastric cancer.Samples and Methods1.SamplesSamples:One hundred and fifty-one samples of gastric cancer tissues were obtained from Department of Surgical Oncology of the First Affiliated Hospital of China Medical University and Liaoning Cancer Hospital during December 2003 to May 2004.Besides,fresh gastric cancer tissues of thirty-six cases were taken and paired normal gastric mucosa taken at least 5 cm away from the cancer margin were collected simultaneously from Department of Surgical Oncology of Liaoning Cancer Hospital during March to September 2007.These samples were placed in liquid nitrogen immediately and then stored at -70℃refrigerator until analysis.All the samples were diagnosed pathologically,and all patients didn't receive either chemical treatment or radiotherapy before operations.The gastric cancer tissues and paired normal gastric mucosas of twenty cases with integrated data were used for experiment.2.Methods (1) Constructing gastric cancer tissue chipTissue chips were made by tissue chip maker(Miroarrayer,Beecher Instruments, USA).Five TMA blocks numbered 1 to 5 containing gastric cancer and precancerous lesions were constructed.Block 1 contains 117 tissue samples;Block 2 contains 109 tissue samples;Block 3 contains 110 tissue samples;Block 4:contains 101 tissue samples;Block 5 contains 124 tissue samples.The TMA blocks were cut continuously and all sections were within 4μm.Sections for immunohistochemical stain were fixed on the slides processed by 0.1%polylysine to prevent slipping.Sections were baked in 60℃for 1 hour,and then continued baking in 58℃for 18 hours.After baking,the sections were kept in room temperature for further use.(2) Immunohistochemical staining for CHFR and mP53 protein expressionTissue chips of gastric cancer and precancerous lesions were immunohistochemical stained by Envision method.Immuno-Bridge kits,rat anti- human CHFR monoclone antibody(working dilution 1:75) and rat anti-human P53 monoclone antibody(ready to use) were bought from Abnova Company and Beijing Zhongshan Jinqiao biothchnique limited company,respectively.All steps were accomplished in accordance with the instructions.PBS(0.01mol/L,pH7.4 used instead of specific antibodies for negative control.Gradingof immunohistochemical stain:the positive staining signals of the CHFR and mP53 protein were yellow brown granules and located in cytoplasm and nucleus respectively.For each sample,two representative high power fields were observed.Positive rate was assessed by the percent of positive cells in all counted 100 cells,which was taken their average value together from two representative high power fields.Positive cells≤20%of total counted cells were determined negative while positive cells＞20%of total counted cells were determined positive.(3) RT-PCR of CHFR and Runx3 genesTotal RNA extracted by using TRIZOL reagent was used for cDNA synthesis.RTPCR was performed with primers of CHFR,Runx3 gene orβ-actin.(4) Methylation -specific PCR for methylation level TIANamp Genomic blood/cell/tissue genomic DNA kit was used to extract DNA. Genomic DNA was modified by sodium bisulfite.After DNA purification,PCR was performed by using methylated and unmethyiated product of CHFR and Runx3 gene as primers.(5) Western blotting for protein encoded by CHFR and Runx3 genesLysis buffer solution was added into tissue at the proportion of 5:1 to extract protein.Protein density was determined by Coomassie brilliant blue.And then12% SDS polyacrylamide gel electrophoresis was performed.Nitrocellulose membrane sheet transferred for 2 hours,and incubated with primary antibody(CHFR1:400,Runx3 1:300) for 2 hours,further incubated with horseradish peroxidase signed secondary antibodies in room temperature for 2 hours.Immunological straps were finally shown with immunoblotting chemoluminescene reagent(ECL reagent).The electrophoretic results were scanned into images.Data were collected by BANDSCAN5.0 software and the ratio of strip density toβ-tubulin density served as index for statistical analysis.3.Statistical analysisThe data was processed using SPSS 13.0 statistical software.Quantitative data were expressed with mean±standard deviation(SD).Data were analyzed by Fisher's exact x~2 test,independent-samples t-test and Spearman rank related test.P values less than 0.05 were considered to be statistically significant,and P values less than 0.01 were remarkably significant.Results1.The expressin of CHFR and mP53 gene coded protein in human gastric cancerThe positive rate of CHFR protein in gastric cancer tissues(49.67%,75/151) was significantly lower than in normal gastric mucosa(85.25%,52/61)(P＜0.05).The down-regulation or loss of mitotic CHRF protein expression in gastric cancer was closely correlated with gender of gastric cancer patients.The negative rate of CHFR protein expression in the female gastric cancer patients was significantly higher than that in the male gastric cancer ones(64.00%vs 43.56%,P＜0.05).The negative rates of CHRF protein expression were also significantly different between gastric cancer patients of BorrmanⅢ+Ⅳand BorrmanⅠ+Ⅱtypes(57.14%vs 34.78%,P＜0.05).In the present study,although CHFR protein expression showed no significant differences among various histological types of gastric cancer,the negative rate of CHRF protein expression was the highest in signet ring cell carcinoma(71.43%,5/7).The negative rate of CHFR protein expression was not correlated with the depth of invasion and lymph node metastasisof gastric cancer.In addition,no correlation was found between the expressionof CHFR protein and mP53 protein expression in gastric cancer(P＞0.05).2.Aberrant expressions of the CHFR and Runx3 gene in gastric cancer tissuesCHFR mRNA expression was down-regulated in gastric cancer tissue (0.2186±0.2113) compared with that in normal gastric mucosa(0.7020±0.2163) and the difference between them was significant(t=7.148,P＜0.001).Among them,CHFR mRNA expression in 14 cases of poorly differentiated gastric cancers tissues (0.1364±1.772) was significantly lower than that(0.4106±0.1574) in 6 cases of well-differentiated group(t=3.276,P＜0.01).Runx3 mRNA expression in gastric cancer (0.1755±0.1877) was significantly lower than that in normal gastric mucosa (0.6195±0.2225),t=6.822,P＜0.001.Among them,Runx3 mRNA expression in 14 cases of poorly differentiated gastric cancers(0.1114±0.1863) was significantly lower than that(0.3250±0.0725) in 6 cases of well-differentiated group(t=t=2.514,P＜0.05).In addition,Runx3 mRNA expression in gastric cancers which infiltrated throughout muscular layer was significantly lower than non-infiltrated group (0.1113±0.1329 vs 0.3660±0.2084,t=3.224,P＜0.05).Runx3 mRNA expression in gastric cancer tissues with lymph node metastasis was significantly lower than that without lymph node metastasis(0.0750±0.1054 vs 0.3263±0.1867,t=3.850,P＜0.01). However,the mRNA expression levels between the CHFR and Runx3 genes showed no significant correlation(P＞0.05).3.The CHFR and Runx3 protein expressions in gastric cancer tissuresData were collected by BANDSCAN5.0 software,the ratio of strip density toβ-tubulin density served as index for statistical analysis.The down-regulation or loss rate of CHFR gene coded protein was 70.00%(14/20).The difference in relative optical density value between gastric cancer and normal gastric mucosa was significant (0.2435±0.2620 vs0.5955±0.2196,t=4.605,P＜0.001).Among them,the level of CHFR protein expression in poorly differentiated group was significantly lower than that in well-differentiated group.Their relative optical density values were respective 0.1143±0.1224 vs 0.5447±0.2573,and the difference between them was significant (t=5.162,P＜0.01).The down-regulation or loss rate of Runx3 protein expression in gastric cancer was significantly higher than that in normal gastric mucosa(65.00%, 13/20 vs 5.00%,1/20).The difference in relative optical density value between gastric cancer and normal gastric mucosa was significant(0.2700±0.3877 vs 0.5860±0.2784, t=2.961 P＜0.05).The relative optical density value of Runx3 protein expression in well differentiated group(0.6930±0.3449) was significant higher than that in poorly differentiated group(0.0893±0.238),and the difference between them was significant(t =4.546,P＜0.01).The relative optical density value of Runx3 protein expression in gastric cancers which infiltrated through muscular layer was significantly lower than non-infiltrated group(0.1485±0.3 vs 0.6360±0.3576,t=2.851,P＜0.05);Runx3 protein expression in gastric cancer with lymph node metastasis was significantly lower than that without lymph node metastasis(0.0683±0.2107 vs 0.5848±0.3887,t=3.916, P＜0.01).4.The aberrant methylation of CHFR and Runx3 promoters in human gastric cancerThe methylation rate of CHFR gene in gastric cancer tissue was 45.00%(9/20).As a control,CHFR methylation was screened for the corresponding normal gastric mucosas of these same patients.No methylation was detected in these tissues.The difference between gastric cancer tissue and normal gastric mucosa was significant (P＜0.001).The CHFR gene methylation rate in poorly differentiated gastric cancer was significantly higher than that in well-differentiated group(P＜0.05).The Runx3 gene methylation rate(65.00%,13/20) in gastric cancer tissue was higher than that(5.00%, 1/20) in normal gastric mucosa(P＜0.001).The aberrant methylation of the Runx3 gene was significantly correlated with differentiation degree,depth of tumor invasion and lymph node metastasis(P＜0.05).5.The correlation of CHFR and Runx3 gene methylation with their mRNA and protein expressionCHFR mRNA expressive levels in 9 gastric cancer samples with CHFR methylation were all down-regulated or lost,while in 11 gastric cancer samples with unmethylated CHFR gene,the mRNA expressions were down-regulated only in 5 samples(r=0.592,P＜0.05).The mRNA expressions in 12 out of 13 gastric cancer samples with the aberrant methylation of Runx3 gene were down-regulated or lost, while in 7 gastric cancer samples with unmethylated Runx3 gene,the mRNA expressive level was down-regulated only in 1 sample(r=0.780,P＜0.001).CHFR protein expressions in 9 gastric cancer samples with aberrant methylation of CHFR gene were all down-regulated or lost,while in 11 gastric cancer samples with unmethylated CHFR gene,only 5 samples were down-regulated(r=0.592,P＜0.05). Runx3 gene coded proteins in 13 gastric cancer samples with aberrant methylation of the gene were all down-regulated or lost,while in 7 gastric cancer samples with unmethylated Runx3 gene,none was down-regulated(r=1).The results of this study suggested that aberrant methylation of the CHFR and Runx3 gene is the main cause of down-regulation or loss of their mRNA or coded protein expression.Conclusions1.The results of the CHRF protein expression in gastric cancer tissues of 151 cases show that the down-regulation or loss of the mitotic CHRF protein expression occurs frequently in gastric cancer.It may involve in the carcinogenesis of gastric cancer and closely correlate with gender or more invasive type of human gastric cancer.2.The methylation rates of CHFR and Runx3 genes in gastric cancer tissues were all significantly higher than those in their normal gastric mucosas,suggesting that they may involve in gastric carcinogenesis and development.In addition,the aberrant methylation of CHFR and Runx3 promoters in gastric cancer tissues is the main cause of down-regulation or loss of their mRNA or coded protein expression.Thus,the detection of CHFR and Runx3 genes will be helpful to early diagnosis of gastric cancer.3.The mRNA expression levels between the CHFR and Runx3 genes showed no significant correlation in gastric cancer tissues,but they were all correlated with differentiation degree of the gastric cancers.Both them may participate the histological differentiation of gastric cancer by various mechanism of action.Besides,down-regulation or loss of the Runx3 mRNA expression in gastric cancer tissures was significantly correlated with the depth of tumor invasion and lymph node metastasis, which can help predicting the patient's prognosis and for better clinic treatment.