The Critical Regions And Spreading Of Methylation Within Runx3 CpG Island In Human Gastric Cancer

INTRODUCTIONRunx3(PEBP2αC/CBFA3/AML2) is a newly discovered tumor suppressor gene, which is involved in the regulation of cellular growth and apoptosis as a member of transcription factors in runt domain family.A lot of research has demonstrated that Runx3 gene is related to many kinds of tumors,especially to gastric cancer these years,so it may be a marker for the diagnosis of gastric cancer and a target for gene therapy.It is now seen,however,that the methylation positive rates reported at home and abroad vary significantly,and are not fully parallel to their expressions.Thus it is proposed that the methylation of different region within Runx3 CpG island has different effect on the silencing of this gene.AIMThis study was designed to investigate the critical regions and spreading of methylation within Runx3 CpG island in human gastric cancer.MATERIAL AND METHODS1,MaterialA total of 26 human primary gastric cancer samples and corresponding non-neoplastic gastric mucosa were resected and stored at-80℃from Feb 2006 to Dec 2006 in Sheng Jing Hospital Attatched to China Medical University.Among these samples,there were 16 males and 10 females,ranging in age from 24 to 80 years with a mean age of 58.7 years.Differentiation extent:there were 17 poorly differentiated (undifferentiated or low differentiated)samples and 9 well differentiated(moderately or high differentiated)samples. Stagings of UICC(1997):there were 7 of stageⅠ,4 of stageⅡ,8 of stageⅢand 7 of stageⅣ.2,Methylation specific PCR(MSP)MSP was used to detect the methylation status of successive six regions ranging from the most 5' region to transcription start site within Runx3 CpG island in the above samples,then the positive rates of all regions were calculated.3,Western blotWestern blot was used to detect the expression levels of Runx3 protein in the above samples,then the gray values of Runx3 protein/the gray values of Tubulin were used as relative expression levels of Runx3 protein for statistical analysis.4,Statistical analysisThe results were processed by x~2-test(or Fisher exact probability test) and t-test of SPSS14.0 software,and P values of less than 0.05 were considered to be statistically significant.RESULTS1,The results of methylation specific PCR(MSP)The methylation positive rates of the above successive six regions calculated by the results of MSP decreased gradually with the regions spreading to transcription start site.In gastric cancer group,the 1st region was 92.31%(24/26), the 2nd region was 88.46%(23/26),the 3rd region was 65.38%(17/26),the 4th region was 61.54%(16/26),the 5th region was 46.15%(12/26),the 6th region was 46.15%(12/26), and in non-neoplastic group,the 1st region was 96.15%(25/26),the 2nd region was 84.62%(22/26),the 3rd region was 50%(13/26),the 4th region was 46.15%(12/26),the 5th region was 19.23%(5/26),the 6th region was 11.54%(3/26).The differences between cancer group and non-neoplastic group arised in the 3rd region,and became significant in the 5th and 6th region(p<0.05).And when the grouping was related to the differentiation extent,the differences were significant in the 3rd-6th regioin between poorly differentiated group and well differentiated group(p<0.05).2,The results of western blotThe relative expression level of Runx3 protein was 0.499±0.106 in gastric cancer group,0.721±0.080 in non-neoplastic group, 0.437±0.053 in poorly differentiated group and 0.617±0.073 in well differentiated group.The differences were significant between gastric cancer group and non-neoplastic group,as well as between poorly differentiated group and well differentiated group(p<0.01).DISCUSSIONRunx3 is a newly discovered tumor suppressor gene,and some research has revealed that hypermethylation within promoter CpG island is one of main patterns for the silencing of this gene.In this study we detected the methylation status of successive six regions within Runx3 promoter CpG island in 26 gastric cancers and corresponding non-neoplastic gastric mucosa by MSP,then the methylation positive rates of all regions were calculated. Since there were no significant differences in the 1st and 2nd region and there were appreciable differences in the 3rd-6th region(the differences in the 5th and 6th region were significant) between these two groups,the regions studied in previous research may be within this segment.The methylation positive rates of the regions within this segment in this study were 42.31-65.38%,which were in accordance with the results reported previously.It is seen that the Runx3 protein is frequently down-regulated in gastric cancers.In this study we detected the expression levels of 26 gastric cancers and corresponding non-neoplastic gastric mucosa by western blot.It was also in accordance with the results reported previously that in this study the expression levels of Runx3 protein in gastric cancer group were lower than those of non-neoplastic gastric mucosa group,and the expression levels of Runx3 protein were related to the differentiation extent of tumor.Now the methylation positive rates reported at home and abroad vary significantly, and are not fully parallel to their expressions.This indicates that the methylation of different region has different effect on the silencing of this gene and there may be critical regions,whose methylations can silence this gene. The results of this study revealed that there were significant differences in the expression levels of Runx3 protein between gastric cancer group and non-neoplastic gastric mucosa group,as well as between poorly differentiated group and well differentiated group(p<0.01),and there were also significant differences in methylation positive rates under this circumstance(there were significant differences in the 5th and 6th region between gastric cancer group and non-neoplastic gastric mucosa group,as well as in the 3rd-6th region between poorly differentiated group and well differentiated group,p<0.05).When the groupings were related to gender,age and clinical stages,there were no significant differences in the expression levels of Runx3 protein,and there were also no significant differences in methylation positive rates between regions under this circumstance.The above results showed the methylation of transcription start site(the 5th and 6th region described in this study) can cause the down-regulation of Runx3 protein,so this site may be critical regions for the methylation of Runx3 gene.It was also seen that the methylation positive rates of successive six regions ranging from the most 5' region to transcription start site in this study decreased gradually,which indicated the methylation may occur in the most 5' region within Runx3 CpG island initially,and then spread to transcription start site step by step.Further analysis showed that methylation positive rates of all regions were not related to gender,age and clinical stages,but the positive rates of the 3rd-6th regions were related to the differentiation extent of tumor,which indicated that the spreading of methylation may be related to the differentiation extent of tumor,except for gender,age,and clinical stages.CONCLUSIONThe methylation spreads from the most 5' region to transcription start site within Runx3 gene promotor CpG island of human gastric cancers,and the spreading is related to the differentiation extent of tumor,except for gender,age and clinical stages.The transcription start site may be critical regions for the methylation of Runx3 gene.