| OBJECTIVE:To investigate the methylation and expression of RASSF2A, pl6 in gastric carcinoma and matched adjacent gastric mucosa tissues, analyze the association between methylation and expression of RASSF2A, pl6. To study their functions in gastric carcinogenesis and relation with clinicopathological parameters in gastric cancers.METHODS :RASSF2A and pl6 CpG island methylation status were tested by MSP(Methylation-specific polymerase chain reaction), their mRNA expression level was measured by semi-quantitative RT-PCR(reverse transcription-polymerase chain reaction), pl6 protein expression was measured by immunohistochemistry in gastric carcinoma and matched adjacent gastric mucosa tissues.RESULTS: (1)The frequency of methylation of gene RASSF2A in gastric carcinoma was 57.14%(16/28) and significantly higher than in adjacent gastric mucosa(x~2=13.46, P<0.01);There was a significant correlation between methylation of RASSF2A and the depth of invasion. The methylation frequency of RASSF2A increased remarkably in line with the progression of the gastric carcinoma(P<0.01);Methylation of RASSF2A was found to be statistically associated with lymph node metastasis(P<0.01);The methylation frequency of RASSF2A increased from well differentiated to poorly differentiated gastric carcinoma, but showed no significance(P>0.05);No significant association of RASSF2A methylation was identified with sex, age of the patients(both P>0.05).(2) The expression level of RASSF2A mRNA in gastric carcinoma wassignificantly lower than that in adjacent gastric mucosa(T=-5.94, JD0.05);No significant association of RASSF2A mRNA was identified with sex, age of the patients(both P>0.05).(3) In 16 patients with the RASSF2A promoter CpG island methylation, 6 showed the loss of RASSF2A mRNA expression, there was a significant correlation between promoter CpG island methylation and mRNA expression(r=-0.452, P=0.016);The expression level of RASSF2A was showed down-regulated in the methylated gastric carcinoma, the difference has significance(t=3.824, P<0.01).(4) The methylation frequency of gene pl6 in gastric carcinoma and adjacent gastric mucosa was 46.43%(13/28), 7.14%(2/28) respectively, there was a significant difference between them;The methylation frequency of gene pl6 in well differentiated gastric carcinoma was significantly lower than in poorly differentiated (P<0.05);The methylation frequency of pl6 increased remarkably in line with the progression of the gastric carcinoma(P<0.05);Methylation of pl6 was found to be statistically associated with lymph node metastasis(F<0.01);There was no significant association with sex, age of the patients(both P>0.05).(5) The expression level of pl6 mRNA in gastric carcinoma was significantly lower than in adjacent gastric mucosa(T=-5.94, P<0.01);Much more frequent in poorly differentiated gastric carcinoma, down-regulation of pi6 mRNA was correlated with differentiation grade (P<0.01);The expression level of pl6 mRNA was significantly lower in gastric carcinoma with lymph node metastasis than that with no lymph node metastasis^-cO.Ol);The expression level of pl6 mRNA was gradually down-regulated with the depth of invasion increased, but showed nosignificance(P>0.05);No significant association of pl6 mRNA was identified with sex, age of the patients(both P>0.05).(6) The expression rate of pl6 protein in gastric carcinoma was 35.71 %(10/28) and significantly lower than in adjacent gastric mucosa(^2=26.53, P<0.01);Much more frequent in poorly differentiated gastric carcinoma, loss of pl6 protein was correlated with differentiation grade (P<0.01);The expression rate of pl6 protein was significant difference between lymph node metastasis and non(P<0.05);The expression rate of pl6 protein was gradually down-regulated with the depth of invasion increased, but showed no significance(jP>0.05);There was no significant association with sex, age of the patients(both i>>0.05).(7) In 13 patients with the pl6 promoter CpG island methylation, 4 showed the loss of pl6 mRNA expression, there was a significant correlation between promoter CpG island methylation and mRNA expression(r=-0.439, F=0.020);The expression level of pl6 was showed down-regulated in the methylated gastric carcinoma, with significant difference(t=4.988, P<0.01).(8).In 13 patients with the pl6 promoter CpG island methylation, 12 showed the loss of pi 6 protein expression, there was a significant correlation between promoter CpG island methylation and protein expression(r=-0.544, F=0.003).(9) Comethylation frequency of RASSF2A and pi 6 promotor CpG island was 32.14%(9/28), there was no significant relation between them(r=0.227, P=0.245).(10) Coexpression rate of RASSF2A and pl6 mRNA was 67.86%(19/28), there was no significant relation between them(r=0.036, />=0.858);The opposite light density value of RASSF2A and pl6 mRNA in gastric carcinoma was 0.827±0.238 and 1.001 ±0.378 respectively, there was no significant relation between them (T=-1.440, F=0.150).CONCLUSIONS: 1, The methylation frequency of RASSF2A and pl6 promoter CpG island was significantly higher in gastric carcinoma than in adjacent gastric mucosa tissues, methylation of RASSF2A and pl6 was correlated with depth ofinvasion, lymph node metastasis, which may play important roles not only in the gastric carcinogenesis but also in the malignant progression. Methylation of RASSF2A and pl6 may be important biomarkers in gastric carcinoma's invasion.2, The expression of RASSF2A mRNA, pl6 mRNA and pl6 protein was lower in gastric carcinoma than in adjacent gastric mucosa tissues, there was a significant correlation between promoter CpG island methylation and mRNA, protein expression. Promoter CpG island methylation may contribute to the low level or loss of RASSF2A mRNA, pl6 mRNA and pl6 protein expression.3, Much more frequent in poorly differentiated gastric carcinoma, down-regulation of pl6 mRNA and pl6 protein was correlated with differentiation grade, the methylation frequency of gene pl6 increased from well differentiated to poorly differentiated gastric carcinoma. Gene pl6 may be important role in evaluation of gastric adenocarcinoma's differentiation.4, MSP is a simple, sensitive, and specific method for determining the methylation status of gene promoter CpG island.
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