| Objective: Gastric cardia adenocarcinoma (GCA) is one of the frequentkind of malignant tumor in the northern China. In resent years, the mortality ofGCA is still increasing in our country. It is hard to achieve in early diagnosisof the tumor so that majority of patients treated in hospital has been in the latestage. Furthermore, early GCA is hard to achieve so that majority of patientstreated in hospital are in the late stage. So far, the pathogenesis of GCA is stillremain unclear.DNA methylation has become one of the most common epigeneticmechanisms of gene regulation. In this study, we investigated the promoterand exon1methylation of insulin-like growth factor binding protein7(IGFBP7)in GCA and corresponding normal tissues, and we tried to explore itscorrelation with carcinogenesis, infiltration, metastasis and pathologicaldifferentiation of GCA. Thus we focused on the study of epigeneticmodulation of IGFBP7expression in GCA, to provide a new theory andexperimental evidence for pathogenesy, gene therapy and immunotherapy,clinical prognosis of GCA.Methods:1Methylation specific PCR (MSP) method was used to examine themethylation status of the5' CpG island of IGFBP7gene in85GCA tumortissues and67corresponding normal tissues.2Reverse transcription polymerase chain reaction (RT-PCR) method wasused to detect the mRNA level of IGFBP7gene in GCA tumor tissues andcorresponding mormal tissues.3Immunohistochemistry method was used to examine the proteinexpression of IGFBP7in GCA tumor tissues and corresponding normaltissues. 4SPSS13.0was applied to analyze the results of experiment.Results:1The relationship between methylation status of IGFBP7and GCA clinicalpathology:1.1The methylation status of IGFBP7promoter:For the promoter site, methylation frequency of IGFBP7in tumorspecimens (52.9%,45/85) was significantly higher than that in correspondingnomal tissues (35.8%,24/67)(P<0.05). Methylation frequencies of IGFBP7gene in moderate and poor differentiation group60.9%(39/64) was muchhigher than that in high differentiation group33.3%(7/21)(P<0.05).Methylation frequencies of IGFBP7in lymph node metastasis group (63.5%)was significantly higher than that in non lymph node metastasis group (36.4%)(P<0.05). Methylation status of IGFBP7gene was not associated with depth ofinvasion, age, and gender (P>0.05).1.2The methylation status of IGFBP7exon1:For the5' UTR of exon1, methylation frequency of IGFBP7in tumorspecimens (35.3%,30/85) was significantly higher than that in correspondingnormal tissues (19.4%,13/67)(P<0.05). The methylation frequencies ofIGFBP7gene was not associated with differential degree, status of lymphnode metastasis, depth of invasion, group of age and gender (P>0.05).1.3Methylation analysis of two site in common:Twenty-six tumor specimens and13corresponding normal tissues showedsimultaneous methylation of IGFBP7in the promoter site and exon1site. Thesimultaneous methylation frequency of IGFBP7did not show any significantdifference between tumor tissues and corresponding normal tissues (P>0.05).Methylation frequency of IGFBP7in the promoter site was significantlyhigher than that in the5' UTR of exon1(P<0.05). In tumor, simultaneousmethylation rate of the two sites was not associated with lymphatic metastasisand pathology grades(P>0.05).2The mRNA expression of IGFBP7:IGFBP7mRNA expression in tumor tissues (0.50±0.33) was significantly lower than that in corresponding normal tissues (0.90±0.29)(t=-3.759,P=0.001). The mRNA expression of IGFBP7was correlated with status oflymphatic metastasis and pathology grades (P <0.05).3The protein expression of IGFBP7:The protein expression of IGFBP7in tumor specimens (26.8%,15/56) wassignificantly lower than that in corresponding normal tissues (80.0%,24/30)(P=0.000). The protein expression of IGFBP7was significantly associatedwith pathological differentiations of GCA (X2=10.957, P=0.001). However, itwas not associated with lymphatic metastasis (P>0.05).4Relationship between methylation status of IGFBP7and its expression ofmRNA and protein:4.1The relationship between methylation status of IGFBP7promoter and itsexpression of mRNA and protein:The mRNA expression of IGFBP7in methylated tumor tissues (0.27±0.06)was significantly lower than that in unmethylated tumor tissues (0.80±0.28)(P<0.05). The positive protein expression of IGFBP7in methylated tumortissues (10%,3/30) was significantly lower than that in unmethylated tumortissues (57.1%,12/21)(P<0.05). IGFBP7protein expression was inverselycorrelated with its promoter methylation status (R=-0.509, P<0.05).4.2The relationship between methylation status of IGFBP7exon1and itsexpression of mRNA and protein:The mRNA expression of IGFBP7in methylated tumor tissues (0.43±0.29)did not show any significant difference from that in unmethylated tumortisssues (0.53±0.35)(P>0.05). The protein expression of IGFBP7inmethylated tumor tissues (23.3%,7/30) also did not show any significantdifference from that in unmethylated tumor tisssues (44.4%,8/18)(P>0.05).The protein expression of IGFBP7was not correlated with its methylationstatus of exon1site(R=-0.220, P>0.05).Conclusions:1Compared to the5' UTR of exon1, IGFBP7promoter site was more likely tobe methylated in GCA and may play an important role in the down-regulation of IGFBP7.2Methylation of IGFBP7in promoter may be one of the mechanisms that leadto the occurrence of GCA.3The methylation status of IGFBP7promoter was not correlated with age,gender and depth of invasion of patients. However, it was significantly relatedto histological type and lymph node metastasis, indicating that promotermethlation of IGFBP7may be associated with tumor prognosis.4There seemed to be a correlation between the low expression of IGFBP7andGCA, and hypermethylation of IGFBP7promoter may be one of the reasonsthat lead to down-expression of IGFBP7in GCA. |
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