The Study Of PpENK And S100A4 And Their Methylation Status In Pathogenesis Of Pancreatic Carcinoma

Pancreatic carcinoma is associated with high malignancy and poor prognosis. Itis the reason that overall 5-year survival rate of pancreatic cancer does not exceed 5ï¼…in most studies, because it is diagnosed too late to have the chance of surgery, whichis still considered as curative therapy for pancreatic cancer today. The incidence ofpancreatic cancer is steadily increased in recent years. Mechanism of pancreaticcancer pathogenesis is still not fully understood which makes the early diagnosis andtreatment difficult.That spurs us to reveal the internal secret of pancreatic cancer andsearch the available pathway in tumor treatment.The human ppENK gene has been localized to chromosome 8, at q23-q24,which have four transcribed exons and three introns.As a neuropeptide transmittergene, the ppENK gene encodes met-enkephalin, which is a topically active inhibitoryfactor to pancreatic cancer that interacts with the opioid growth factor receptor. TheS100A4 gene was initially cloned from the mouse adenocarcinoma cell lineCSML100.As a gene,its expression is associated with the metastasis potential oftumors. S100A4, which is a member of the S100 subfamily and has been localized tochromosome 1q21, has been shown to participate in many biological phenomenasuch as tumor cell invasion and metastasis.DNA methylation plays an important role in regulating the gene transcription,and its role in carcinogenesis has been a topic of considerable interest in the last fewyears. Alteration in DNA methylation is common in a variety of tumors as well as indevelopment. So methyltransferase inhibitors have been developed as strategies oftumor therapy. The aim of this study was to explore the association of hypermethylation ofppENK and hypomethylation of S100A4 with pancreatic carcinoma, and to identifythe effects of demethylating agent on pancreatic cell lines.Partâ… . The study of ppENK and S100A4 and their methylation status inpathogenesis of human pancreatic carcinoma tissues.Methods:Human tissues of pancreatic cancer and normal pancreas were used1.ppENK methylation status was detected in the tissues of pancreatic carcinomaand normal pancrease by MSP. 2The expression of S 100A4 protein was investigatedby immunohistochemistry. 3.S100A4 methylation status was detected in pancreaticcarcinoma and normal pancreatic tissue by COBRA.4. Fisher's Exact Test was usedto analyze:â‘ The association of methylation status of ppENK gene or S100A4 withtumor stage, size, location, sex, age, smoking, drinking et al.â‘¡The association ofhypomethylation of S100A4 gene with methylation of ppENK gene.Results:1.90.3ï¼…(28/31) methlation ofppENK was detected in the pancreatic carcinomatissue in 31 patients, but it was not seen in normal pancreatic tissue. There were nocorrelation between methlated ppENK with clinicopathological features of pancreaticcarcinoma such as with tumor stage, size, location, sex, age, smoking and drinking et al.2. S100A4 protein expression was positive in 27of 32 (87.1ï¼…). 3.The expression ofS100A4 was a significant association with tumor differentiation (p=0.017). There wasno significant association between S100A4 labeling and other clinicopathologicalfeatures. 4. In the total 31 human specimens, 22 hyomethylation was observed at the(+331) CpG site, and there was a significant association between S100A4 labeling and(+331) CpG site hyomethylation (OR=16.8, p=0.017). Hypermethylation of S100A4was detected in normal pancreatic tissues. 5. (+331) CpG site hyomethylation was a highly significant correlation with CA199 (p=0.011).6. S100A4 (+331) CpG sitehyomethylation was a highly significant correlation with ppENK methylation (p=0.019).Summary:1.The Methlated ppENK rate was 90.3ï¼…in pancreatic cancer tissues and therewas no correlation between methlated ppENK and clinicopathological features. 2.Thetotal positive rate of S 100A4 is 87.1ï¼…in pancreatic cancer tissues and the expressionof S100A4 was a significant association with tumor differentiation. 3.The S100A4hypomethylation rate was 71.0ï¼…in pancreatic cancer tissues and there wascorrelation between hypomethylation rate and CA199 level. 4.Hypomethylation of(+331) CpG site in the first intron of the S100A4 gene is associated with the stainingof S100A4 expression in pancreatic cancer tissues. 5.There was correlation betweenS100A4(+331) CpG site hyomethylation and ppENK methylation.Partâ…¡The study of ppENK and S100A4 and their methylation status inpathogenesis of human pancreatic cancer cell linesMethods:Five pancreatic carcinoma cell lines (Panc-1,Pupan-1,Aspc-1,PC3,SW1990)were used:1.ppENK gene was assayed by reverse transcriptase polymerase chain reaction(RT-PCR). 2.ppENK methylation status was detected in pancreatic carcinoma celllines by MSP. 3.S100A4 gene was assayed by reverse transcriptase polymerase chainreaction (RT-PCR). 4. S100A4 gene (+331) CpG site methylation status was detectedin pancreatic carcinoma cell lines by COBRA. 5.To analyze the association ofhypomethylation of S 100A4 gene with and methylation of ppENK gene by Fisher'sExact Test. 6.Two pancreatic carcinoma cell lines(Panc-1,Aspe-1) were treated with5-aza-dC in different concentrations. The growth of cell was measured by MTT. 7. Apoptosis and cell cycle was analyzed by flow cytometry through PI stain. 8.Thechanges of ppENK methylation status in Aspc-1 and Panc-1 cells were analyzed aftertreated by 5-aza-dC.Results:1.There was ppENK mRNA expression in normal pancreatic tissue, but not infive pancreatic carcinoma cell lines (Panc-1,Pupan-1,Aspc-1,PC3,SW1990).2.Methlated ppENK was detected in 100ï¼…(5 of 5 ) pancreatic cancer cell lines.Methlated ppENK is associated with ppENK mRNA expression in pancreaticcarcinoma cell lines and normal pancreatic (p=0.018). 3.There are S 100A4 mRNAexpression in five pancreatic carcinoma cell lines (Pane-1,Pupan-1,Aspc-1,PC3,SW1990), but not in normal pancreatic tissue. 4.Hypomethylation of the S100A4gene was detected in 100ï¼…(5 of 5) pancreatic cancer cell lines at (+331) CpG site.Hypomethylation of (+331) CpG site is associated with S 100A4 mRNA expression inpancreatic carcinoma and normal pancreatic (p=0.048). 5.S100A4 (+331) CpG sitehyomethylation was a significant correlation with ppENK methylation ((p=0.048)).6.The proliferation of Aspc-1 tended to decrease significantly (1.608±0.219) treatedwith 5-aza-dC(1.0uM) at 96h, compared with the control (2.644±0.247),the similarchange can be seen in Panc-1 but the time at 120h. 7.The apoptotic rates of Pane-1and Aspc-1 induced by 5-aza-dC (1.0uM) were 31.57±6.76ï¼…and 16.64±8.22ï¼…comparing with those cells without treatment by 5-aza-dC(3.21±1.43ï¼…, 3.82±1.71ï¼…). 8.The G₁ phase was increased concomitant with decreasing in the percentageof S phase only in Panc-1 cell line (p＜0.05), but not in Aspc-1 cell line. 9. Withtreatment by 5-aza-dC, methlated ppENK was not detected and the ppENK mRNAexpression reversed.Summary:1.In five pancreatic carcinoma cell lines, there are methlated ppENK,but not ppENK mRNA expression. Methlated ppENK is associated with ppENK mRNAexpression. 2.In five pancreatic carcinoma cell lines, There are S100A4 mRNAexpression and hypomethylation at (+331) CpG site. Hypomethylation of (+331)CpG site is associated with S100A4 mRNA expression. 3.S100A4 (+331) CpG sitehyomethylation was a significant correlation with ppENK methylation. 4.Aftertreated with 5-aza-dC, Methlated ppENK was not detected and the ppENK mRNAexpression reversed. 5.After treated with 5-aza-dC,there were changes of cellgrowth,cell apoptosis and cell cycle.Conclusions:1.S100A4 activation and ppENK inactivation by change of their methylationstatus play an important role in pancreatic carcinogenesis. 2.S100A4hypomethylation and ppENK methylation are important molecular event todistinguish pancreatic carcinoma tissue from normal pancreatic tissue. 3.There iscorrelation between S100A4 (+331) CpG site hyomethylation and ppENKmethylation. 4.The expression of S100A4 is a significant association with tumordifferentiation. 5.There is correlation between S100A4 (+331) CpG sitehypomethylation and CA199 level. 6.The influence of cell growth,cell apoptosis andcell cycle may contribute to the change of ppENK methylation status.