| Objective: The incidence of pancreatic cancer is increasing year by year in the world,with high degree of malignancy,insidious onset,rapid progression,and poor prognosis.Regrettably,the specific mechanism of malignant development of pancreatic cancer has not yet been clarified.In the early stage of the study,screening in clinical samples revealed that there was hypermethylation of CpG island in the promoter region of Bone morphogenetic protein-2(BMP-2)gene in patients with pancreatic cancer.Based on this,We hypothesized that hypermethylation of the CpG island in the promoter region of the BMP-2 gene may result in down-regulation of tumor suppressor gene and causes pancreatic cancer.To further confirm our hypothesis,we applied a variety of methods to detect BMP-2 methylation rate in clinical samples,and constructed a BMP-2 gene overexpressing PANC-1 pancreatic cancer model to reverse verify the relationship between hypermethylation of CpG islands in the promoter region of BMP-2 gene and pancreatic cancer in vitro,and exploring its possible mechanisms.Methods:(1)Pyrosequencing(quantification of methylation rate): extraction of cancer tissues,adjacent tissues and peripheral blood DNA of patients with pancreatic cancer(ductal adenocarcinoma),after bisulfite conversion,purification and PCR amplification,pyrosequencing analysis,determination of the methylation rate of CpG island in the promoter region of BMP-2 gene(Comparison of peripheral blood and adjacent tissues in healthy controls);(2)Methylation-specific PCR(methylation characterization): extraction of whole blood DNA from 48 patients with pancreatic cancer(ductal adenocarcinoma),After bisulfite conversion,purification and methylation-specific PCR(MSP),the methylation status of BMP-2 gene was determined by agarose gel electrophoresis;(3)Quantitative analysis of serum BMP-2 in pancreatic cancer,pancreatitis patients and healthy control by ELISA;(4)H&E staining and immunohistochemistry(IHC)of patients with pancreatic cancer analysis of the difference in expression of BMP-2 protein in cancer tissues and adjacent tissues;(5)The pancreatic cancer cell line over-expressed BMP-2 gene was constructed and the cell proliferation difference was detected by CCK8 method;(6)Scratch test and transwell invasion test respectively explore the migration and invasion ability of two cell lines;(7)The lysed proteins of pancreatic cancer tissues,adjacent tissues and constructed cells were extracted for Western,to verify the possible pathway proteins.Results:(1)Pyrosequencing results show that the CpG island methylation rate of the BMP-2 gene promoter region was high in patients with pancreatic cancer compared with the control group;(2)Methylation specific PCR(MSP)amplification and agarose gel electrophoresis showed that the methylation rate of pancreatic ductal adenocarcinoma was as high as 77.1%(37/48);(3)ELISA results showed that serum BMP-2 concentration(89.14±19.42 pg/mL)was significantly lower in patients with pancreatic cancer than that in normal controls(102.33±10.54 pg/mL)(P=0.012),and was lower than that in patients with pancreatitis(101.66 ±22.29 pg/mL)(P=0.034);(4)Immunohistochemistry showed that the expression of BMP-2 in pancreatic cancer tissues was significantly lower than that in adjacent tissues;(5)CCK8 assay showed that the rate of empty cell proliferation was significantly faster than that of PANC-1 cells overexpressing BMP-2;(6)The results of scratch test and transwell invasion showed that the migration and invasion ability of the cells in the empty group were stronger than those in the overexpressed BMP-2 group;(7)Western blot results showed that the expression of related proteins in Smad and PI3K-Akt two signaling pathways in pancreatic cancer tissues was lower than that in adjacent tissues.The expression of two key receptors(BMPR2 and BMPR1A)in the Smad pathway in the overexpressed BMP-2 cells was higher than that in the no-load control group.Conclusions:(1)Hypermethylation of CpG island in the promoter region of BMP-2 gene was found in pancreatic ductal adenocarcinoma patients;(2)Overexpression of BMP-2 gene can inhibit the proliferation,migration and invasion of pancreatic cancer cells;(3)Hypermethylation of CpG island in the promoter region of BMP-2 gene affects the malignant behavior of pancreatic cancer may through Smad and PI3K-Akt signaling pathways. |
PDF Full Text Request