Study Of The Correlation Between Deficiency Of DNA Mismatch Repaire System And Pancreatic Carcinogenesis

ObjectPancreatic cancer is one of the most malignant tumors, and in recent years its incidence raises so high that it seriously threatens human's life and health. Because there is few specific clinical characters of pancreatic cancer , with high degree of malignancy, it metastasis very easily, and most patients have developed into the advanced stage when they come to visit the doctors. Therefore, how to improve the early diagnosis rate, how to evaluate the prognosis of pancreatic cancer and how to select therapeutic regimen according to its abnormal changes at molecule and gene level, which have become the key point for improving the prognosis of pancreatic cancer.In approaching years, many reseachers have found another important carci-nogenesis mechanism, that is the deficiency of DNA mismatch repair system (MMR) , which can induce tumorigenesis. Their studies show that the transform of mismatch repair genes may result in microsatellite instability ( MSI) , which cause the mutation and accumulation of oncogene and anti - oncogene and lead to the activation of oncogene and deactivation of anti - oncogene, then cell can-ceration and tumor generation finally.DNA mismatch repair gene lies upstream the oncogene and anti - oncogene. They were first confirmed in the hereditary nonpolyposis colorectal cancer ( HNPCC) , composed of a serial of specific enzyme molecules repairing DNA base mispairing. Their normal expression can maitain the integrity and stabilty of genetic materials , guaranteeing the fidelity of DNA replication. In following studies, MMR was aproved not only related to carcinogenesis of herediary carcinoma as HNPCC but also some sporadic carcinomas. hMSH2 and hMLH1 arethe widely investigated mismatch repair genes at present.Microsatellite instability (MSI) means the genetic instability characterized by length alterations within simple repeated sequences. MIS is an important phenomenon of the abnormal expression of DNA mismatch repair gene. Many experiments have demonstrated that MSI is present in HNPCC, some sporadic colorectal cancer, endometrial cancer, gastric cancer, ovarian cancer and so on, which are caused by the abnoral expression of mismatch repair gene. More studies on some sporadic colorectal cancer and gastric cancer further indicate that there is close relationship among MSI, loss of hMLHl protein expression and hypermeth-ylation of hMLHl promoter. Pancreatic cancer belongs to HNPCC correlated tumor and it maybe more associated with MMR. Because pancreatic cancer is charactered by rare early discovery , bad prognosis, low exairesis rate and few samples, there has been few report on the correlation between MMR and pancreatic carcinogenesis at present. However , whether there is relationship between deficiency of DNA mismatch repair system and panreatic carcinogenesis? Is the deficiency of MMR caused by mismatch repair gene promoter methylation? Whether it can provide direct basis for the evaluation of pancreatic cancer and the selection of therapeutic perscription by investigation on the correlation between MMR and pancreatic cancer?In our study, we have applied many methods of molecular biology to detect the state of MSI in pancreatic cancer, and the protein expression of mismatch repair gene hMLHl and hMSH2 and their promoter'methylation, in order to explore the correlation between deficency of MMR and pancreatic carcinogenesis and its clinical characters, trying to find a more effective genetic marker, which helps give the directing information for the early diagnosis, treatment and prognosis of pancreatic cancer, and provide the direct basis for the next gene treatment.Materials and Methods1. Samples:The fresh surgical samples of pancreatic carcinoma, totally 35 samples,were all obtained from the First Affiliated Hospital of DaLian Medical Univercity (2002 ~2004). After pathological diagnosis, they were kept in the refrigerator at - 80X1. The cases included pancreatic carcinoma and adjacent normal tissues.2. DNA extraction;Genomic DNA was prepared using phenol/chloroform extraction, followed by ethanol precipitation, quantitation , then stored at - 20^.3. Microsatellite Analysis:Microsatellite alterations were analyzed by PCR using the five microsatellite markers (BAT25, BAT26, D2S123, D5S346, and D17S250). PCR products were confirmed without mixed band by agarose gel electrophoresis, and amplified by SSCP. After heat denaturation, electrophoresed in a denaturing 6% polyacrylamide gel for 14 - 16h, then stained with ethidium bromide (EB) for 30 -45min. The gel was dried on a filter paper and directly visualized under UV illumination.4. Immunohistochemistry:Blocks of paraffin - embedded tumors and normal tissues were cut into 5 -m slices and fixed in cold acetone for 30 min, then washed in PBS. Afer that, sections were blocked in the non - special blood serum for 10 min, and incubated at 37 C for 30 min using a monoclonal antibody against hMLHl and hMSH2, then add their second antibody. Finally, the samples were microwaved at high power four times (5 min each) in citrate buffer and stained by horseradish per-oxidase(HRP) -DAB.5. Western blot;The total protein was extracted from clear supernatant liquid after tissue ho-mogenation, then quantited by Coomassie brilliant blue. SDS -PAGE was performed and the protein was transferred onto the nitrocellulose filter. After incubated with the monoclonal antibody against hMLHl or hMSH2 and goat anti - rat IgG tagged by horseradish peroxidase(HRP) ,and finally stained with DAB.6. Promoter methylation analyses:By Methylation - Specific PCR ( MSP) , the hMLHl and hMLH2 gene promoter was determined by chemical treatment with sodium bisulfite and stored at-20t. The primer sequences were accordance with the literature. PCR was performed for one cycle of 95 °C for 5 min, followed by 35 cycles of 95 °C for 45s, 60°C for 30 s, and 72°C for 30 s, and a final 5 - min extension at 72°C. Each PCR products (5 ui) mixed with sample buffer (ljxl) was loaded onto 1. 8% agarose gel electrophoresis (stained with 0. 5|xg/ml ethidium bromide), and directly visualized under UV illumination.7. Assessment standard:Tumors with instability in two or more markers are defined as MSI - H;tumors with instability in one marker are defined as MSI - L;and tumors in which none of the markers exhibit MSI are defined as MSS. The immunohisto-chemistry results are judged by the idea that if the tumor cells that exhibited an absence of nuclear staining while in the presence of non - neoplastic cells with nuclear staining were considered loss of protein expression . Amplification of methylation( M) and non - methylation( U) at the same time by SSCP, The presence of a visible PCR product in those lanes marked M indicates the presence of methylated genes of hMLHl and hMSH2;the presence of product in those lanes marked U indicates the presence of unmethylated genes.8. Database is set up according to the clinical data and pathological characteristic.9. Statistical analysis:The Student's t test , One - Way ANOVA, The PearsonX2 test or Fisher's exact probability test were used to analyze the data by SPSS for Windows Release 11.0 (P-value <0.05 was considered statistically significant) .Results1. Incidence of MSI in pancreatic cancer;The respective incidence of MSI in the 5 microsatellite markers BAT25> BAT26^D2S123^ D5S346A D17S250 were 5/35 ( 14% K6/34 (18%), 10/35 (29% ) ^7/35 ( 20% K5/33 ( 15% ) 0 Among the 35 pancreatic cancer analyzed, 7(20%) were MSI-H, 14(40%) were MSI-L, and 14(20%) were MSS. No MSI was present in normal tissues and there was statistical differencebetween them.2. Expression of hMLHl and hMSH2 in pancreatic cancer;Immunohistochemistry results showed that hMLHl expression were normalor a litter lower in pancreatic carcinoma tissues with MSS;while always absent or significantly lower in pancreatic carcinoma tissues with MSI. hMSH2 were mainly expressed in the glomeruli pancreatici, seldom in the gland alveolus nearby;while expressed in the acinar cell of pancreatic cancer . There is no abnormal hMSH2 expression or partly low expression either in the pancreatic cancer with MSI - H or MSI -1 or in the normal tissue.3. Detection of hMLHl and hMSH2 protein level:Western blot confirmed the immunohistochemistry'results: in the 35 cases pancreatic carcinoma tissues , 21 showed low expression or absent expression of hMLHl protein (60% );while hMSH2 protein expression were not different a-mong normal tissues or carcinoma tissues with MSI - H or MSI - L.4. Methylation of hMLHl and hMSH2 gene promotersThe incidence of hMLHl promoter methylation was 57.1% (20/35) in the 35 cases pancreatic carcinoma, whereas no methylation in the normal tissue, and there was statistical significance between them (P<0.05);while methylation of hMLH2 protmotor was present in only one case of pancreatic cancer.5 The relationship between hMLHl expression and tumorous clinical patho - logical characteristic.The significant correlation was observed between the loss rate of hMLHl protein expression and differentiation, lymph node metastasis or tumorigenesis site of pancreatic cancer (P <0.05) , whereas no difference was found between the loss rate and age, gender or tumor size of pancreatic cancer (P >0.05) .Conclusion1. There is MSI pathway in the genesis of pancreatic cancer, and the high detection rate suggests MSI be the frequent event during pancreatic carcinogene-sis and development, it can be considered one of the diadynamic criteria for pancreatic cancer. And the loss of hMLHl expression may lead to MSI.2. Methylation of hMLHl promotor is associated with MSI and loss of hMLHl protein expression, which play an important role in the pancreatic carcino-genesis. Thus, hMLHl promotor methylation maybe a critical mechanism of the pancreatic carcinogenesis.3. The deficiency of MMR system is mainly due to the methylation of mismatch repair gene promotor, not gene mutation.4. The aberrant expression of hMLHl may result in pancreatic carcinogenesis , and the methylation of hMLHl promotor is the crucial mechanism for hMLHl gene inactivation, furthermore, there is correlation between the expression of hMLHl and clinical pathological characteristic of pancreatic cancer.