Research Of Human Pancreatic Carcinoma Related Gene-mucin 4

1 BackgroundThe morbidity of pancreatic cancer is increasing year by year. In the United States, there are approximately 29,200 cases of cancer of the exocrine pancreas diagnosed each year and about 28,900 died from the disease. Cancer of pancreas is the fourth most common cause of cancer death in the United States and Japan, which has exceeded gastric cancer. In Shanghai, morbidity of pancreatic cancer is 6.0/100,000 in males and 5.5/100,000 in females during 2000. Both incidence and death rates develop 50% from 70s. In decades, scientists do hard work to control pancreatic cancer but have little achievements. It is difficult to make diagnose in early stage. Most of the diagnoses are too late to get the best therapy. Ninety percent of patients wihtin the first year after diagnose. Since 1960, the relative 5-year survival rate for pancreatic cancer has increased from 3% to 5%. Because of these , the pancreatic cancer is called 'the King of the Cancer' ,or 'the cancer of the 21^st Century' In order to investigave biological characteristics of pancreatic cancer, and seek novel adjuvant treatment for this disease, scientists have been paying more and more attention on molecular biological research of pancreatic cancer in recent years.On the level of molecule, occurrence and evolution of pancreatic cancer is result of expression of gene. Generally, the genes that are altered in the development of cancer fall into three broad categories: oncogenes, the presence of which may cause cellular transformation; tumor-supperssor genes, the absencer of which may cause cellular transformation; and mutator genes.The existing in vestigations prave the occurrence and evolution of pancreatic cancer is closely relased to manifold gene. These oncogenes include: ras(K-ras,N-ras), Cer-bB(Cer-b-B1, Cer-bB2), myc, raf, met, DR-a and ATT, et. The tumor-suppressor genes involve P53, RB, APC, DCC, P16, et. Translation-suppressor genes involve nm23.Occurrence and evolution of pancreatic cancer is a polygenic disease, so many gene linked to pancreatic cancer await us to discover and investigate. Along with swiftdevelopment of molecular biology, gene chip, a biological technique make it possible to detect a large numer of genes.Microarray is the technique that a large numer of genes are arranged orderly on the carrier, such as glass chip or something in high density. Date are obtained by examining the signal of fluorescence, and analyzed and compared by computer software. A great lot of genes can be examined rapidly, accurately, and effectively in one experiment. With the technique of gene clip,we shall. discover plenty of genes, which express differently in pancreatic carcinoma,by investigating gene expression profiles. Analysis of the functions of these differently expressed genes can provote the research on molecular biology of pancreatic cancer, and help us to discover novel diagnosis markers. It is important to early diagnose and treatment. Jin-gang,Tan zhijun and colleagues screened pancreatic cancer related genes in six samples by microarray technique. They found 301 genes exprssing diffrently in more than 5 samples of 6, 166 genes expressed highly in tumor tissues. MUC4 gene is involved.Mucin 4 was first isolated from a cDNA library of human trachea. The gene was located to human chromosome 3q29. Mucin 4 is a member of mucin family. All the family members of mucin share a consensus structure: a N-terminal mucin repeats, the first EGF-like region, a N-glycosylation site, the secondary EGF-like region, a hydrophobic transmembrane region and the C terminal cytoplasm region. The EGF-like region is a Cys rich region and share high homogy with EGFR ligand. Mucin protein might interact with.C-erb family members and regulate the cell growth. Based on our previous study of expression pattern of cDNA microarray, we put forward our research to estimate the value of detecting mucin 4 expression and explore the molecular mechanism of mucin 4.2 Objective1) Research of mucin4 gene expression profile changes in human pancreatic carcinoma using method of immunohistochemistry and RT-PCR ,to discuss its potential roles in genesis and development of pancreatic carcinoma.2) Reveal the function of mucin 4 using sense and antisense over-expressionsystem. The cell cycle and apoptosis were employed to estimate the influence and the possibility of therapy target.3) Explore the interaction protein pattern of mucin 4 using yeast two hybridization systems. Reveal the molecular mechanism of mucin 4 pathway. Found novel gene marker for pancreatic diagnosis and novel target for pancreatic cancer therapy.3 Materials and Methods1) 35 samples of pancreatic cancer, 12 samples prancreatic benign tumor and 5 samples of chronic pancreatitis were detected mucin 4 expression using a rabit anti human mucin 4 antibody with ABC IHC kit. 12 sample of fresh pancreatic cancer and two pancreatic cancer cell lines were detected mucin 4 expression using RT-PCR. The difference were valued by x² test.2) Construct sense and anti-sense over-expression plasmids, verify the expressin of MUC4 gene in pancreatic cancer line BXPC, and detect the influence toward the cell cycle and apoptosis.3) The pLex A Matchmarker yeast two hybrization system was employed to detected the interaction protein of mucin 4. the ORF of mucin 4 was cloned into pLex A. The bait pLex A-mucin 4 plasmid was used to screen a premade human fetal brain cDNA- pB42AD library. The positive clone were isolated by a plate with X-gal and without Leu. After verified by mate test, all independent clones were retrieved by PCR with pB42AD consensus primers and identified by sequence analysis.4 Results1) 24 out of 35 samples present positive signals of mucin 4 in the IHC using a rabit anti human mucin 4 antibody. There are 6 samples with a strong positive, 14 samples with middle positive and 4 sample with weak positive. While 2 out of 10 benign pancreatic tumor sample present weak mucin 4 expression and no expression was detected in 5 chronic pancratitis samples. The tissues beside tumor were not deteced the expression of mucin 4. T-test reveal that pancreatic cancer tissues with a higher expression of mucin 4 comparing to both benign pancreatic tumor and chronicpancratits (P<0.01). However, we have not found a close linkage between mucin 4 expression and pancreatic characterizations such as size, transplant and TNM stage. 6 out of 12 pancreatic cancer samples and two pancreatic cancer cell lines were detected mucin 4 expression. And Not singal was detected in all corresponding tumor side tissues.2) Both sense and anti-sense over-expression plasmids were constructed and verified by DNA sequence analysis. Both of them were used to transfected pancreatic cancer line BXPC. 24h after plasmids transfected, the cell cycle and apoptosis were detected on a FCM. Sense over-expression of mucin 4 had not influence on cell cycle and cell apoptosis ( apoptosis cell: sense over-expression 8.25+1.26, control 2.01+0.97; S stage cell:. Sense over-expression 33.57+2.51; control 34.01+2.76) Antisense over-expression of mucin 4 had no influence on cell cycle, but it resulted in cell apoptosis (apoptosis cell: antisense over-expression 22.31+5.82 ; control 2.01+0.97 ; S stage cell antisense over-expression 33.70+3.23; control 34.01+2.76).3) 125 positive clones were isolated from 1000000 screened library plamids. 56 presentation clones were sequenced and 16 independent clones were isolated. 3 (NM-002295 (RPSA), NM-175573 (ADRM1) and NM₀04068 (AP2Ml))out of 16 positive clones are tumor related cell surface protein. 6 out of 16 (NM-002796(PSMB4) , NM-005348, (HSPCA), NM₀07126 (VCP), NMJM8442 (IQWD1), NM₀15440 (methylenetetrahydrofolatedehydrogenase) , NM₀06155 (septin2) ) belong to reported tumor related proteins.and the others might be the background of yeast two hybrization system.5 Conclusion:l)The expression of mucin 4 was detected in two pancreatic cancer cell lines—BXPC and S988. Mucin 4 transcripts of pancreatic cancer are higher than that of chronic pancratitis and the tissues beside tumor. So the expression pattern of mucin 4 might be a useful tumor marker in diagnosis of pancreatic cancer. IHC might be a better method for diagnosis of pancreatic cancer.2)Over-expression of mucin 4 antisense mRNA result in apoptosis of pancreatic...