The Expression Of BTG-2 In The Pancreatic Cancer And Its Function On Pancreatic Cancer Cell Growth Inhibition

1 BackgroundNowadays, the pancreatic cancer is still a sort of lethal disease. Surgical excision is the sole curative means all the same. Unfortunately, the total rate of surgical resection is very low, and median survival is just 17-20 months after curative resection. At the time, when operations are given, 40% of patients have been in local late stage, and 50% of patients have been with metastases. In some medical centers, curative resections are given to minority of patients in early stage who have been through the high selection. Though the five-year survival rate of these patients may rise to 15%-25%, the treatment of pancreatic cancer still faces severe challenge on the whole. Because of these, the pancreatic cancer is called "the King of the Cancer" , and "the Pertinacious Fortress of the 21st Century" . In order to investigate biological characteristics of pancreatic cancer, and to seek novel adjuvant treatment for this disease, scientists have been paying great attention on molecular biological research of pancreatic cancer in these years.Analysis of the functions of these differently expressed genes can promote the research on molecular biology of pancreatic carcinoma, and can help us to discover more gene markers or protein markers of pancreatic cancer. This is very significant to prophylaxis, diagnosis, and treatment of pancreatic cancer.BTG2, human homological gene of PC3/TIS2 and a new member of anti-proliferation genes, is concerned with the cell growth andpolarization, and regulate DNA renovation. As gene of restraint, BTG-2 can modulate β-catenin albumen inside the cell, and regulate the conduction approach of Wnt protein, thus function on cell proliferation, withering adjustment and tumor formation. Secreted from Wnt coding, glycoprotein and specificity receptor in the exterior of cell, interact on the completion of the signal conduction through a series of phosphorylation or phosphorylation removal of albumen in their backward position. Combined with receptor—protein in cell membrane, ligand Wnts introduce the signal inside the cell, then result in excess phosphorylation of dishellved protein in cytoplasm, and further activate the glycogen synthase kinase 3(GSK-3), and prevent dissociated β-catenin albumen, being phosphorylated and degraded by the ubiquitin-proteasome system. Accumulated to a fixed quantity in cytoplasm, β - catenin albumen, compound with lymphoid enhancing factor /T cell factor consequently activate various of gene transcription in the backward position, such as C-myc, cyclin D1, which induce excess proliferation and malignant transform. In normal condition, β - catenin albumen in cytoplasm/karyon, remain at the lower level through protease degradation which is attributed to phosphorylation of APC, dependent GSK-3β. Wild-degradation of β - catenin albumen, induce the abnormal accumulation in the cell, which bring the various tumor at a high level of frequency. Qiao found among the 43 cases of the pancreas cancel, 65.1% of the cases have shown the accumulation of β - catenin albumen in cytoplast, and accumulation of β - catenin albumen in plasm relevant to the high expression of cyclin-D1. He also found that patients live a short life when they are getting β- catenin albumen accumulated in plasm. According to the above, Wnt signal conduction approach and β-catenin albumen, are proved contributed to the pancreas cancel. Lowy' s study showed the expression of β- catenin albumen, exist in all the7 cases of examined carcinoma in situ, 4 of which have the expression in karyon. Thus the abnormal expression of 0 - catenin albumen related to the appearance of pancreas cancel is considered. Therefore, Wnt approach to its abnormal activation is the element to the occurrence and growth of the pancreas cancel, and low expression of BTG-2 can activate Wnt signal approach, which speed up the proliferation, control and death of pancreas cancel cells, are further identified.On the basis of these, we put forward our tentative plan and research objective on this investigation. In an attempt to provide theoretical rationale and experimental foundation for early stage diagnosis and gene therapy, we figure on screening out pancreatic cancer related genes in a large scale using by microarray technique, and doing further research on the functions of filtrated genes.2 Objective1) Assay expression of BTG-2 gene in clinic resection samples of pancreatic carcinoma and in cell lines of pancreatic carcinoma, and discuss its potential role in genesis and development of pancreatic carcinoma.2) Discuss the potentiality of constructing the eukaryotic expression vector of BTG-2 gene.3) Study and research the effect of recombinent plasmid of BTG-2 on biological traits of pancreatic carcinoma cell line Panc-1 in vitro and in vivo, and discuss the gene therapeutic possibility of BTG-2 gene.3 Materials and Methods1) Detect expression of BTG-2 gene in pancreatic carcinoma tissues and adjacent normal tissues using method of RT-PCR, and expression of BTG-2 gene in pancreatic carcinoma cell line Panc-1, SW1990 and Patu8988 using the same method. Detect expression ofBTG-2 gene mRNA in pancreatic carcinoma tissues using in situ hybridization. Detect expression of PCNA antigen and detect cell apoptosis using TUNEL in these samples. Study and research relationship between expression of BTG-2 gene and proliferation and apoptosis of pancreatic carcinoma cells in these samples.2) Construct the eukaryotic expression vector of recombinent plasmid pcDNA3. l-BTG-2 containing BTG-2 gene using the incision enzymes of Kpn I and Not I .3) The eukaryotic expression vector of BTG-2 gene was introducted into cultural Panc-1 cells by liposome-mediated transfection, then cells were screened by G418. The expression of BTG-2 gene mRNA in control cells with transfection of empty vector and in experimental cells with transfection of eukaryotic expression vector with BTG-2 gene was detected by using the method of Northern blot. The expression of 0 -catenin protein in these two groups was detected by using the method of Western Blot. Scale growth curve of these two group cells, and detect the competence of proliferation of these two group cells by using MTT method. Inoculate 4-week-old Balb/c nude mouse with control cells with transfection of empty vector and experimental cells with transfection of eukaryotic expression vector with BTG-2 gene, to observe the growth of tumors. Detect expression of BTG-2 gene mRNA using in situ hybridization, and cell apoptosis using TUNEL in these graft tumor tissues. To discuss the gene therapeutic possibility of BTG-2 gene in pancreatic carcinoma.4 Results1) BTG-2 mRNA expresses in pancreatic carcinoma tissues and adjacent normal tissues, and in two pancreatic carcinoma cell lines Panc-1 and SW1990, but not in Patu8988. Comparative content of BTG-2 mRNAin pancreatic carcinoma tissues was 0. 7520±0. 1650, and in adjacent normal tissues was 0.5810+0.1580. There was significant variance between two groups (P=0. 004). Hybridization in situ showed that the expression of BTG-2 mRNA was mostly in cytoplasm in pancreatic carcinoma, a few mesenchymal cells expressed this gene. 32 patients with pancreatic carcinoma were divided into two groups, according to the difference of PCNA results. One group (+~++) was 20, and the other 12. The comparative content of BTG-2 in the former group was 0.8932 + 0.1220, and the comparative content in the latter was 0.6636+0.1227. There was significant variance between two groups (P=0. 007). In situ analysis of cell apoptosis by TUNEL showed that a small amount apoptosis cells in pancreatic carcinoma tissues with high expression of BTG-2, and apoptosis staining did not appeared in several samples with low or moderate expression of BTG-2.2) Eukaryotic expression vector of recombinent plasmid pcDNA3. l-BTG-2 was certificated duble-enzyme incision. The result showed that recombinent plasmid contained the sequence of BTG-2 gene. The further result of sequencing showed that the sequence possessed 98% homology to the sequence of known BTG-2 gene.3) The expression of BTG-2 gene mRNA in control cells with transfection of empty vector and in experimental cells with transfection of eukaryotic expression vector with BTG-2 gene was detected by using the method of Northern blot. The results approved that transfection with BTG-2 gene could repress the expression of BTG-2 mRNA in carcinoma cells significantly. The expression of P -catenin protein in these two groups was detectedby using the method of Western Blot. The results approved that transfection with BTG-2 gene could down-regulate the expression of P -catenin protein. Cells with transfection of BTG-2 gene were selected by G418 for two weeks. The growth curve of experimental and control cells showed that transfection with BTG-2 could reduce speed of cell growth. The result of competence of proliferation of these two group cells by using MTT method showed that transfection with BTG-2 could reduce the competence of proliferation significantly (P=0. Oil). Compared with control group, the repression rate of proliferation competence in experimental group was 25.25%. Inoculate 4-week-age Balb/c nude mouse with control cells with transfection of empty vector and experimental cells with transfection of eukaryotic expression vector with BTG-2 gene. The result showed that there was no significant viriance (P=0. 266) in volume of graft tumor between two groups (experimental 32.39+8.42mm3, control 34.48+11.49 mm3). There was significant viriance (P=0. 009) in eventual volume of graft tumor between two groups (experimental 57.13 + 17.84mm3, control 198. 73±73. 52 mm3). Situ hybridization showed that BTG-2 mRNA assumed high-expression in experimental graft cells comparing with control cells. The result of TUNEL showed that the apoptosis cells increased significantly in experimental graft cells comparing with control cells.5 Conclusions1) Compare with expression in pancreatic carcinoma tissues, BTG-2 mRNA expresses significantly high in adjacent normal tissues. This gene also expressed in two pancreatic carcinoma cell lines Panc-1 and SW1990. Expression of BTG-2 is related to decrease of proliferation competence and increase of apoptosis.