The Preliminary Study On The Mechanism Of Pancreatic Cancer Metastasis

ObjectivesPancreatic cancer is the 5th leading cause of cancer-related death ,and its incidence is increasing in China. The 5-year survival rate of pancreatic cancer is less than 3% because of local invasion and regional lymph node metastasis. E-ven lots of patients had lost the cure chance when diagnosis, and how to handle metastasis is the key to improving prognosis. There are 3 steps to metastasis at least; attachment, infiltrate, and deprivation. In the past, the progress of tumor cells invading, transition and emigration vessels was regarded as the rate-limiting step of metastasis. Welch proposed the rate-limiting step be that of metastasis tumor cells growing in the metastasis location in 1999. Respecting the role of tumor suppressing gene in rate limiting-step, we propose that we could handle tumor metastasis by recovering the expression of tumor suppressing gene. So we plan to research the mRNA and proteins expressions of matrix metalloproteinase-2 ( MMP-2) , matrix metalloproteinase-9 ( MMP-9) , tissue inhibition protein-ase-1 (TIMP-1} and tissue inhibition proleinase-2 (TIMP-2) in normal pancreas and pancreatic cancer, investigate the upset status of gene regulation in pancreatic cancer, and recover the expression of KiSS-1 gene in pancreatic cancer.Methods1. Research on mRNA expressions of KiSS-1, MMP-9, MMP-2, TIMP-1 and TIMP-2 genes in pancreatic cancerWe collected 39 cases of pancreatic cancer, 9 cases of normal pancreas in Primary hospital of ShenYang Military Region and the 2nd affiliated hospital of China Medical University. There's no difference of sex and age between two groups. The mRNA expressions of MMP-2, MMP-9, TIMP-1, TIMP-2, and KiSS-1 were detected by hybrizaton in situ and RT-PCR analysis. Results were showed by Average ± Standard, and analyze the Average between several groups by SPSS 11.0 software.2. Research on protein expressions of metastin, MMP-9, MMP-2, TIMP-1 and TIMP-2 in pancreatic cancerWe detected protein expressions of MMP-2, MMP-9, TIMP-1, TIMP-2, and metastin by immunochemistry and Western Blot. Results were statistical with H test, U test and x2 test by SPSS 11.0 software.3. Research on clone KiSS-1 gene and producing the plasmidWe Cloned ORF of KiSS-1 gene from human normal pancreas, and analysis its grand by BLAST with network, and produced pcDNA3/KiSS-l plasmid, and screened plasmids. Then amplified and detected proteins by PCR and analysis their rank.4. Research on KiSS-1 gene's effect on AsPC-1 cellAfter transforming, extracting and purring pcDNA3 and pcDNA3/KiSS-l plasmid, we transfected plasmids into AsPC-1 cell, and screened cells by G418 method, and checkup neo gene. We observed cells with morphology, detected its growing and in vitro invasive ability by MTT assay, growth curve, double-deck agar formation test, Boyden chamber, and sensed gene expression by RT-PCR and Western Blot assay.Results1. Positive hybridization signals of purpose genes ( MMP-2, MMP-9, TIMP-1 , TIMP-2, and KiSS-1 gene) in normal pancreas were seen in histiocyte endochylema, and also seen in pancreatic cancer. But hybridization signals of KiSS-1 , TIMP-1, and TIMP-2 in pancreatic cancer were weaker than those in normal pancreas, and signals of MMP-2 and MMP-9 in pancreatic cancer werestronger. The expression of purpose genes had not correlation with sex, age, size of tumor and cell differentiation grade (P > 0. 05). The mRNA expressions of MMP-9, MMP-2 in pancreatic tissues were higher than those in normal, and MMP-9 expression were significantly ( P < 0. 05 ). The mRNA expressions of TIMP-1, TIMP-2 and KiSS-1 in pancreatic tissues were lower than those in normal, and TIMP-1 wasn't significantly among them (P >0.05). The mRNA expressions of MMP-9, MMP-2 in positive lymph nodes patients were higher than those in negative lymph nodes patients significantly ( P < 0. 05) , and TIMP-2 and KiSS-1 expression in positive lymph nodes patients were significantly lower than those (P <0.05). The mRNA expression of MMP-9, MMP-2 and TIMP-1 in metastasis patients had no differentiations with those without metastasis patients significantly ( P <0. 05 ) , and there were no TIMP-2 and KiSS-1 gene expression in metastasis patients (P<0.01) , and MMP-2, MMP-9 expression in stage IA . The more MMP-9 and MMP-2 mRNA expression, the more TNM stages. There were significant difference in MMP-9 mRNA expression between stage Ⅰ A, Ⅰ B, Ⅱ B and stage Ⅳ, and in MMP-2 mRNA expression between stageⅠ A, Ⅰ B, It A and stage　Ⅳ ( P <0. 05). However, there's no correlation between TIMP-1 mRNA expression and TNM stage. The fewer TIMP-2 and KiSS-1 mRNA expression , the more TNM stages, and there were significant difference in TIMP-2 expression between stage Ⅰ A, Ⅱ A, Ⅱ B and stage Ⅳ, and KiSS-1 gene expression in stage Ⅰ A, Ⅰ B, Ⅱ A and Ⅲ, stage Ⅳ ( P < 0. 01). Results showed transduction level of tumor transition promoting genes ( MMP-2, MMP-9) were higher than those of normal pancreas, but those of tumor suppression genes (TIMP-1, TIMP-2, KiSS-1 ) were lower, and there were significantly differences in MMP-9, TIMP-1 and KiSS-1. The transduction levels of MMP-2, MMP-9 in negative lymph nodes were higher than those in positive, but those of TIMP-2 and KiSS-1 were lower. We couldn't detect the expression of KiSS-1 and TIMP-2 in pancreatic cancer tissues without metastasis.2. Expression and stain grade of metastin, MMP-2, MMP-9, TIMP-1, T1MP-2A and KiSS-1 in cancer tissues were not correlated to the TNM stage, tumor size, and differeciation grade by immunity class method ( P > 0. 05 ). Metastin expression was decent with positive lymph nodes and metastasis (P <0.05). Positive expression of metastin was correlated with TNM stage ( P < 0.01). Protein expression of MMP-9 was higher in cancer tissues than that in normal pancreas. Expression and stain grade of MMP-2, MMP-9 in positive lymph nodes were higher than those without lymph nodes metastasis ( P > 0. 0 5 ) . Although Protein expressions of MMP-9, MMP-2 in metastasis were higher than those without metastasis, there's no correlation between them (P>0.05). Protein expressions of MMP-9, MMP-2 increased with TNM stage (P <0. 05). Protein expression of TIMP-1 had no correlation with TNM stage, metastasis and positive lymph nodes ( P > 0. 05 ). Protein expression of TIMP-2 in positive lymph nodes were lower than those negative lymph nodes, but there's no statistic significance ( P > 0. 05 ) , and had correlation with metastasis ( P = 0. 026 ). Positive protein expression was decent with TNM stage ( P < 0. 05 ). Results shows protein expression of tumor transition promoting genes ( MMP-2, MMP-9) were higher significantly than those of normal pancreas, but those of tumor suppression genes (TIMP-1, TIMP-2, KiSS-1) were lower, and there were significantly differences in MMP-9, MMP-2, TIMP-2 and KiSS-1. The protein expressions of MMP-2, MMP-9 in negative lymph nodes were higher than those in positive , but those of TIMP-2 and KiSS-1 were lower.3. Three mRNA bands extracted from normal pancreas were seen clearly, and amplified DNA by RT-PCR, and its conduction located in 453 bp. After culture to recombine, there were 30 bacterial clones, and 18 white clones among them, which means they were recombines. There are 3 clones could be amplified 453 bp DNA by RT-PCR from 6 clones. Purpose fragment was seen after being cutted by enzymes that mean the gene had inserted carrier successfully. By BLAST assay, the whole gene fragment was the as 99% same as the gene cloned from melanoma formally. We transfected KiSS-1 gene into AsPC-1 cells successfully.4. We amplified neo gene fragment (670 bp) from PK1 and PK2 clone which were screened from transfected AsPC-1 cells formally, but no neo gene fragment was found in AsPC-1 cells. Lot's of mRNA were detected in PK1 and PK2 cells but only a few in nul-lransfected and non-transfected AsPC-1 cells. There was no difference in growing speed, clone formation ability before and af-ter transfection, which means transfection had no effection on growth. Matrigel migratory ability and invasion exponent of transfection cells decreased significantly (P<0.01), and invasiveness-suppressing rate reached 50-60% , which was higher than nul-plasmid transfection cell ( P > 0. 05 ). Transfected cells showed an increase in expression of KiSS-1 gene and metastin, but a decrease in mRNA and protein of MMP-9.Conclusion1. We cloned gene code rank from human normal pancreas, and constructed the eukaryon expression plasmid -pcDNA/KiSS-1, which provided research bases of the protein function.2. Transcription and translation level of tumor transit related genes ( MMP-2, MMP-9, TIMP-1, TIMP-2, KiSS-1) in pancreatic cancer and normal pancreas were located and detected by hybrizaton in situ, immunity histochemistry, Western blot and RT-pancreatic cancer analysis.3. MMP-2, MMP-9, TIMP-1, TIMP-2, KiSS-1 gene and proteins in pancreatic cancer and normal pancreas were expressed, and their coherent expression in pancreatic cancer was correlate with malignant action (lymph nodes and other organ metastasis) , and there were no correlation between their expression and sex, age, tumor size and defferenciation grade.4. Transduction and translation level of tumor suppressing genes (TIMP-1, TIMP-2, KiSS-1) expression was decent with TNM stage and lymph nodes metastasis. But as to the tumor promoting genes( MMP-2, MMP-9) , it's on the contrary.5. Matastasis of pancreatic cancer correlated with the disequilibrium of protein expressions of metastasis trigger genes and metastasis suppressing gene.6. It's safe and available to transfect KiSS-1 gene to AsPC-1 cells by lipido-some, and the transfection process didn't effect the function and morphous of the cells.7. The cells transfected KiSS-1 gene expression plasmid had no influence in reproductive activity, and invasion capability decreased. The expressions of...