Effect Of Silencing Matriptase On Cellular Growth Of Human Pancreatic Carcinoma Cell Line SW1990 And Xenograft In Nude Mice

Part One:The study of silencing Matriptase on cellular growth and matastasis in the pancreatic carcinoma cell line SW1990.Objective:To study the effect and mechanism of silencing Matriptase on cellular growth in human pancreatic carcinoma cel line SW1990.Methods:According to the sequences of coding regions NM₀21978.3 of Matriptase in Genbank and the relevant literature, synthesized the Matriptase si RNA and the experiment was divided into four groups : negative si RNA（A group）, 12.5 u M（B group）, 25 u M（C group）, 50 u M Matriptase-si RNA（D group）. The expression of Matriptase at m RNA level at 24 hours after transfection was evaluated by real time PCR;and the expression at protein level at 24 hours was evaluated by Western blot. The cell proliferation was detected by CCK-8 at 24 and 48 hours;The cellular migration was detected by cell wound scratch assay at 24 hours after transfection. The cellular invasion was detected by transwell assasy at 24 hours after transfection. The expression of u PA/pro-u PA、AKT、p-AKT、E-cadherin at the protein level was examined at 24 hours after transfection.Results:The expression of Matriptase at the m RNA level were 1、0.44±0.05、0.28±0.04、0.25±0.07,The expression of Matriptase at the protein level were 0.74±0.06、0.49±0.04、0.34±0.01、0.24±0.05. Individually. Compared with cells transfected with A group, the enzymatic activity of Matriptase and the expression of MMP-9 were both decreased, and the proliferation and migration and invasion ability of the cells transfected with Matriptase-si RNA were decreased significantly.Conclusion:Down-regulation of Matriptase inhibits the proliferation,migration and invasion of pancreatic cancer SW1990 cells, and this result may due to the down-regulated enzymatic activity of Matriptase and MMP-9. The mechanism maybe through inhibiting the activation of u PA, then inhibits the phosphorylation of AKT, and decreases the expression of MMP-9.Part two:The effect of silencing Matriptase on cellular growth of xenograft in nude miceObjective:To study the effect and mechanism of silencing Matriptase on cellular growth of the xenograft in nude mice.Methods :（1） Matriptase-sh RNA lentivirus was produced and transfected to pancreatic cancer cell line SW1990 for stable knockdown the expression of Matriptase. Then using the inverted fluorescence microscope and flow cytometry analyzer for detecting transfection effect. Stable transfected cells was sorted by flow cytometry sorting instrument, Western blot detects the effect of interference.（2） xenograft in nude mice of pancreatic cancers: four weeks of female BALB/c nude mice were divided into two group, empty vector group（sh NC group） and transfection group（sh MT1 group）, every three days testing the body weight and the tumor size of each mouse, the tumor volume is about 500 mm3, 4.8% chloral hydrate anesthesia is injected into each mouse, take pictures of animal imager. Cervical dislocation method executed mice, subcutaneous tumor stripping, remove capsule, and record the weight of tumor. Finally, the tumor volume and weight of the mice in the two groups were analyzed.（3） Western blot and IHC test the expression of Matriptase and MMP-9 of the two groups.Results:Compare to the group of NC, the body weight, tumor size,the expression of Matriptase,MMP-9 of sh RNA-MT1 group were decreased significantly, but the expression of E-cadherin was not changed.Conclusion:Down-regulation of Matriptase inhibits the growth and metastasis of xenograft in nude mice, the mechanism maybe contact to the MMP-9.