Inhibiting Metastasis Of Pancreatic Cancer Cells Silenced By SiRNA KAI1 Gene

Backgroud and Aim: Pancreatic cancer is one of the most common malignant tumors with poor prognosis and early metastasis. When diagnosed, 80% patients have metastasized. And in the past 40 years the incidence of pancreatic adenocarcinoma has increased one to five fold and the average life-time is 5 months with overall 5-year survival rate of 1- 4%. Up to now, no effective methods have been found for diagnosis and treatment of the pancreatic cacner. The disastrous results might be associated with carcinogenesis, proliferation, infiltration and metastasis in the pationet with pancreatic cancer, involving with multiple stages and different genic abnormity. Therefore, it is important theoretical and clinic application value for explaining the metastasis mechanism of pancreatic cancer from the pointview of molecular level.KAI1/CD82 is a metastasis suppressor which belongs to a member of the structurally distinct family of cell membrane glycoproteins, transmembrane 4-protein superfamily. Like other TM4SF members, KAI1 functions in cell-cell and turmor cell-extracellular matrix interactions, thereby potentional influncing the ability of the cancer cells to invade tissue and to metastasize. Guo et al.first reported that KAI1 is associated with metastasis of pancreatic cancer in Cancer Res.in 1996. The inhibiting metastatic mechanism in pancreatic cancer cell regulated by KAI1 is deeply investigated depending on our previous results in the gene. In our present study, the high expression of KAI1 gene was blocked by siRNA techniques in pancreatic cancer T3 cell line. We hope our study coule promote not only a theoretic basement on inhibition of growth and metastasis in pancreatic cancer, but also a new idea and method on clinical therapy of pancreatic cancer etc by means of the gene.Methods: Four sequences of siRNA were designed based on the KAI1 gene sequence using online RNA interfering design software and built lentivirus vector. Then vectors were used to transfect T3 cells by liposome 2000 and virus titer was tested. The experiment of using RNAi lentivrus particle to infect objective cells was carried out according to experimental design. The expression of mRNA of objective protein was tested by real-time PCR in order to judge the interference effects of different targets . The ability of cell migration was tested among the experimental group,blank control group and negative control group by Transwell. The expression of MMP-9, ICAM-1 andβ-catenin was detected among the experimental group, blank control group and negative control group by immuno- fluorescence.Results: 1. .The expression of KAI1 in T3 after the siRNA interference showed that there was a significant difference between experimental group and negative control group (p<0.05). No significant difference have been identified between blank-vector control group and negative control group (p>0.05). 2. The ability of experimental group silenced by siRNA was significantly reinforced compared with negative control group and blank-vector control group(p<0.05), but there was not difference between negative control group and blank-vector control group(p>0.05). The high expression of MMP-9, ICAM-1 andβ-catenin from Wnt signal transduction in the pancreatic cancer T3 cell line after KAI1 geng blocked by siRNA was obviously increased using immunofluorescence.Conclusion:RNAi technology can inhibit the expression of KAI1 gene in human pancreatic cancer cell line T3 effectively. The high expressing of MMP-9 and ICAM-1 in the pancreatic cancer T3 cell line after gene blocked by KAI1 siRNA was obviously increased. It enhances the adhession between pancreatic cancer cells and basement membrance by increasing the expression of ICAM-1 after KAI1 gene silence. It also promotes the decomposition of basement membrance and leads to the elevation of cell migration ability of pancreatic cancer by increasing the expression of MMP-9 after KAI1 gene silenced by siRNA. The expression ofβ-catenin from Wnt signal transduction also increased in the pancreatic cancer T3 cell line after KAI1 gene silenced by siRNA. In a word, KAI1 gene inhibites the metastasis of pancreatic cancer by reducing the expression of MMP-9, ICAM-1 andβ-catenin of pancreatic cancer cells. The study established the theoretic and experimental foudations for the basic research and clinical treatment of KAI1 gene.