A Pilot Study Of Effects Of CD97-siRNA Transfection On Biological Characteristics In Gastric Cancer Cell Line MKN45 And The Underlying Molecular Mechanism

INTRODUCTIONStomach cancer remained the fourth most common malignancy in the world in 2007,with one million new cases.Nearly 70%of new cases occured in developing countries.Generally,stomach cancer rates are about twice as high in men as in women. About 800,000 people worldwide died from stomach cancer in 2007.It ranks first among all causes of death from cancer in China,with an annual mortality rate of approximately 25.2 per 100.000.Cancer of the stomach is difficult to cure unless it is found in an early stage.The main treatments for stomach cancer are surgery,chemotherapy,and radiation therapy. Often the best approach uses two or more of these treatment methods.Detection of two or more than two tumor markers together will increase the rate of early diagnosis of stomach cancer.At present,CEA and CA19-9 are the two main tumor markers of stomach cancer.So,discovery of novel tumor marker is one of the research trend of tumor.Despite the recently reduced mortality rates due to both earlier detection and improved therapy,gastric cancer is still the second leading cause of cancer-related death worldwide.The development of new treatments is therefore crucial for improving the survival rates of gastric cancer patients.In the last few years,dedifferentiation markers and apoptotic factors have attracted considerable attention as possible targets for the diagnosis and therapy of cancer.CD97 is a member of a subfamily of classⅡG-protein-coupled receptors referred to as epidermal growth factor seven-transmembrane(EGF-TM7).CD97 protein possesses a unique hybrid structure that consists of varying numbers of N-terminal EGF-like domains coupled to a seven-span TM7 domain by a mucin-like spacer.In normal tissues,abundant expression of CD97 is only detected in macrophages and dendritic cells except for microglial cells,and in some T and B cells.To date,CD97 has been found abundant expression in several epithelial cancers,such as thyroid, gastrointestinal,pancreatic ductal and oral squamous cell carcinoma.Various evidence shows that CD97 plays an important role in tumor dedifferentiation,migration, invasiveness and metastasis.However,the role of CD97 on gastric carcinoma cell growth and the underlying mechanism remain to be elucidated.Part 1 Effects of CD97-siRNA transfection on biological characteristics in gastric carcinoma cell line MKN45Objective:To investigate the effect of CD97 gene silencing by small interfering RNA on proliferation,apoptosis,migration and invasion of gastric carcinoma cell line MKN45 in vivo and in vitro.Methods:1.Gastric carcinoma cell line MKN45 was adopted,the pairs of CD97-siRNA were designed according to the CD97 gene sequence and synthesized chemically.2.Forty-eight hours after CD97-siRNA transfection,the expression of CD97 at mRNA level was detected with RT-PCR.72 hrs after transfection,the expression of CD97 at protein level was detected with Western blot and Immunocytochemistry assays,MTT assay and FCM were used to evaluate the change of proliferation and apoptosis,and Transwell assay was used to evaluate the change of ability of migration and invasion in gastric carcinoma cells.3.15 nude mice were divided into 3 groups averagely.Group normal control: subcutaneous implant of gastric carcinoma cells with normal gastric carcinoma cells. Group negative control:subcutaneous implant of gastric carcinoma cells with nonsilencing RNA.Group inhibition:subcutaneous implant of gastric carcinoma cells with CD97-siRNA transfection.4 weeks after implantation,the morphology, histopathology and tumor inhibition rate were evaluated on every group.Results:1.Forty-eight hours after CD97-siRNA transfection,RT-PCR revealed that CD97 expression decreased 70.12%at mRNA level,and 72 h after CD97-siRNA transfection, Western blot revealed that CD97 expression decreased 66.35%at protein level.2.Seventy-two hours after CD97-siRNA transfection,MTT revealed that the rate of cell proliferation decreased 52.45%compared to the normal control group,and FCM revealed that the percentage of G1 stage cells of normal control,negative control and transfection group were 14.48±3.45%,15.33±2.13%and 39.79±3.05%respectively;the percentage of S stage cells were 64.17±3.36%,60.35±5.21%and 28.56±1.33% respectively.These data showed that CD97-siRNA restrained the conversion of G1→S stage.FCM revealed that the percentage of apoptotic cells of normal control,negative control and transfection group were 7.24±0.35%,6.88±0.58 and 28.18±0.39% respectively.3.Seventy-two hours after CD97-siRNA transfection,Transwell assay revealed that the number of migration and invasion cells decreased 62.54±11.32%and 67.13±14.03% respectively.4.Tumor formation can be found in every nude mice.Tumor tissue mean weights of the normal control and negative control group were 5.255±0.571g and 5.218±0.547g respectively.However,tumor tissue weight of CD97-siRNA transfection group was 2.125±0.232g.Tumor growth was inhibited significantly(p<0.05) and the inhibition rate was 58.1%.Conclusion:1.The status of CD97 expression can be used as a marker to evaluate the ability of proliferation,migration and invasion of gastric cancer,and this molecule can be used as an important target to suppress the recurrence and metastasis of gastric cancer.2.RNAi technique can be used to suppress the expression of CD97 gene.3.CD97-siRNA may be used as gene therapy means for gastric cancer.Part 2 A pilot study of the underlying molecular mechanism of CD97-siRNA transfection-induced biological characteristics in gastric carcinoma cell line MKN45Objective:To study the underlying molecular mechanism of CD97-siRNA-induced cell cycle arrest, apoptosis,and suppression of migration and invasion of gastric carcinoma cell line MKN45 in vitro.Methods:1.Western blot assay was used to determine the change of level of protein of Cyclin D1,CDK4 and p27 after CD97-siRNA transfection.2.Western blot assay was used to determine the change of level of protein of caspase family after CD97-siRNA transfection.3.Western blot assay was used to determine the change of level of protein of Bid,Bad,Bcl-2 and Bcl-X_L after CD97-siRNA transfection.4.Western blot assay was used to determine the change of level of protein of MMP-7 and TIMP-1 after CD97-siRNA transfection.5.Western blot assay was used to determine the effect of CD97-siRNA transfection on PI3K/AKT signal pathway.Results:1.Western blot assay revealed that CD97-siRNA transfection down-regulated the level of expression of Cyclin D1 and CDK4,and up-regulated the level of expression of p27 in MKN45 cells significantly.2.Western blot assay revealed that CD97-siRNA transfection down-regulated the level of expression of procaspase-9,-3,and up-regulated the level of expression of cleaved-caspase-3 significantly,however,had no significant influence on the level of expression of procaspase-8 in MKN45 cells.These data showed that CD97-siRNA triggers apoptosis of gastric cancer cells largely through the intrinsic pathway.3.Western blot assay revealed that CD97-siRNA transfection down-regulated the level of expression of Bcl-2 and up-regulated the level of expression of Bad significantly, however,had no significant influence on the level of expression of Bcl-x1 and Bid in MKN45 cells.4.Western blot assay revealed that CD97-siRNA transfection up-regulated the level of expression of TIMP-1,down-regulated the level of expression of MMP-7 in MKN45 cells significantly.5.Western blot assay revealed that CD97-siRNA transfection decreased the amount of phosphorylated PI3K and AKT in MKN45 cells significantly.These data showed that CD97 enhance the cancer growth probably through PI3K/AKT signal transduction pathway.Conclusion:1.The CD97 receptor enhance the cancer growth probably through PI3K/AKT signal transduction pathway.2.CD97-siRNA transfection may down-regulate the level of expression of CD97 gene, inactivate the PI3K and AKT,up-regulate the level of expression of p27, cleaved-caspase-3,Bad and TIMP-1,and down-regulate the level of expression of cyclin D1,CDK4,procaspase-9,-3,Bcl-2 and MMP-7,furtherly result in cell cycle arrest,apoptosis,and suppression of migration and invasion of gastric carcinoma cells.