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Biological Function Of REG1B Promoting Proliefration, Migration And Invasion In Human Colorectal Cancer Cell HCT116

Posted on:2016-10-03Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z R LiuFull Text:PDF
GTID:1224330479492220Subject:Physiology
Abstract/Summary:PDF Full Text Request
Objective: Colorectal cancer(CRC) is one of the most prevalent human cancer worldwide, with about 1 million new cases annually. It is estimated that more than 500 thousand people will die from CRC every year. Despite advances in both earlier diagnosis through screening and better treatment modalities which have decreased the mortality in the past decades, there is still no effective method for increasing the overall survival rate of affected patients. In order to improve the horrible prognosis, identifcation of new targets of molecular therapy, especially those that are indicative of proliferation and invasion is urgently needed. 1.To investigate the expression and distribution of the REG1 B protein in the colon cancer tissues to discuss their clinical significance. To detect the expression of the mRNA and protein of REG1 B in the colon cancer cell lines sw480,sw620 and HCT116 to screen the appropriate research tools. 2.To construct and screen the targeting specificity of REG1 B interference plasmids, laying the foundation for subsequent research. 3.To explore the influence of REG1 B gene interference on colon cancer, cell proliferation, apoptosis, cell cycle, cell migration and invasion of colorectal cancer cells. 4.To explore the molecular mechanism of REG1 B gene silencing cell migration and invasion of colorectal cancer cells, the activity of MMP-2 and MMP-9 was detected. Co-immunoprecipitation analysis was used to verify the REG1 B protein interacting with IL-6 protein, and identified if both of them existing interactions under physiological conditions. Methods: 1.We collected 30 cases of colon cancer samples and their matched normal tissues to make paraffin specimens. IHC were used to detected the expression and distribution of the protein of REG1 B in the samples. Their clinical significances were analyzed.Real-Time RT-PCR, western blot and immunofluorescence method were used to detect the expression of the mRNA and protein in colon cancer cell lines sw480,sw620 and HCT116. 2.The REG1B-shRNA expression vector GV102 was constructed and sequenced to detect the correctness of the interference sequence. Lipofectamine 2000 was used to transfect HCT116 cells. The interference efficiency was detected using a fluorescence microscope, real time RT-PCR and western blot. The sequence with highest interference efficiency was screened. 3.Lipofectamine 2000 was used to transfect HCT116 cells. A cell count growth curve and cck-8 kits were performed to measure the cell proliferative changes. Flow cytometry was analyzed to detect the changes of cell apoptosis and cell cycle after REG1 B was knocked down. Wound healing analysis was performed to detect the cell migration changes. The transwell chambers were used to detect the changes of cancer cell migration and invasion after REG1 B was silenced. 4.The gelatin zymography assay was performed to analysize the activity of MMP-2 and MMP-9. HCT116 cells were analyzed for protein interaction between REG1 B and Il-6 proteins by co-immunoprecipitation and Western blot using REG1 B and Il-6 antibody. Results: 1.The expression of the protein of REG1 B was higher in the colon cancer tissues than in the normal tissues. Its expression mainly located in the epithelial cells of the intestinal crypt and it was correlated with the degree of tumor differentiation. The expression of the mRNA and protein of REG1 B was higher in the colon cell lines SW620 and HCT116 than in the cell line sw480. 2.The targeting specific REG1 B interference plasmid was constructed successfully. The interference efficiency of three plasmids reached 34.4%, 84.2%and 90%, respectively. The third shRNA inhibition rate was the highest. 3.The silence of REG1 B gene can inhibit the proliferation of colon cancer cell line.The cells were arrest at G0/G1 and the percentage of S-phase cells were decreased.The analysis of apoptosis showed that the silence of REG1 B gene did not increase the death of the cells compared with the control group. REG1 B gene silencing significantly reduced the migration and invasion of colon cancer cell line. 4.The activity of MMP-2 and MMP-9 were inhibited after REG1 B was silenced. The protein expression of Il-6 can be detected in the protein complexes of co-immunoprecipitation of REG1 B antibodies with total protein of HCT116 cells. The protein expression of REG1 B can be detected in the protein complexes of co-immunoprecipitation of IL-6 antibodies with total protein of HCT116 cells. Conclusions: 1.The expression of the protein of REG1 B was high in the colon cancer tissues. The expression of the mRNA and the protein were higher in SW620 and HCT116 cells than in SW480 cells.There were higher transfection efficiency in HCT116 cells, so the HCT116 cells were selected as a follow-up research tool. 2.The REG1 B interference plasmids were constructed and screened successfully. 3.REG1 B gene silencing can inhibit the proliferation of colon cancer cells, and this function may work by affecting cell cycle, but no correlation with apoptosis. After REG1 B was silenced, cell migration and invasion was reduction. Therefore, REG1 B gene can promote the migration and invasion of colon cancer cells. 4.The level of MMP-2 and MMP-9 was deduced after REG1 B was silenced. There is a physical interaction between REG1 B and Il-6 in HCT116 cells. Therefore, REG1 B can be used as a potential target of gene therapy.
Keywords/Search Tags:colorectal cancer, REG1B, RNAi, co-immunoprecipitation
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