| Part I Expression and significance of Slug and E-cadherin in colorectal carcinomaObjective:To Explore the expression and the prognostic significance of Slug and E-cadherin in colorectal carcinoma tissues.Methods:the expression of Slug and E-cadherin in65colorectal carcinoma tissues and20normal colorectal tissues by SP Immuno-histochemistry, Analyzed the relationship between the expression level of Slug and E-cadherin and the clinical pathological features and the prognosis of patients.Results:The abnormalities expression rate of Slug in colorectal cancer tissues was47.1%,while in normal colorectal tissues the expression rate was12%, with statistically significant difference beween two groups(p<0.01); The abnormalities expression rate of E-cadherin in colorectal cancer tissues was55.4%,while in normal colorectal tissues the expression rate was8%, with statistically significant difference beween two groups(p<0.01). Both positive expression rate were correlated with tumor infiltration depth, differentiation, lymph node metastasis, with statistically significant difference(P<0.05). In the correlation analysis, Slug expression was not correlated with Ecadherin expression.Conclusions:the expression of Slug and E-cadherin may correlate with occurrence,development and metastasis of colorectal carcinoma and The expression of Slug and E-cadherin can be regarded as valuable indicators for evaluating biological behavior and the prognosis of colorectal carcinoma. Part Ⅱ Construction and identification of a lentiviral vector mediated RNA interference of Slug geneObjective To construct lentiviral vector of Slug RNA interference (RNAi) of human.Methods:Towards the Slug gene sequences siRNA in human coloncancer cell:three siRNA interference sequences of Slug targeting gene were designed for constructing recombinant plasmid shRNA, respectively.A small amount of shRNA lentiviral vectors of three kinds of plasmid-shRNA was constructed and then transfected into human coloncancer cells HCT116, respectively. Slug expressions of mRNA and protein were detected by FQ-PCR and Western blotting, respectively. Then the effects of gene silence were determined for screening the effective target. Constructing pGC-Slug-GFP, to construct the recombinant plasmid using the selected target sequence of siRNA for constructing a large number of pGC-Slug-GFP, and then detecting the titer of virus.Results:The lentiviral vector pGC-Slug-GFP for Slug gene was constructed successfully.Strong green fluorescence was observed in293T cells under fluorescent microscope after co-transfection of the cells with the3plasmids of lentiviral vector. The virus in the supernatant reached a titer of2×107TU/ml. The transfection efficiency of the collected virus exceeded90%in293T cells with a multiplicity of infection. Western blotting identified the presence of Slug expression in the transfected HCT116cells.Conclusion The lentiviral vector for Slug has been successfully constructed with a high yield of lentivirus, which facilitate further investigation of the roles of Slug gene in the development and progression of colorectal cancer. Part Ⅲ Effects of Slug RNA interference by lentivirus on proliferation and apoptosis of colon carcinoma cells HCT116Objective:To investigate the inhibitory effect of Slug gene short interfering RNA (siRNA) on proliferation and cell cycle of human colon carcinoma HCT116cell line.Methods:SiRNA targeting Slug gene was constructed into lentivirus vector, and SiRNA targeting Slug was transfected in to colon cancer cell line HCT116in vitro. Non interference group, negative siRNA group and RNA interference group were set up. Cell proliferation was measured by MTT test and colony formation assay. Cell cycle was detected by flow cytometry with annexin V/PI. Scratch healing experiment was performed to measure the cell mobility. Trnaswell assay was used to assess the ability of cell invasion.Results:After transfection Slug siRNA Flow cytometry test the percentage of cells in G1phase cells (52.3±0.6) higher than the negative control group (45.1±0.3)(P<0.05).the proliferation rate of HCT116cells was deceased by MTT assay and colony formation assay.. The percentage of cells at G1phase(52.3±0.6) in RNAi group was increased compared to negative control group(45.1±0.3)(P<0.05). scratch healing experiments showed significantly decreased cell mobility (P<0.05).The cell invasion ability compared withCON and NC group was significantly reduced.Conclusion:Direct inhibition of Slug expression by siRNA significantly suppresses the proliferation of HCT116cells in vitro, indicating a new approach of gene therapy for colon carcinoma. |
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