Effects And Mechanism Of ADAM17 ShRNA On Apoptosis Of HT29 Colon Cancer Cells

PART 1 Effect and mechanism of ADAM17 on the invasion of HT29 human colon cancer cells Objectives To observe the effects of ADAM17 on the invasion of hunman colon cancer cell lines HT29 in vitro and investigate the mechanism.Methods The expression of ADAM17 protein was silenced by slow virus transfection,The expression of ADAM17 mRNA was detected by real-time quantitative PCR,The ability of cell migration was detected by wound healing assay,The experiments in cell migration and invasion were performed in vitro.The expression of apoptotic protein MMP-2?MMP-9 and ERK werer detected by Western blot.Results The expression of ADAM17 protein in HT29 cells was significantly inhibited?P<0.01?;The res?lts showed that the migration and invasion ability of the cells were significantly inhibited after the expression of the silence ADAM17 compared with control group?P<0.01?;When the expression of ADAM17 were silenced,the expression of MMP-2?MMP-9 decreased significantly,p-ERK1/2 decreased significantly compared to the blank control group?P<0.01?,but the expression level of ERK1/2 was not significantly different compared to the blank control group?P>0.05?.conclusion 1 Silent ADAM17 gene can inhibit the migration and invasion of HT29 human colon cancer cells.2 ADAM17 may inhibit the expression of MMP-9 and MMP-2 by inhibiting ERK pathway activation,thus affecting cell migration and invasion ability.Part 2 Effect and mechanism of ADAM17-shRNA promotes apoptosis of HT29 colon cancer cells Objectives To investigate the inhibition effect of silence of ADAM17 gene on the proliferation and apoptosis of colon cancer HT29 cells and its possible mechanism.Methods HT29 cells were divided into 3 groups:cells of interference group were transfected with recombinant lentivirus vector,cells of negative control group were transfected with negative recombinant lentivirus vector,and cells of blank control group were treated with PBS.The expression of ADAM17 mRNA was detected by real-time PCR,the expressions of caspase3,protein kinase B?Akt?,Glycogen synthase kinase-3??GSK3??,Phospho-protein kinase B?P-Akt?,Phospho-Glycogen synthase kinase-3??P-GSK3??protein were detected by Western blot method,the cell proliferation was detected by MTT assay,and the apoptosis rate and the cell cycle were detected by Annexin V-FITC/PI cell death detection kit.Results Compared with the control group and the negative control group,the interference group was related to low expressions of ADAM17 mRNA and its protein,low optical density OD value at the same time point?24h,48 h,and 72 h?,high apoptosis rate,the G₀/G₁ phase cell percentage increased,proportion of G₂/M phase cells decreased?P<0.05?,high expression level of caspase3 protein?P<0.05?,but low expression levels of p-Akt and p-GSK3?protein?P<0.05?.The expression level of Akt and GSK3?was not significantly different compared to the blank control group?P>0.05?Conclusion 1 silence ADAM17 gene can inhibit colon cancer cell proliferation and induce apoptosis,at the same time can be blocked the cells in G0/G1 phase 2 silence ADAM17 genes may be by inhibiting the activation of Akt/GSK3?beta pathway,play to the role of the induction of apoptosis and reg?lation of cell cycle...