Overexpression Of OTUD1 In HCT116 Cells Inhibits

ObjectiveWith the rising incidence of colorectal cancer and the limited treatment,one of the ways to improve the survival rate of patients with colorectal cancer is early prediction and early intervention.Due to the current predicted recurrence and metastasis of colorectal cancer is not yet effective Therefore,this study aims to investigate the role and mechanism of ovarian tumor domain-containing protease 1(OTUD1)gene in proliferation and invasion of human colon cancer cell line HCT116.MethodsSelected low expression OTUD1 human colon cancer cell line,using lentivirus packaging technology overexpression OTUD1 Gene,empty plasmid as a control,puromycin screening stable transfected colon cancer cell lines constructed.Transfection efficiency was detected by reverse transcription PCR and Western-Blot.Cell migration and invasion assay test two groups of cells migration and invasion ability changes.Cell proliferation experiments and Flow cytometry was used to detect the changes of cell proliferation in both groups.Clone formation assay was used to detect the clonogenic capacity of both groups of cells.Western-Blot analysis of related protein molecular level changes,to explore its possible mechanism.Statistical Analysis Statistical analysis using SPSS software package(Standard Edition 17.0),the results are x ± s,two-tailed t-test for the analysis of relevant experimental data,each experiment repeated at least three times.P<0.05 for the difference was statistically significant.Results1.The expression of OTUD1 in HCT116 and HT29 was lower in 6 colon cancer cells,HCT116 cells as the main research object.Application of lentivirus packaging technology overexpression OTUD1 gene mining.The infection efficiency wasdetected by RT-PCR and Western-Blot.According to the experimental results,HCT116-OTUD1 cells at mRNA and protein levels were significantly higher than the control group.1.Transwell cell migration and invasion experiments were used to compare HCT116-OTUD1 cells and control group.HCT1 16-Control cells migration and invasion ability,according to the experimental results show that the number of cell migration in the control group cell migration was 98.40 ± 8.08,the experimental group was 30.40 ± 4.39,t = p = 0.000;invasion experiment control group number Was 68.00 ± 4.85,and the experimental group was 19.20 ± 4.87,p = 0.000.It indicated that over-expression of OTUD1 gene inhibited the migration and invasion ability of HCT1 16 cells.2.Using cell proliferation experiments,flow cytometry test compared before and after overexpression of two groups of cells proliferation force.According to the results of CCK-8 cell proliferation experiment,the cell proliferation rate of control group was 13.61 ± 0.76 at 72h,the experimental group was 11.34 ± 0.62,t = 3.991,p=0.016;the cell proliferation rate of control group was 16.45 ± 0.60 at 96h According to the results of flow cytometry,the positive rate of EDU in control group was 34.00 ±1.93,the experimental group was 21.50 ± 1.45,t = 8.967,p = 0.000;the two groups were 12.41 ± 0.96,t = 6.161,p = 0.004;Experiments show that overexpression of OTUD1 gene can inhibit cell proliferation.3.The use of cell cloning formation assay to detect cell cloning ability.According to the experimental results show that the control group.The number of clones formed was 112.67 ± 13.43 in the experimental group and 150.00 ± 9.54 in the experimental group,t = 3.926,p = 0.017,indicating that overexpression of OTUD1 gene can inhibit the clonogenic capacity of HCT116 cells.4.The result of Western-Blot showed that the expression of epithelial marker E-cadherin was up-regulated and the expression of mesenchymal marker Vimentin was down-regulated,indicating that the EMT effect of HCT116 cells was weakened and the invasion ability was inhibited after overexpression of OTUD1 gene.This study examined the two groups of cells p-Akt,p-Erk1/2 protein levels were down to varying degrees,and Akt,Erk1/2 no significant change,indicating over-expression of OTUD1 gene MAPK/Erk and PI3K/Akt signal Pathway activation was significantly inhibited.ConclusionThe mRNA expression of OTUD1 in HCT116 cells was the lowest in 1,6 colon cancer cell lines.Overexpression of OTUD1 can affect its ability of migration and invasion by attenuating the EMT effect of cells.Overexpression of OTUD1 can inhibit cell proliferation,which may be related to the inhibition of the activation of MAPK/Erk and PI3K/Akt signaling pathway.