The Role Of CCDC8 Gene In Breast Cancer Cells MDA-MB-231 And The Relationship Between ANKRA2, LKB1 And 14-3-3σ

ObjectiveRecently, breast cancer has a younger trend, and most triple negative breast cancer are young patients, triple negative breast cancer is considered to be result of multi-stage, multi-factor and multi-gene interaction, we select the triple negative breast cancer cell line MDA-MB-231, knockdown of CCDC8 gene by si-RNA of MDA-MB-231,and observe the influence of proliferation and invasive ability on MDA-MB-231 cell, and probing the relationship between CCDC8 with ANKRA-2, LKB-1 and 14-3-3 in this cell line.Methods1, Take CCDC8 RNA 944,1860,1979 as the target to synthesize three segments of siRNA, interference fragment and transfected into MDA-MB-231 cells by liposome method, while set a the negative control group.2, By the mean of fluorescence and RT-PCR screening of stable transfection cell line.3, MTT detected CCDC8-1860 that stably transfected group changes in proliferation ability with negative control group.4, With the method of Cell scratch test identify the migration of CCDC8-1860 group and negative control group.5,By the mean of flow cytometry analysis the change of cell cycle and apoptos rate of stably transfected group CCDC8-1860.6,Each group cells proliferation ability and population were analyzed with the flat cell cloning experiment.7, Real-time PCR and Western blotting detection of CCDC8-1860 in MDA-MB-231 cells induced by CCDC8, ANKRA-2, LKB-1 and 14-3-3 genes DNA and protein expression.Results1, RT-PCR detection shows that 1860-siRNA can effectively inhibit CCDC8-mRNA (p<0.05).2.MTT assay showed that knockdown of CCDC8 by si-RNA resulted in a significant decrease in the proliferation of the MDA-MB-231 cell line(p<0.05);3,Cell scratch test showed knockdown of CCDC8 by si-RNA resulted in a significant decrease in the migration rate of the MDA-MB-231 cell line(p<0.05);4,Flow cytometry showed that migration rate the apoptosis rate was increased significantly and cell cycle arrest in G1 phase in CCDC8-siRNA transfected MDA-MB-231 cells (p<0.05);5, The results of plate cell cloning experiment showed that the rate of colony formation was reduced and the ability to proliferate was decreased in CCDC8-siRNA transfected MDA-MB-231 cells, (p<0.05);6, Qt-PCRand Western blotting results showed that:A,Knockdown of CCDC8 by si-RNA, CCDC8 gene and protein expression were decreased; B, Knockdown of CCDC8 by si-RNA, and negative control group compared with the experimental group ANKRA2 gene and protein expression were increased; C, Knockdown of CCDC8 by si-RNA, LKB1 gene and protein expression were increased; D, Knockdown of CCDC8 by si-RNA,14-3-3 σ protein expression were no obvious change.Conclusions1, knockdown of CCDC8 by si-RNA resulted in a significant decrease in the proliferation, the migration rate and invasive ability of the MDA-MB-231 cell line. but promote the apoptosis of MDA-MB-231 breast cancer cells.2, CCDC8 gene expression was lower in MDA-MB-231 breast cancer cells rusult to a significant increased in the gene and protein expression of ANKRA2 and LKB 1; CCDC8 inhibited while the expression of 14-3-3 gene and protein expression were no obvious change.