Overexpressed ZIC1 Suppressed The Growth Of Breast Cancer Via Targeting Survivin

Objective:ZIC1 gene and Survivin gene are considered to be a potential tumor suppressor gene and an oncogene,respectively,and play important roles in the occurrence and development of breast malignancies.This study aims to confirm the role of ZIC1 gene in breast cancer,analyze the correlation between ZIC1 gene and Survivin gene expression in breast cancer,and explore the molecular mechanism of ZIC1 gene in breast carcinoma.Methods:(1)120 cases in Shanghai changhai hospital were collected to detect the levels of ZIC1 and Survivin expression of breast cancer and corresponding adjacent tissues,and analyzed the relationship of ZIC1 and Survivin expression by IHC.(2)Select six human breast cancer cell lines,and a normal mammary epithelial cell line,with regular culturing,and then used western blotting technique to detect the expression of ZIC1 and Survivin;(3)Transfected rLV-ZsGreen-PGK-Puro or rLV-Zic1-PGK-Puro into MDA-MB-231 and SK-BR3 breast cancer cell lines,and then used the fluorescence microscope and western blotting technology to assess the transfection efficiency,and analyze the relationship between ZIC1 and Survivin protein;(4)MTT method was uesd to detect the proliferation ability of MDA-MB-231 and SK-BR3 breast cancer cell after ransfecting rLV-ZsGreen-PGK-Puro or rLV-Zic1-PGK-Puro;(5)The tablet cloning test was used to detect the lone formation efficiency of MDA-MB-231 and SK-BR3 breast cancer cells after ransfecting rLV-ZsGreen-PGK-Puro or rLV-Zic1-PGK-Puro;(6)Flow cytometry was used to detect the cell cycle distribution of MDA-MB-231 and SK-BR3 breast cancer cells after ransfecting rLV-ZsGreen-PGK-Puro or rLV-Zic1-PGK-Puro;(7)Flow cytometry was used to detect the apoptosis of MDA-MB-231 and SK-BR3 breast cancer cells after ransfecting rLV-ZsGreen-PGK-Puro or rLV-Zic1-PGK-Puro;(8)Minute ? Mitochondria Isolation Kit was used to extract mitochondria and cytoplasm protein in breast cancer cells;(9)The JC-1 kit was used to detect the purified mitochondrial membrane potential of MDA-MB-231 and SK-BR3 breast cancer cells;(10)Western blotting technique was used to detect the expression of related proteins in the MDA-MB-231 and SK-BR3 breast cancer cells;(11)Collect and injected stable transfected cells into the right side of the back subcutaneous in nude mice.Observe and record the growth of the mass change.At 39 days,obtained the tissues and used immunohistochemistry technique to detect theexpression of Zic1 and Survivin.Results:(1)ZIC1 was expressed only in the cytoplasm,whereas Survivin was expressed both in the nucleus and cytoplasm.ZIC1 expression in tumors was significantly lower than that of normal tissues,opposite to Survivin.ZIC1 expression in tumors was significantly lower than that of Survivin,with a significant negative correlation.(2)ZIC1 expression in breast cancer cell lines was lower than that of normal breast epithelial cell,opposite to Survivin.After transfecting,Survivin expression was also significantly decreased(P < 0.05).(3)The growth of breast cancer cell with high expression of ZIC1 was blocked in G1 phase,and its cell proliferation activity,clone formation ability and level of mitochondrial membrane potential was decreased obviously,along with Cyto-C protein to be released from the mitochondria into the cytoplasm,and the apoptosis rate was increased significantly(P < 0.05).(4)Tissues of "ZIC1" group were significantly smaller than the "Vector" group.The expression level of ZIC1 was significantly higher in “ZIC1” group,and Survivin was significantly lower than that in “Vector” group.(5)With high expression of ZIC1,the levels of phosphorylation of Akt,mTOR and P70S6 K were significantly decreased,CyclinD1 expression was also decreased obviously and P21 and P27 was increased.In addition,Bcl-2 and Bcl-xL was decreased obviously,the level of phosphorylation of Bad was decreased,Bad expression was raised obviously,and Caspase9 and Caspase3 was activated.Conclusions:(1)ZIC1 expression in invasive breast cancer was significantly lower than that of normal breast tissues,opposite to Survivin,and there was a significantly negative correlation between ZIC1 and Survivin.(2)The level of Survivin was significantly reduced due to high ZIC1 expression.(3)With high ZIC1 expression,proliferation and clone formation efficiency was inhibited by blocking the growth of breast cancer cells in the G1 phase.(4)With high ZIC1 expression,mitochondrial membrane potential was significantly decreased and Cyto-C was released from the mitochondria into the cytoplasm,and then apoptosis was promoted.(5)The tumor growth rate in "ZIC1" group was significantly lower than "Vector" group,and Survivin could be suppressed continuously with high ZIC1 expression.(6)The activation of the Akt/m TOR signaling pathway was significantly inhibited with high ZIC1 expression in breast cancer cells,while the mitochondrial apoptotic pathway was promoted.