PGC-1β Regulates Breast Tumor Hepatocyte Apoptosis Through MTOR Signaling Pathway

Cancer is one of the most severe diseases for human.Many scientists have done their best works on cancer therapy research.As immortalized cells,cancer cell has the ability of abnormal growth,differentiation and apoptosis.Apoptosis can eliminate some bad cells which may havepotential risk,and keep cellular balance for our body.If the regulation mechanisms of apoptosis were broken,disease or cancer,even death would occur.Apoptosis is a necessary for regulating living body development.Cell apoptosis in our body could be regulated accurately,but apoptosis in cancer cells was inhibited.At present,the elucidation of the mechanisms and signal pathway involved in apoptosis and discovery of the molecules to induce cell apoptosis and block apoptosis-resistant factor would be the final goal of clinical research in cancer.Like normal cell,cancer has active energy metabolism during the process of tumor formation and metastasis.During the process,peroxisome proliferative actives receptor γ coactivator-1(PGC-1)gene plays an important function.PGC-1 gene family includes PGC-1α、PGC-1β and PRC,which are similarity in component and structure.There is an interaction role existing among three subunits.PGC-1β is related with adipognesis,muscle damage repair,vascular proliferation and cell apoptosis.Mammalian Target of Rapamycin(mTOR)is important signal pathways in regulating cell proliferation,which can integrate multiple stimulating signals stimulate outside the cell,therefore affect different aspects of cell activity.It has been reported that mTOR was involved in regulating apoptosis induced by PGC-1β.But the detailed regulating relationship between mTOR and PGC-1βand underlying mechanism remain unclear.To investigate the molecular mechanisms that regulate PGC-1β mediated cell apoptosis by mTOR signal pathway will be ann important issue of the project.Breast cancer is the most common female malignant tumor,which is one of the severe diseases threating to women’ s health.The main reason for breast tumor occurence is that the breast tissues have breast cancer stem cells.To investigate regulation mechanisms involved in apoptosis of breast cancer stem cells will help us design a better way to inhibit the growth of breast cancer,which will provide a new pathway for curing breast cancer.In this study,we will take breast cancer stem cells as an object,and set the regulation mechanisms of cell apoptosis controlled by PGC-1β and mTOR signal pathway as research goal.First,the breast cancer stem cells were isolated and characterized;second,the knockdown and over-expression vector for PGC-1β were constructed,then the vectors were transfected into the breast cancer stem cells,followed by the detection of the change of index.The detailed experimental contents are as follow:1)Isolation and characterization of the breast cancer stem cells:Fresh tumor tissues from the patients suffered breast cancer were collected from surgical rooms.The tissue samples were mechanically sheared and enzyme-digested to get single cell suspensions.Cancer cell surface antigens were analyzed by Flow cytometry analysis,and cell growth curve was established by MTT;qPCR was employed to detect the expression of stem cell-specific genes Sox2 and Nanog in the cancer cells;Immunofluorescence staining was performed to exam the expression of breast cancer-specific proteins BCL-2,progesterone receptor(PR),VEGF,Ki-67 and E-Cad;Nude mouse tumorigenicity was used to detect the cells oncogenecity.The results showed that we have successfully cultured and established human breast cancer stem cells in the state of sphere growth.The stem cells had a CD44+/CD24-phenotype as characterized by Flow cytometry analysis,expressed high levels of Sox2 and Nanog genes,and was positive for staining of Bcl-2,PR,VEGF,Ki-67,and E-Cad.The cells were also shown to have strong oncogenecity.This study demonstrated that human breast tumors contained CD44+/CD24-cancer stem cells which are capable of self-renewal,and can be long term cultured and expanded in vitro.2)Construction of over-expression and knockdown vectors of PGC-1β:By the comparison of normal tissue and breast cancer tissue,we find that PGC-1β was highly expressed in breast cancer tissue.To study the function of PGC-1β,we constructed the vector of PGC-1β eukaryotic expression vector pcDNA3.1/PGC-1β and PGC-1βknockdown vector pGenesil/shPGC-1β.The vectors would help us to investigate the function of PGC-1β in regulating the proliferation and apoptosis of cancer cells.3)Apoptosis analysis of human breast cancer stem cells transfected with the over-expression and knockdown vectors of PGC-1β:mTOR signal pathway could integrate several stimulating signal from outside of cell,which has an important function in regulating cell signal pathway involved in cell proliferation.In this experiment,we first detected the change of mitochondrial genome regulated by PGC-1β;then detected the change of ATP and AMP influenced by PGC-1β in the cells;Western blot was used to analyses the change of mTOR related protein in the cells transfect with the vectors for PGC-1β over-expression and knockdown;Flow cytometry was used to analyse the apoptosis of the cells transfect by the vectors for PGC-1β over-expression and knockdown.The results demonstrated that PGC-1β could enhance mitochondrial genome expression,the over-expression of PGC-1β can increase cell the expression of AMP and AMPK;meanwhile the phosphorylation of mTORCl related protein is up-regulated,and the cell apoptosis is decreased when the cells were transfected with PGC-1β over-expressing vector;All the results above are opposite when the cells were transfected with PGC-1β knockdown vector.The results demonstrated that PGC-1β has the function of regulating the apoptosis of breast cancer cells under the cooperation of mTOR signal pathway.Conclusion:In this thesis,we firstly isolated and characterized breast cancer stem cells from breast cancer tissue,then we constructed the vector of PGC-1β eukaryotic expression vector pcDNA3.1/PGC-β and PGC-1β knocdown vector pGenesil/shPGC-1β.Finally the vectors were transfected into breast cancer stem cells,and the change of some genes about cellular metabolism and mTOR related protein was detected.The resluts demonstrated that PGC-1β coulde increase the level of phosphorylation in mTORCl related proteins and decrease the level of phosphorylation in mTORC2 related protein,and down regulate the apoptosis of breast cancer cells under the cooperation of mTOR signal pathway.