| Background&Obiective: Breast cancer is one of the most common malignant tumor of female that accounts for approximately 22 % of all female malignant tumor,which is one of the important diseases endangering women's health.Triple—negative breast cancer denotes tumors that do not express the estrogen receptor(ER),progesterone receptor(PR),and human epidermal growth factor receptor 2(HER-2)genes.Triple-negative breast cancer was commonly seen in premenopausal patients with younger,and it is not treatable by radiotherapy and chemotherapy,easy early visceral metastases,brain metastases,5-year survival rate less than 15%.Triple negative breast cancer is lack of effective treatment,and it is one of problem demanding prompt solution in the field of cancer research.Shp2 molecules are one of the members of the family of protein tyrosine phosphatase that encoded by e gene PTPN11,which is the first to be sure for the human proto-oncogene phosphatase.Shp2 is a special kind of tyrosine phosphatase that compared to negative regulatory role of other kinds of phosphatase dephosphorylation of inhibition signal transduction,abnormal activation of Shp2 molecules involved signal amplification reaction that produced by a variety of growth factors and hormones in the cells inside and outside,Shp2 has played a positive regulation related to cell survival and proliferation signal transduction pathways.Previous studies showed that the Tumor contains a small amount cells that have infinite proliferation potential and self-renewal ability and drive the formation and growth of Tumor,the Cancer Stem Cell(CSC)or Tumor Initiating Cell(TIC).Which is primary cause of tumor cells treatment resistance and relapse and metastasis.Therefore the further study of tumor stem cells are expected to fundamentally change the treatment of tumor and provide new targets and methods for cancer therapy.Triple negative breast cancer is less chemosensitive,higher relapse and metastasis,worse prognosis.Biomarkers and therapeutic targets of recurrence and drug resistance is the current research hot spot of triple negative breast cancer.This project intends to explore Stem cell phenotype and clinical significance of Shp2 by a variety of classic molecular biology and cell biology technology.To reveal relationship between Shp2 and stem cells,and explore relationship between Shp2 and 5–Fu resistance in vitro cell research.At the same time the results should be validated with tissue chip samples ofbreast cancer,and association of Shp2 with clinical features and prognosis of triple negative breast cancer would be systematic analyzed.The value of Shp2 would be estimated as the prognostic marker and clinical guidance value.The result would be provide new molecular targets of triple negative breast cancer diagnosis and treatment.Methods:1.To Construct interference expression vector sh RNA-Shp2 of Shp2 gene and negative control interference expression vector sh RNA-PC3.1,cell morphology and transfection efficiency would be observed in 24 hours after transient transfection MDA-MB-231 cells with liposome transfection.2.Shp2 was examined by means of Western blot and Real-time PCR.3.Migrating and repairing ability of MDA-MB-231 cells were evaluated by wound healing assay and Transwell migration testing,invasiveness of breast cancer cell was examined by Matrigel-Transwell invasion assay,To investigate biological behavior of Shp2 resistance to chemotherapy by flow cytometry.4.After tumor stem cells were cultured by Spheroid experiment in vitro,the change of the cancer stem cell phenotypic markers was examined by the Real-time PCR under down-regulated expression of Shp2,thus further investigate the relationship between expression of Shp2 and triple negative breast cancer recurrence and metastasis.5.The Shp2 molecules was examined by means of immunohistochemical staining integrated chip,combined with the clinical prognosis,SPSS18.0 was used for data analyses.Results: 1.Shp2 gene was transfected into cell lines MDA-MB-231 of triple negative breast cancer and expression down-regulated.2.Self-renewal ability of Breast cancer stem cell decreased significantly when Shp2expression was interfered in the Spheroid and chemotherapy resistance experiment.3.Migrating,repairing and invasiveness in vitro ability of triple negative breast cancer cell lines decreased significantly when Shp2 expression was interfered in wound healing assay,Transwell migration testing and invasion assay in vitro.4.Apoptosis was detected by means of PI/Annexinv double staining and flow cytometry(FCM)analysis respectively after cell lines MDA-MB-231 by interfering Shp2 and control cells were treated with 5-Fu.The results showed that chemosensitivity of triple negative breast cancer cell would be influenced by Shp2 expression down-regulated and the apoptosis of cell induced by chemotherapeutic drugs.5.Based on the result of Shp2 in the immunohistochemical staining chip,the higher the Shp2 expression,the worse patients' overall survival and disease-free survival.Conclusion: Shp2 promoted regulation of triple negative breast cancer stem cells and Shp2 expression are closely related with prognosis.The results showed that expression of Shp2 was significantly increased.The high expression of Shp2 implies poor prognosis.Chemotherapy resistance and self-renewal ability of triple negative breast cancer cell maybe partly due to Shp2 In vitro and the effect of 5-Fu would be enhanced when Shp2 was interfered.To sum up,Shp2 may play very important roles in the regulation of triple negative breast cancer stem cells,may be prognostic marke and potential clinical therapeutic targets of triple negative breast cancer. |
PDF Full Text Request