The Impact Of Tumor Microenvironment And Epigenetic Mechanism On Tumor

PART ONE EXPRESSION AND SIGNIFICANCE OF CHEMOKINE`S RECEPTOR: CXCR7/RDC1 IN HUMAN BREAST CARCINOMA CELLSObjective : To investigate the expression and significance of CXCR7/RDC1 in human breast carcinoma cells.Methods:The mRNA and protein expression of CXCR7 in human breast cancer cells (MCF-7, ZR-75-30, MDA-MB-435S, MDA-MB-453, MDA-MB-231,and HBL-100) were detected with the application of RT-PCR and Western-Blot, respectively.Results : The expression levels of CXCR7 varied significantly between six cell lines. CXCR7 mRNA can be detected in all of the six breast cancer cell lines, and CXCR7 protein expression was statistically prominent in ER positive cell lines than in ER negative cell lines(P<0.01).Conclusion : CXCR7/RDC1 may play a role in occurence, development and prognosis of breast cancer. PART TWO THE EFFECT OF MATURE ADIPOCYTE ON BIOLOGICAL BEHAVIOUR OF BREAST AND PROSTATE CANCER CELLObjective:To study the effect of mature adipocyte on biological behaviour of cancer cells:breast and prostate.Methods:collect the mature adipocyte conditioned-medium and hypoxia adipocyte conditioned-medium(0.1%O2), use them to treat tumor cells. Migration assay and Invasion assay were performed to detect the metastasis capacities; Use Immunofluorescence to find the change of cell`s morphology; Real-time-quantitive-PCR and ELISA were used to detect the chemokines secreated by hypoxia adipocyte; Western blot and Real-time-quantitive-PCR were performed to detect the expression of upregluated hypoxia induced factor; cell proliferation ability was determinded by MTT assay.Results:①adipocyte conditioned-medium stimulates the tumor cells migration and invasion capacities, promote tumor cell proliferation and change the cell`s morphology, more significant under hypoxia adipocyte conditioned-medium ;②The expression level of HIF-1α,Glut-1,VEGF on adipocyte were upregulated during hypoxia 12hr, 24hr, 48hr detected by western blot and Real-time-quantitive-PCR;③The level of CCL5 mRNA increase on hypoxia adipocyte and CCL5 protein were detected increase in the supernant of hypoxia adipocyte.Conclusion:Mature adipocyte serve as a highly active endocrine cell in microenvironment secretes numerous factors, including growth factors, cytokines, hormone-lile molecules, extracellular matrix proteins that may play an important role in influencing tumor behavior and prove to be critical for tumor survival, growth and metastasis in tumor. PART THREE PCDH10 IS A FUNCTIONAL TUMOR SUPPRESSOR FOR BREAST CANCER AND FREQUENTLY METHYLATED IN PRIMARY TUMORSObjective: To investigated the role of PCDH10 in breast carcinogen- sis and identified promoter hypermethylation as a major mechanism of inactivation of this gene.Methods:we used semiquantitative reverse-transcription (RT)-PCR and methylation-specific PCR (MSP) detected PCDH10 downregulation or silencing in breast cancer cell lines; We further examined PCDH10 methylation in breast primary tumor and adjacent non-tumor, together with normal breast tissue samples as controls using MSP,BGS; after Pharmacological demethylation treatment, RT-PCR to detect the reexpression of PCDH10 and MSP,BGS to analyse the change level of methylation; colony formation was used to investigate the effects of ectopic PCDH10 expression on the growth of tumor cells in vitro.Results:Promoter methylation of PCDH10 was frequently detected in breast cancer cells and primary breast tumors, but not in any non-tumor cell line and seldom weakly methylated in normal epithelial tissues, which were further confirmed by high-resolution bisulfite genomic sequencing (BGS). Pharmacological demethylation restored PCDH10 expression, indicating a direct epigenetic inactivation. Ectopic expression of PCDH10 resulted in significant inhibition of colony formation in breast cancer cells. Conclusion:our results demonstrated that PCDH10 is frequently silenced by epigenetic mechanism in a tumor-specific way. It may play a role in breast carcinogenesis and could also serve as a specific biomarker for breast cancer.