MiR-125a Rs12976445 Has A Correlation With Survival In Breast Cancer Patients

Part Ⅰ Association between miR-125 a rs12976445 and survival in breast cancer patientsObjective : Micro RNAs(miRNAs)act as an oncogene or a tumor suppressor by negatively regulating target genes.Genetic variants in miRNA genes confer susceptibility to cancer and risk of death in cancer patients.The aim of this study was to investigate whether miRNA polymorphisms were associated with survival in breast cancer patients.Methods: 1.We retrospectively collected data of breast cancer patients after surgical resection of the primary tumor between 2001 and 2003 。 A total of 196 breast cancer patients were included in the final analysis after excluding patients with a history of cancer,other than breast cancer,or without survival status or clinical information.2.Genomic DNA was extracted from five sections of each FFPE tissue specimen using QIAamp DNA FFPE Tissue Kit.All SNPs were genotyped using polymerase chain reaction-ligation detection reaction(PCR-LDR)method 。 Codominant,recessive and dominant models were used for each SNP.Besides,Relative statistical analyses were also applied for these SNPs.Results: 1.Five miRNA polymorphisms(miR-26a1 rs7372209,miR-125 a rs12976445,miR-218 rs11134527,miR-423 rs6505162,and miR-608 rs4919510)were genotyped in196 breast cancer patients.2.We found that miR-125 a rs12976445 was significantly associated with survival in codominant,recessive,and dominant models.However,only association under the codominant model remained significant after adjustment for lymph node metastasis,TNM stage,estrogen receptor,and progesterone receptor.Furthermore,this effect remained in stratification analysis.Conclusions: In conclusion,our results provide evidence that miR-125 a rs12976445may serve as a prognostic biomarker for breast cancer.Further large-scale studies are required to confirm these findingsPart Ⅱ The role of oncostatin M on human breast cancer cellsObjective:To study the inhibition effects of oncostatin M(OSM)gene on human breast cancer cell line(michigan cancer foundation-7,MCF-7).Methods : 1.MCF-7 cells were infected with the optimized dose of retrovirus expressing OSM.Expression of the target gene was determined by reverse transcription-polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA).2.Methyl thiazolyl tetrazolium(MTT)assay was employed to examine growth inhibition effect of OSM gene on MCF-7 cells.Cell apoptosis was determined by flow cytometry.The morphologic changes of nucleus was detected by Hochest staining,in addition,the expression of apoptosis related genes in tumor cells were further detected by RT-PCR.Results:1.After make use of retrovirus infections to MCF-7 cells,we assure that 100 MOI is the optimized dose in the fluorescence microscope,and the ratio of infection could be up to 90%;2.After make use of retrovirus infections to MCF-7 cells,we carried on experiments to test the expression of OSM protein using RT-PCR and ELISA.It is shown that OSM was highly expressed in the group with OSM infection in m RNA level and protein level,respectively.And the index of MCF-7 Cell proliferation is apparently lower than the control group without OSM infection analysised by MTT assay.Besides,Cell apoptosis assay by flow cytometry showed that the rate of Cell apoptosis increased significantly and Hochest staining showed us the nuclear disruption and nucleolysis.3.After make use of retrovirus infections to MCF-7 cells,we collect cells and extract RNA with Trizol,then carried out experiments to analysis RNA simples with RT-PCR.The result told us that the transcription expression of Bax,caspase 3 had a visible increase with the infection of OSM;however,Bcl-2 decreased apparently.Conclusion:1.OSM gene could significantly inhibit the growth of MCF-7 cells and induce its apoptosis.2.The mechanism of OSM gene have effects on MCF-7 cells might be related to the upregulation of Bax and Caspase-3,and the downregulation of Bcl-2.Part Ⅲ Zoledronic acid inhibits angiogenesis by downregulating VEGF expression through STAT3 and ERK pathwayObjective : The reason why zoledronic acid can inhibit angiogenesis remains unknown.So this paper wanted to carry experiments to explore the mechanism of zoledronic acid inhibiting angiogenesisMethods: 1.Effect of zoledronic acid on angiogenesis was evaluated in zebrafish embryos.Transgenic(flk:GFP)zebrafish embryos were treated with various concentration of zoledronic acid for 24 hours.Then representative photographs are listed to show the subintestinal vessels2.Brd U incorporation assay,transwell assay,capillary-like formation,RT-PCR and Elisa assay were used to determine the phenotypics of HUVEC cells.Results: 1.zoledronic acid inhibits vessel formation in the zebrafish embryo;2.zoledronic acid decreases the proliferation,chemotactic motility and capillary structure formation of HUVECs in Vitro;3.zoledronic acid decreases ERK1/2 and STAT3 signaling in HUVECs;4.zoledronic acid decreases VEGF expression.Conclusions: In conclusion,our results provide evidence that zoledronic acid can inhibit angiogenesis by downregulating VEGF expression through STAT3 and ERK pathway...