Effect Of Rab25 On Biological Functions In Human Breast Cancer Cells MCF-7

【Objectives】:Thawing the breast cancer cells (MCF-7) with the different expression levels of Rab25 gene prepared by the formor research group, discover the active proliferation of different cells and the influence to forming new transplantation tumors of nude mice. To investigate the effect of different expression levels of Rab25 gene on biological functions in breast cancer cells.【Methods】:1.Thawed the human breast cancer cells with the different expression levels of Rab25 gene prepared by the formor resrarch group, including original MCF-7, MCF-7 transfected eukaryotic expression plasmid pDNR-Dual-Rab25, MCF-7 with Rab25 RNA interferenced and MCF-7 transfected empty plasmid (short as Original, Up-regulated, Down-regulated and Empty), enlarged the number to be stand-by.2.Polymerase chain reaction (PCR) to examine the real expression of Rab25 for the series groups.3.MTT reduction assay to examine the active proliferation of the series cells.4.Gathered the all the researched breast cancer cells in exponential phase of grwth and transplanted them to the subcutaneous tissues of nude mice, then observeed them intently for 40 days. Recorded the state of the transplantation tumors.5.Measured the expression of Rab25 mRNA in the researched transplantation tumors by real-time quantitative PCR.【Results】:1.Observed the cell appearance in the Original, the Up-regulated and the Empty group had no significant difference in morphology under the light microscope. They had regular shape and growed as monolayer. The shape of the cells were polygonal or fusifourm with round or elliptic nucleus and translucent cytoplasm. While the Down-regulated group cells with irregular-shaped increased their bodies, and the intercellular were wadened. The transparent of cytoplasm were also brightness down. The dendrites of the cell increased and became short and thick. The adhesion levels of the cell decreased either. It indicated that the cell growth activity were in a downward trend.2.Rab25 gene could be checked out in the Original , the Up-regulated and the Empty groups of MCF-7, but no expression in The Down-regulated group.3.In the researched breast cancer cell lines,the proliferation rate and invasion ability increased after the transfection of Rab25 ,while decreased after the interference of Rab25 gene. The difference was sinificant between experimental groups and control groups (p<0.05).4. Successfully established the nude mice model with the transplantation tumor of breast cancer. By monitoring changes in the tumor volume in nude mice to establish their growth curve. We found that the tumor volume increased over times,and the Up-regulated group`s tumor volume was much larger than the other three, the difference was significant (p <0.01). We collected the final tumors from each group and weighed, the tumors from the Up-regulated group were heavier than the Original group and the Empty plasmid group , while the Down-regulated group tumors` weight were ligehter than the Original group and the Empty plasmid group,and both of the differences were significant (p <0.01). But the Original group and the Empty plasmid group compared transplantation tumor weight was similar, and the difference was not statistically significant (p> 0.05). These prove that the Rab25 Up-regulated group compared with Rab25 interference group, the original cells and empty vector group, the tumorigenicity in nude mice were significantly increased.5. Through real-time quantitative PCR detected the transplantation tumors in nude mice,we found that the Up-regulated group Rab25 mRNA expression was significantly increased, while the interference group tumor Rab25 mRNA expression was significantly reduced. Established two standard curves, the relative quantitative analysis showed that the Rab25 mRNA expression in Up regulated group for the Original group of 4.986-fold, while the Down-regulated group Rab25 mRNA expression in Original group was only 0.082 times.【Conclusions】:1.Rab25 could be checked out in the Original, Up-regulated and Empty groups of MCF-7 cells, but no expression in the Down-regulated group.2.The study found that the proliferation rate and the tumorigenicity in nude mice increased with the up-regulation of Rab25 gene, while decreased with the down-regulation of Ran25 gene. Rab25 played important role in the growth, proliference and invasion of breast cancer cells,which provided new ideas for the basical research and clinical application in breast cancer.