The Influcence Of Stathmin SiRNA Expression Vectors On Biological Behaviour In MCF-7 Cells

Microtubules (MTs) are a major component of the eukaryotic cytoskeleton and have been assigned many functional roles, such as intracellular trafficking, protein synthesis, intracellular signaling, chromosome segregation during mitosis and cell fate determination. MTs are also involved in specific myocardial cell functions, including regulation of contraction, ion channel function, receptor recycling, and sarcomere structure.Cellular microtubules are always in the process of polymerization and depolymerization. The transition between the stable and disassembled MT forms is regulated by two main classes of MT regulators: MT-associated proteins (MAPs), which have the ability to polymerize and stabilize MTs, and Oncoprotein 18 (Op18)/stathmin family members, especially the MT-destabilizing protein families.Stathmin, also known as oncoprotein 18 (Op 18), p18, p19,19K, metablastine, Lap 18, prosolin, and pp21/pp23, is an 18 kDa ubiquitously expressed cytosolic protein that destabilizes microtubules through tubulin sequestration and/or promotion of microtubule catastrophe. Stathmin was first identified as a cellular phosphoprotein overexpressed in leukemia, and has since been associated with highly proliferative breast, ovarian and prostate cancer cells. Furthermore, stathmin is often overexpressed in breast cancer cell lines harboring mutant p53.Therefore, we hypothesized that repression of stathmin by using small interfering RNA (siRNA) may partially influence the morphology of microtubules and the proliferation, apotosis, and migration capability of breast cancer MCF-7 cells with highly expressed stathmin.First, we constructed the expression plasmid of stathmin siRNA, transiently transfected breast cancer MCF-7 cells. The transfective efficiency was normalized by observing the GFP expression under fluoroscopy. mRNA and protein expression of the stathmin gene in the transfected cells was detected by RT-RNA and Western blot, respectively. Our study showed that the expression level of stathmin mRNA and stathmin protein in the transfected cells was markedly decreased compared with controls.Then, the effect of stathmin on proliferation of MCF-7 cells was detected by MTS assay. The growth of MCF-7 cells treated with stathmin siRNA was significantly inhibited. In order to evaluate the effect of stathmin siRNA expression vector on cell apoptosis in breast cancer MCF-7 cells in vitro, AO/EB and Western blot analysis were used. AO/EB assay showed that transfection of stathmin siRNA expression vector induced significantly apoptosis of MCF-7 cells. Western blot analysis indicated that the level of the apoptosis-associated proteins (caspase-3,9) was increased.The effect of stathmin siRNA expression vector on the migration of MCF-7 cells was investigated using wound healing assay in vitro. Our study indicated that stathmin siRNA expression vector could inhibit the migration of MCF-7 cells.To analyze the microtubule network, indirect immunofluorescence staining was performed. We found that cells transfected with stathmin siRNA showed long and bundled microtubule polymers, whereas cells transfected with scrambled-control siRNA showed short and diffuse microtubule polymers. Furthermore, The fluorescence intensity was greater for cells transfected with stathmin siRNA compared with controls.These findings support the hypothesis that repression of stathmin by using small interfering RNA (siRNA) inhibits the growth and migration of human breast cancer MCF-7 cells. Down-regulated stathmin could induce cell apoptosis and microtubule polymerization in vitro. Our research supports the concept that silencing stathmin may represent a novel therapeutic strategy in the treatment of breast cancers.