The Effct Of Pokemon On The Invasion And Migration Ability Of MCF-7 Cells

Objective:1.To establish a recombinant lentiviral vector that stably express pokemon in MCF-7 cells and to assess the effect of pokemon on the invasion and migration of MCF-7 cells.2.To investigate the expression and significance of Pokemon protein in breast cancer tissues.Methods:The experiment included two parts.Firstly,Pokemon-c DNA was constructed by RT-PCR. The pokemonc-DNA was ligated with GV165 vector,and then three plasmids(GV165/ Pokemon、p Helper 1.0、p Helper 2.0)were co-transfected into 293-T cells. The supernatant containing the lentivirus particles was harvested to determine the virus titer. MCF-7 cells were infectd with the lentiviral vectcr expressed high pokemon protein(pokemon group) and empty vector(NC group)respectively,then The MCF-7 cell line with stably ectopic pokemon expression was established.The transfection efficiency were assessed by fluorescence microscopy. Western blot was used to screen GFP-pokemon fusion protein. Transwell and wound healing analysis were utilized to determine the effect of pokemon on the invasion and migration of MCF-7 cells.Secondly,the expression of pokemon was detected by using immunohistochemistry staining method in the composite tissue chip,which contains 185 cases breast cancer tissues and 23 cases of breast cancer adjacent tissues.Then, the correlation between pokemon expression and clinicopathological parameters was analysed.Results:The first part:1.The recombinant lentiviral vector containing pokemon gene was constructed successfully:1)The target gene fragment was cloned by RT-PCR,and the recombinant lentivural vector GV165-pokemon was constructed through the method that the pokemon gene and GV165 vector were digested with restriction enzyme、ligated and transformation.The above result was verified by restriction analysis and gene sequencing.2) The supernatant containing the lentivirus particles was collected after the three vector(GV165/ Pokemon、p Helper 1.0、p Helper 2.0)was co-transfected into 293 T cells for 48 hours.3)The diluted lentiviral vector was transfected into 293 T cells for 24 hours,then The virus title was detected by RT-PCT,and the result of the detection was 2×108TU/ml.2.The MCF-7 cell line with stably ectopic pokemon expression was established:1)The expression of GFP(green fluorescence)can be observed by fluoresence microscopy in the two groups cells( the pokemon group and NC group).2)The green fluorescence was mainly distributed in the nucleus of the MCF-7 cell infected with pokemon expression vector,while in the NC group its expression was mainly in the cytoplasm.3)western blot result showed that the expression of GFP-pokemon fusion protein can be detected in the sixth clone that was infected with pokemon expression vector,while it just detected the expression of GFP in the NC group.3.The number of cells reached in the lower chamber of the transwell little room in the pokemon group was significantly less than NC group,which was statistically significant(P<0.05), suggesting pokemon gene can inhibit the MCF- 7 cell invasion ability.4.Would healing experimental results showed that the cell migration distance has no obvious change after 8 h、32 h between pokemon group and NC group, suggesting that pokemon gene have no obvious effect on the migration ability of MCF-7 cells. The second part:1.The expression of pokemon was mainly distributed in the nucleus of breast cancer tissues.The pokemon expression in the breast cancer tissus was significantly higher than in the breast cancer adjacent normal tissues.2.The expression of pokemon protein is correlateed with tumor type of breast cancer,while it is irrelevant with age、node metastasis、nuclear grade.Conclusion: 1. Pokemon could suppress the invasion ability of MCF-7 cells. 2.pokemon protein was overexpressed in the breast cancer tissues,and its expression was significantly higher in the ductal breast cancer tissus than in the lobular breast cancer tissues.