The Effect Of COX2 Expression On The Proportion Of CD133~+CD44~+ Cells In PC3 Cells Of Prostate Cancer In Vitro

OBJECTIVE: To investigate the effect of abudance of COX2 on the proportion of CD133~+CD44~+ cells in PC3 cells of prostate cancer,providing experimental evidence basis for clinical research and development of new drugs to treat prostate cancer.Materials and methods: PC3 cells of logarithmic growth phas-e was used in experiments.(1)to observe the effects of different concentrations of celecoxib on the expression of COX2 and proporti-on of CD133~+CD44~+ cells of PC3 cells: The experiment was divid-ed into blank group,DMSO group,25 μmol/L celecoxib group,50 μmol/L celecoxib group,(1)The expression of COX2 protein was detected by Western Blot in each group;(2)The PGE2,which is one of the downstream products of COX2,was detected by ELISA in each group;(3)The proportion of CD133~+CD44~+ cells in PC3 cells were detected by flow cytometry in each group.(2)To observe the effects of different concentrations of celecoxib on the number of microspheres formed and their proportion of CD133~+CD44~+ cells in PC3 cells:(1)The experiment was divided into DMSO group,25 μmol/L celecoxib group,50 μmol/L celecoxib group,PC3 cells were cultured in serum-free medium and treated with celecoxib for 2 weeks using ultra-low adhesion cell culture plates.Under low magnification microscope,the number of microspheres formed in P C3 cells(at least 50 cells were defined a microsphere);(2)The proportion of CD133~+CD44~+ cells in the 0,50 μmol/L celecoxib treate dmircrosphere were measured by flow cytometry.(3)To observe t he effect of over-expression of COX2 on the proportion of CD133~+CD44~+ cells in PC3 cells: the experiment was divided into blank group,empty vector group and COX2 over-expression group,COX2 over-expression plasmid was constructed and transfected into PC3 cells.(1)The expression of COX2 protein was detected by Western Blot in each group;(2)The PGE2 was detected by ELISA in each group;(3)The proportion of CD133~+CD44~+ cells in PC3 cells were detected by flow cytometry in each group.RESULTS:(1)(1)The results of Western Blot showed that the relative optical density of COX2 protein compared to β-actin protein in the blank group,DMSO group,25μmol/L celecoxib group and 50μmol/L celecoxib group were 1.06±0.05,1.07±0.07,0.58± 0.04 and 0.18±0.03 respectively.Compared with blank group or DMSO group,the expression of COX2 protein was decreased after treatment with celecoxib in PC3 cells.The difference was statistical-ly significant(P < 0.05).(2)The results of ELISA showed that the expression level of PGE2 in the blank group,DMSO group,25μmol/L celecoxib group and 50μmol/L celecoxib group were 139.0±4.7,132.0±2.6,85.0±2.6 and 60.0±4.1pg/ml respectively.Compared with blank group or DMSO group,the expression of PGE2 was decreas-ed after treatment with celecoxib in PC3 cells.The difference was statistically significant(P < 0.05).(3)The results of flow cytometry showed that the proportion of CD133~+CD44~+ cells in the blank group,DMSO group,25μmol/L celecoxib group and 50μmol/L celecoxib group were 1.38±0.02%,1.27±0.02%,1.04±0.03%,0.59± 0.01% and 0.59±0.01% respectively.Compared with the blank grou p,DMSO group,the proportion of CD133 + CD44 + cells was decr-eased after the celecoxib treatment.The difference was statistically sig-nificant(P < 0.05).(2)(1)Under low magnification microscope,the DMSO group,25μmol/L celecoxib group and 50μmol/L celecoxib group,the number of microspheres in each field of vision was 12.0±0.9,6.1±0.91 and 3.0±0.9 respectively.The results showed that cel-ecoxib inhibits the number of microspheres in PC3 cells.(2)The res-ults of flow cytometry showed that the proportion of CD133~+ CD44~+ cells in the DMSO group and 50μmol/L celecoxib group we re 40.4±0.6%,17.3±0.47% respectively.The difference between the two groups was statistically significant(P< 0.05).(3)(1)COX2 over-expression vector was constructed successfully.(2)The results of Western Blot showed that the relative expression level of COX2 protein compared to β-actin protein in the blank group,empty vector group and COX2 over-expression group were0.67±0.02,0.64± 0.04 and 1.16±0.03 respectively.COX2 protein in COX2 over-expression group was higher than that in blank group and empty vector group.The difference was statistically significant(P<0.05).(3)The resul-ts of ELISA showed that the expression level of PGE2 in the blan-k group,empty vector group and COX2 over-expression group wer-e 140.0±6.5,145.0±3.8 and 233.0±3.2 pg/ml respectively.PGE2 in COX2 over-expression group was higher than that in blank group a nd empty vector group.The difference was statistically significant(P < 0.05).(4)The results of flow cytometry showed that the propor-tion of CD133~+CD44~+ cells in the blank group,empty vector group and COX2 over-expression group were 1.06±0.05%,1.17±0.04% and 2.04±0.08% respectively.The proportion of CD133~+CD44~+ cells in COX2 over-expression group was higher than that in blank group and empty vector group.The difference was statistically significant(P < 0.05).Conclusion:(1)Though inhibitting the expression of COX2,celecoxib can reduce the proportion of CD133~+ CD44~+ cells in PC3 cells which is a marker for cancer stem cells.(2)Up regulation of COX2 expression can increase the proportion of CD133~+ CD44~+ cells in PC3 cells.