| Objective : To detect the effect of progesterone on the proliferation,apoptosis and FOXO1 expression in ovarian cancer cells with different PR expression,to explore the molecular mechanism of progesterone in ovarian cancer and to provide theoretical basis for the treatment of ovarian cancer.Methods: Experimental group: control group(progesterone concentration 0 ?mol/L)and experimental group(progestin concentration 6.25,25,100 ?mol/L).Two well-developed ovarian cancer cells,HO-8910(high PR expression)and SKOV3(low PR expression),were studied and the following experiments were performed:1.Ovarian cancer cells were treated with different concentrations of progesterone(6.25,25,100 ?mol/L),the control group was added with equal amount of progesterone-free culture fluid.After 48 hours,cell morphology was observed by the inverted microscope.2.Different concentrations of progesterone(6.25,25,100?mol / L)were used to treat the two cells respectively,the control group was added with equal amount of progesterone-free culture fluid.The proliferation of each group was detected by CCK8 assay at 24 h,48h and72 h respectively.3.Two ovarian cancer cell lines,HO-8910 and SKOV3,were treated with different concentrations of progesterone(6.25,25 and 100?mol / L)respectively,the control group was added with equal amount of progesterone-free culture fluid.After 48 hours,Annexin V-FITC / PI double staining Cytometry was used to detect the apoptosis in each group.4.Two well-prepared ovarian cancer cell lines,HO-8910 and SKOV3,were treated with different concentrations of progesterone(6.25,25,and 100?mol/L),the control group was added with equal amount of progesterone-free culture fluid.The cells were incubated for48 h and Western blot was performed to detect the expression of PR and FOXO1 protein in each group.Results:1.The cell morphology observed under the inverted microscope after different concentrations of progesterone for 48 hours: the ovarian cancer cells HO-8910 and SKOV3 in the control group were similar to the epithelioid cells,showing round,fusiform and polygonal cells in a compact arrangement with some cells extending pseudopodia.In the experimental group(6.25,25 and 100?mol/L)cell gap increased,adherent cells decreased,floating cells increased,some cells broken,interstitial cell debris can be seen.The larger the progesterone concentration,the more obvious the characteristics of the above microscope,the same concentration of progesterone for the same time,the above microscopic characteristics of HO-8910 cells were significantly greater than that of SKOV3 cells.When the concentration of progesterone reached 100?mol/L,the adherent cells of HO-8910 became spindle-shaped while the outline of SKOV3 cells was unclear,resulting in the phenomenon of multiple cell fusion.2.Proliferation inhibition rate(%)of progesterone in control and experimental groups(6.25,25,100 ?mol/L)at different times:(1)HO-8910 cells:(24h)0.50±1.08?15.35±1.13?25.56±0.90?43.65±1.74,(48h)-0.15±1.39?24.07±0.63?43.61±3.42?64.20±1.39,(72h)-1.07±1.66?43.05±2.27?62.08±1.00?73.09±1.41;(2)SKOV3cells:(24h)5.8±1.08?8.46±1.18?9.96±0.69?13.95±2.70,(48h)-0.15±1.39?13.46±1.04?16.15±1.19?25.84±2.04,(72h)0.43±1.24?16.87±0.66?26.87±2.38?32.24±1.14.Proliferation inhibition rate comparison: progesterone can inhibit proliferation of HO-8910,SKOV3 cell,prolonged action time at the same concentration and increased concentration at the same time,the inhibition effect is enhanced,and there is a time-effect,dose-effect relationship on cell proliferation inhibition rate.(1)The inhibitory rate of HO-8910 cells after treatment with different concentrations of progesterone at the same time was significantly different between the experimental group and the control group,among experimental groups(P<0.05).After the same concentration of progesterone for 24 h,48 h,and 72 h,the inhibition rate of the experimental group at each time period was statisticallysignificant(P<0.05);(2)the same concentration of progesterone for SKOV3 cells after 24 h,48h,and 72 h,the inhibition rate of the experimental group was statistically significant(P<0.05).At the same time,different concentrations of progesterone were used.After 24 hours,the inhibition rates of the control group and 100 ?mol/L group were compared,and after 48 hours and 72 hours,the inhibition rates of the control group and the experimental group were compared,there was statistical significance(P<0.05).The inhibitory rate of the control group and the 6.25,25?mol/L group for 24 h was compared,the difference was not statistically significant(P>0.05);(3)The same effect concentration and time,the inhibition rate of HO-8910 cells and SKOV3 cells was statistically significant(P<0.05),and the inhibitory effect of progesterone on HO-8910 cells was significantly stronger than that of SKOV3.3.Apoptosis rate(%)of progesteronein the control group and experimental group(6.25,25,100 ?mol/L)after 48 hours:(1)HO-8910 cell : 1.75 ± 0.22,5.26 ± 0.20,16.00 ± 0.40,42.50± 3.96.With the increase of progesterone concentration,the apoptotic rate increased.The apoptotic rate of each concentration group was compared,and the difference was statistically significant(P <0.05).(2)SKOV3 cells : the apoptotic rates were 1.44 ±0.55,2.75 ± 0.66,4.11 ± 0.47,6.96 ± 0.41,There was no significant difference in apoptosis rate between control group and 6.25?mol/L group,25?mol/L group and 100?mol/L group(P> 0.05),the apoptotic rate among the other concentrations was statistically significant(P<0.05).(3)The apoptosis rate of HO-8910 was higher than that of SKOV3 at the same concentration for 48 h,the difference of apoptosis rate between the two cells was statistically significant(P <0.05).4.After 48 hours of progesterone treatment,the expression of FOXO1 in HO-8910 and SKOV3 cells increased.With the increase of progesterone concentration,the expression of FOXO1 increased.The expression of FOXO1 in experimental groups and control group was compared.The difference was statistically significant(P<0.05).In HO-8910 cells,there was a statistically significant difference in the expression of FOXO1 between the experimental groups(P<0.05).In SKOV3 cells,there was no significant difference in the expression of FOXO1 between 6.25?mol/L group and 25?mol/L group,25?mol/L groupand 100?mol/L group(P>0.05).There was no significant difference in the expression of PR-B and PR-A protein between HO-8910 and SKOV3 cells compared with the control group.Conclusion:1.Progesterone shows a time-effect and dose-effect relationship on the proliferation inhibition of HO-8910 and SKOV3 cells,and pro-apoptotic in a dose-response relationship.Progesterone significantly inhibites proliferation and promotes apoptosis in HO-8910 cells compared with SKOV3 cells.2.Progesterone can promote the expression of FOXO1 in HO-8910 and SKOV3 cells.The higher the progesterone concentration,the higher the expression of FOXO1.Progesterone may inhibit proliferation and promote apoptosis of ovarian cancer cells by affecting the expression of FOXO1.3.The concentration of progesterone does not affect the protein expression of PR-B and PR-A in HO-8910 and SKOV3 cells. |
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