| Objective:To understand the effects of the proliferation in vitro and the cell cycle of estrogen and progesterone on human endometrial carcinoma JEC cell lines, and to explore effects of estrogen and progesterone on the expressions of p57kip2 and Cyclin E in JEC cells,providing a theoretical basis for the further study of the occurrence and development of endometrial carcinoma.Methods:Human endometrial carcinoma(EC) JEC cells selected at the middle level were cultured in vitro. The experiment was divided into two parts:(1)treated with β-estradiol of high, medium and low concentrations(10-6, 10-8, 10-10 mol/L) as the estrogen treatment group, the JEC cells were only treated with the culture medium without β-estradiol in it as the control group.(2)treated with progesterone of high, medium and low concentrations(10-4, 10-6, 10-8 mol/L) as the progesterone treatment group,the JEC cells were only treated with the culture medium without progesterone in it as the control group. After 24, 48, 72,96 hours, MTT was used to test the growth state of JEC cells. After 24, 48, 72 hours, light and electron microscope were used to observe morphological and ultrastructural changes,western blot(WB) was used to test the protein expressions of p57kip2 and Cyclin E in JEC cells. After 72 hours, flow cytometry(FCM) was used to test the cell cycle distribution changes of JEC cells. After 24, 72 hours, real-time fluorescence quantitative polymerase chain reaction(RT-q PCR) was used to test the m RNA expressions of p57kip2 and Cyclin E in JEC cells.Results:MTT results show:(1) when β-estradiol was in medium and low concentration range,it was promoting JEC cells’ growth(P<0.05), while in the high concentration, it was inhibiting JEC cells’ growth(P>0.05).(2) When progesterone was in medium and low concentration range, it was promoting JEC cells’ growth(P<0.05),while in the highconcentration, it was inhibiting JEC cells’ growth(P<0.05).Light microscope results show:(1) the shapes of JEC cells were mostly long oval with prominent nucleoli. The distribution density of JEC cells in medium and low concentration groups of β-estradiol were higher than those in the control group. The density distribution of cells in high concentration group was lower than in the control group. There were no significant changes in cell morphology in all groups.(2) The distribution density of JEC cells in medium and low concentration groups of progesterone were higher than those in the control group. The density distribution of cells in high concentration group was lower than in the control group. There were no significant changes in cell morphology in all groups.Electron microscope results show:(1) With the increase of the concentration ofβ-estradiol, the microvilli and secretory vesicle numbers of JEC cells decreased.(2) With the increase of the concentration of progesterone, the microvilli and secretory vesicle numbers of JEC cells decreased.FCM results show:(1) After 72 hours, β-estradiol obviously increased the cell percentage at the S phase of cell cycle and decreased at the G0/G1 phase(P<0.05).(2) After72 hours, progesterone slightly increased the cell percentage at the G0/G1 phase of cell cycle(P>0.05).WB results show:(1) JEC cells were treated with β-estradiol, comparisons of different concentrations of the same time, at 24 hours, the protein expression of p57kip2 is the lowest in the medium concentration group, and the highest in the high concentration group(P<0.05), the protein expression of Cyclin E is the lowest in the high concentration group, and the highest in the medium concentration group(P<0.05); at 48 and 72 hours,along with the increase of β-estradiol concentration, the protein expressions of p57kip2 and Cyclin E increased(P<0.05). Comparisons of different time periods of the same concentration: in the low concentration group, the protein expression of p57kip2 is the lowest in the 48 hours, and the highest in the 72 hours, in the high and medium concentration groups, along with the time passed, the protein expression of p57kip2increased(P<0.05); in the medium and low concentration groups, along with the time passed, the protein expression of Cyclin E was decreased(P<0.05), in the high concentration group, the protein expression of Cyclin E is the lowest in the 24 hours, and the highest in the 48 hours(P>0.05). There was no correlation between the protein expressions of p57kip2 and Cyclin E(P>0.05).(2) JEC cells were treated with progesterone,comparisons of different concentrations of the same time, at 24 hours, the protein expression of p57kip2 is the lowest in the medium concentration group, and the highest in the high concentration group(P<0.05), the protein expression of Cyclin E is the lowest in the medium concentration group, and the highest in the low concentration group(P<0.05);at 48 and 72 hours, along with the increase of progesterone concentration, the protein expression of p57kip2 increased(P>0.05), while the protein expression of Cyclin E decreased(P<0.05). Comparisons of different time periods of the same concentration: along with the time passed, the protein expression of p57kip2 increased in all the concentration groups(P<0.05); in the low concentration group, the protein expression of Cyclin E was the highest in the 24 hours and the lowest in the 48 hours(P<0.05), in the high and medium concentration groups, along with the time passed, the protein expression of Cyclin E decreased(P<0.05). The positive expression rate of p57kip2 and Cyclin E was negatively linear regression relation(P<0.05).RT-q PCR results show:(1) JEC cells were treated with β-estradiol, comparisons of different concentrations of the same time, at 24 and 72 hours, along with the increase of theβ-estradiol concentration, the m RNA expressions of p57kip2 and Cyclin E all increased(P<0.05). Comparisons of different time periods of the same concentration: in all concentration groups, the m RNA expression of p57kip2 is higher in 72 hours than in 24hours(P<0.05); in the high and medium concentration groups, the m RNA expression of Cyclin E is lower in 72 hours than in 24 hours(P<0.05), in the low concentration group, the m RNA expression of Cyclin E is higher in 72 hours than in 24 hours(P>0.05). There was no correlation between the m RNA expressions of p57kip2 and Cyclin E(P>0.05).(2) JEC cells were treated with progesterone, comparisons of different concentrations of the same time, at 24 and 72 hours, along with the increase of concentration, the m RNA expression of p57kip2 increased(P<0.05), while the m RNA expression of Cyclin E decreased(P<0.05).Comparisons of different time periods of the same concentration: in all concentration groups, the m RNA expression of p57kip2 is higher in 72 hours than in 24 hours(P<0.05),while the m RNA expression of Cyclin E is lower in 72 hours than in 24 hours(P<0.05).The positive expression rate of p57kip2 and Cyclin E was negatively linear regression relation(P<0.05).Conclusion:The m RNAs and proteins of p57kip2 and Cyclin E in the occurrence and development of EC are affected by estrogen and progesterone. With the increase of concentration andthe extension of time, the JEC cells become the state of proliferation inhibition from the state of proliferation promoting. Along with the extension of time, the m RNA and protein expression of p57kip2 increase in the high and medium concentration groups of estrogen,while the m RNA expression of Cyclin E decreases, in the medium and low concentration groups, with the extension of time, the protein expression of Cyclin E decreases as well.Along with the concentration increase of progesterone and the extension of time, the m RNA and protein of p57kip2 increase, while both m RNA and protein of Cyclin E decrease in the high and medium concentration along with the extension of time of progesterone. |
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