Authors: Wang Jie, Kang Gangjin, Yuan Xuefeng, Xu Manhua, Jiang Yan, Luo Bo
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Human LECs can express telomerase activity, which participates in the formation of cataract.It is reported that estrogen can increase the expression of telomerase activity in human endometrial cancer and breast cancer cells and play an important role in promoting proliferation and anti-apoptosis, but whether estrogen exerts its role on human LECs is still unclear.
This study aimed to investigate whether β-estradiol (β-E2) can increase the telomerase activity of human LECs and the influence of β-E2 on proliferation and apoptosis of human LECs.
Human LECs line was cultured and passaged in vitro, and 1×10-6 mol/L β-E2 was added in the medium for 0, 6, 12, 24 and 48 hours, and reverse transcription PCR was used to determine the optimal time of the expression of human telomerase reverse transcriptase (hTERT) mRNA in the cells.Cultured cells were divided into five groups.The cells in the blank contol group were cultured in the routin medium.Ethanol of 0.1% was added in the solvent control group, and 1×10-8, 1×10-7 or 1×10-6 mol/L β-E2 was added in the medium in different contents of β-E2 groups, respectively.The relative expression level of hTERT mRNA in different groups was detected by reverse transcription PCR.Telomere repeat amplification protocol (TRAP)-ELISA was employed to determine the telomerase activity.The proliferative value of the cells was assayed by cell counting kit-8, and the apoptosis rate of the cells was examined by Hoechst33258 staining.
The optimal time of β-E2 to rise the expression of hTERT mRNA (absorbance) was at 24 hours under the 1×10-6 mol/L.The relative expression levels of hTERT mRNA in the cells were 0.477±0.015, 0.712±0.013 and 0.914±0.031 in the 1×10-8, 1×10-7 and 1×10-6 mol/L β-E2 group, which were signifincatly higher than 0.428±0.010 in the blank control group and 0.426±0.010 in the solvent control group(all at P<0.05). The telomerase activity values (absorbance) were 0.711±0.015, 0.941±0.010 and 1.249±0.047 in the 1×10-8, 1×10-7and 1×10-6 mol/L β-E2 group, which were higher than 0.535±0.013 in the blank control group and 0.543±0.013 in the solvent control group (all at P=0.000). The proliferantive values of the cells (absorbance) were significantly raised in the 1×10-8 mol/L β-E2 group compared with 1×10-7 and 1×10-6 mol/L β-E2 group (both at P=0.000), and no significnant difference was found in the proliferetive values between the blank control group and the solvent control group (P=0.718, 0.856). The apoptosis rates of the cells in the 1×10-8, 1×10-7 and 1×10-6 mol/L β-E2 group were lower than those in the the blank control group and the solvent control group (all at P=0.000), and there was no significant difference between the blank control group and the solvent control group (P=0.777). No obvious correlation was found between the HLECs preliferative values and hTERT mRNA expression levels or telomerase activity values (r=-0.299, P=0.278; r=-0.157, P=0.576). However, significantly negative correlations were seen between apoptosis rates and hTERT mRNA expression levels or telomerase activity values (r=-0.975, P=0.000; r=-0.981, P=0.000).
β-E2 can increase the activity of telomerase in human LECs, and high dose of β-E2 can inhibit apoptosis, but it dose not promote proliferation.