Regulation of circadian clock gene BMAL1 in trophoblast dysfunction through promotion of ROCK1
Objective:To analyze the effect of circadian clock gene named brain and muscle arnt-like protein 1(BMAL1)on the Ras homologue genomic member A(RhoA)/Rho-associated coiled-coil kinase 1(ROCK1)signaling pathway and the regulatory mechanism of biological functions in trophoblast cells.Methods:The trophoblast cell line JEG3 cells were selected and transfected with BMAL1 overexpression plasmid.The cells were then treated with 1 μmol/L,2 μmol/L,and 5 μmol/L of ROCK1 inhibitor Y27632,and divided into control group,BMAL1 group,BMAL1+1 μmol/L Y27632 group,BMAL1+2 μmol/L Y27632 group,and BMAL1+5 μmol/L Y27632 group,respectively.Real-time quantitative PCR(qRT-PCR)was performed to detect the mRNA expression levels of BMALA,RhoA,ROCK1,as well as epithelial-mesenchymal transition(EMT)markers as N-cadherin,Vimentin,and transcription factors Snail,Slug.Western blot was used to detect the protein expression levels.Cell proliferation activity was assessed using the CCK8 assay.Flow cytometry was performed to examine the cell cycle,apoptosis,and intracellular reactive oxygen species(ROS)levels.Results:Compared to the control group,the expression of ROCK1 mRNA in BMAL1 group was significantly increased(P＜0.01),while the increase in RhoA mRNA expression was not statistically significant(P＞0.05).Also,the mRNA expression levels of N-cadherin,Vimentin,and Snail were significantly increased in the BMAL1 group when compared with the control group(P＜0.01).At the protein level,the expression of ROCK1,RhoA,and Snail proteins in the BMAL1 group was significantly increased when compared with the control group(P＜0.05).After the addition of the ROCK1 inhibitor Y27632,the protein levels of Vimentin and Snail were significantly decreased(P＜0.05).In addition,compared with the control group,the BMAL1 group showed a significant increase in cell viability(P＜0.001),while the BMAL1+Y27632 group all showed a significant decrease in cell viability when compared to the BMAL1 group(P＜0.01).The results of flow cytometry showed that the cell cycle was transformed from G0/G1 phase to S+G2/M phase in the BMAL1 group,accompanied with non-significant reduced total apoptosis rate of cells(P＞0.05)and significant reduced intracellular ROS level(P＜0.01)when compared with the control group.Compared with the BMAL1 group,the early apoptosis rate and total apoptosis rate in both BMAL1+1 μmol/L Y27632 group and BMAL1+2 μmol/L Y27632 group were significantly increased(P＜0.05),and the mean fluorescence intensity of cellular ROS was increased in the BMAL1+1 µmol/L Y27632 group with no statistical significance(P＞0.05).The mean fluorescence intensity of cellular ROS was significantly higher in BMAL1+2 μmol/L Y27632 group than that in BMAL1 group(P＜0.001).Conclusions:The circadian clock gene BMAL1 was involved in the regulation of trophoblast cell proliferation,DNA synthesis,apoptosis and oxidative stress levels by promoting ROCK1 expression.
Brain and muscle arnt-like protein 1Rho-associated coiled-coil kinase 1TrophoblastRecurrent spontaneous abortion
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