生物钟基因BMAL1通过促进ROCK1调控滋养层细胞功能障碍的研究

Regulation of circadian clock gene BMAL1 in trophoblast dysfunction through promotion of ROCK1

目的 分析生物钟基因脑和肌肉组织芳香烃受体核转运蛋白类似蛋白1(BMAL1)对Ras同源物基因组成员A(RhoA)/Rho相关激酶1(ROCK1)信号通路的影响以及对滋养层细胞生物学功能的调控机制。方法 选择滋养层细胞系JEG3细胞,根据转染BMAL1过表达质粒以及添加1μmol/L、2μmol/L、5μmol/L ROCK1抑制剂Y27632,分为对照组、BMAL1组、BMAL1+1μmol/L Y27632组、BMAL1+2μmol/L Y27632组、BMAL1+5μmol/L Y27632组。实时荧光定量PCR(qRT-PCR)检测BMAL1、RhoA、ROCK1以及上皮-间充质转化(EMT)标记物N-cadherin、Vimentin和转录因子Snail、Slug的mRNA表达水平;Western blot检测蛋白表达水平;CCK8法检测细胞的增殖活力;流式细胞术分别检测细胞周期、凋亡以及胞内活性氧(ROS)水平。结果 JEG3细胞过表达BMAL1后,与对照组相比,ROCK1 mRNA表达显著增加(P<0.01),RhoA mRNA表达增加但差异无统计学意义(P>0.05)。与对照组相比,BMAL1组N-cadherin、Vimentin、Snail的mRNA表达水平显著增加(P<0.01)。在蛋白水平,BMAL1组ROCK1、RhoA及Snail蛋白表达较对照组显著增加(P<0.05),Y27632处理可以显著降低Vimentin及Snail蛋白表达(P<0.05)。此外,与对照组相比,BMAL1组细胞活力显著增加(P<0.001),BMAL1+Y27632各浓度组较BMAL1组细胞活力显著降低(P<0.01)。流式细胞术结果显示,与对照组相比,BMAL1组细胞周期由G0/G1期向S+G2/M期转化,细胞总凋亡率有降低趋势但差异无统计学意义(P>0.05),胞内ROS水平显著降低(P<0.01);与BMAL1组相比,BMAL1+1μmol/L Y27632组和BMAL1+2μmol/L Y27632组细胞早期凋亡率及总凋亡率显著增加(P<0.05),BMAL1+1μmol/L Y27632组细胞ROS平均荧光强度增加但差异无统计学意义(P>0.05),BMAL1+2μmol/L Y27632组细胞ROS平均荧光强度较BMAL1组显著增加(P<0.001)。结论 生物钟基因BMAL1通过促进ROCK1表达参与调控滋养层细胞增殖、DNA合成、凋亡及氧化应激水平。

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 1μmol/L,2μmol/L,and 5μmol/L of ROCK1 inhibitor Y27632,and divided into control group,BMAL1 group,BMAL1+1μmol/L Y27632 group,BMAL1+2μmol/L Y27632 group,and BMAL1+5μmol/L Y27632 group,respectively.Real-time quantitative PCR(qRT-PCR)was performed to detect the mRNA expression levels of BMAL1,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+1μmol/L Y27632 group and BMAL1+2μmol/L Y27632 group were significantly increased(P<0.05),and the mean fluorescence intensity of cellular ROS was increased in the BMAL1+1μmol/L Y27632 group with no statistical significance(P>0.05).The mean fluorescence intensity of cellular ROS was significantly higher in BMAL1+2μmol/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.