摘要
目的探讨石胆酸在转录后水平对PPARαmRNA稳定性的调控。方法构建PPARα3'UTR荧光素酶报告基因载体并转染HepG2细胞,比较两种浓度石胆酸处理后荧光素酶活性的变化。结合生物信息学预测,确定石胆酸诱导下差异表达的miRNAs及其在3'UTR上的潜在结合位点,对结合位点进行突变(Mut1、Mut2和Mut1+Mut2),比较石胆酸处理后Mut1、Mut2和Mut1+Mut2荧光素酶活性的变化。利用Westernblot和RT-qPCR检测两种浓度石胆酸处理下信号通路的激活及其下游转录因子基因表达水平,比较不转染对照组和瞬时转染过表达转录因子的PPARα蛋白表达,确定参与石胆酸调控PPARαmRNA稳定性的信号通路和转录因子。结果与对照组相比,100μmol/L石胆酸诱导3'UTR1和3'UTR2片段荧光素酶活力均显著下降超过50%(P<0.01)。miRNAPCRarray筛选发现石胆酸诱导miR-21和miR-22差异表达,100μmol/L石胆酸诱导miR-21表达上调2.35倍(P<0.05)。定点突变3'UTR1中两个预测的miR-21位点,构建了Mut1、Mut2和Mut1+Mut2报告基因载体。相对于对照组,石胆酸下调3'UTR1荧光素酶活性51%,而Mut1、Mut2和Mut1+Mut2分别被下调37%、39%、13%。Westernblot显示石胆酸磷酸化ERK1/2,激活ERK1/2信号通路;100μmol/L石胆酸上调转录因子EGR1基因表达水平5.83倍(P<0.01);瞬时转染过表达EGR1显著下调PPARα蛋白水平(P<0.05)。结论石胆酸通过激活肝细胞中ERK1/2信号通路,诱导早期生长应答因子EGR1和miR-21的表达上调,使miR-21作用于3'UTR调控区,降低PPARαmRNA的稳定性,从而减少PPARα基因和蛋白表达水平。
Objective To investigate the regulatory effects of lithocholic acid(LCA)on nuclear receptor peroxisome proliferator-activated receptor-alpha(PPARα)mRNA stability at the post-transcriptional level.Methods PPARα3'UTR luciferase reporter gene vectors were constructed and transfected into HepG2 cells to observe the changes in cellular luciferase activity in response to LCA treatments.Bioinformatic prediction and miRNA PCR array technique were used to identify the differentially expressed miRNAs induced by LCA and their potential binding sites on the 3'UTR.The binding sites(Mut1,Mut2 and Mut1+Mut2)were mutated to compare the changes in cellular luciferase activity following LCA treatment.Western blotting and RT-qPCR were used to detect the activated signaling pathway and the expression levels of its downstream transcription factors in LCA-treated cells.The changes in PPARαprotein expression level were detected in the cells following overexpression of the transcription factors.Results Treatment with 100μmol/L LCA significantly reduced luciferase activity of PPARα3'UTR1 and 3'UTR2 in HepG2 cells by more than 50%(P<0.01)and induced significant upregulation of miR-21 and miR-22,especially the former(by 2.35 folds,P<0.05).Two predicted miR-21-binding sites in the 3'UTR1 were mutated to construct Mut1,Mut2 and Mut1+Mut2 reporter gene vectors.LCA treatment down-regulated 3'UTR1 luciferase activity by 51%,while Mut1,Mut2,and Mut1+Mut2 were down-regulated by 37%,39%,and 13%,respectively.LCA caused ERK1/2 phosphorylation and activation of the ERK1/2 signaling pathway,and treatment with 100μmol/L LCA upregulated the expression of transcription factor early growth response 1(EGR1)by 5.83 folds(P<0.01).Transient overexpression of EGR1 significantly decreased cellular PPARαprotein levels(P<0.05).Conclusion LCA reduces PPARαmRNA stability and thus decreases PPARαmRNA and protein expressions in hepatocytes by activating the ERK1/2 signaling pathway and upregulating EGR1 and miR-21,which targets 3'UTR regulatory region of PPARαmRNA.
作者
庞碧滢
黄娜娜
黄晓霞
李馨
熊文婷
孔波
姚焱
PANG Biying;HUANG Nana;HUANG Xiaoxia;LI Xin;XIONG Wenting;KONG Bo;YAO Yan(School of Life Science,Guangzhou University,Guangzhou 510000,China)
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2023年第12期2086-2094,共9页
Journal of Southern Medical University
基金
国家自然科学基金(81973376)
广州大学研究生“基础创新”项目(2021GDJC-M24)。
关键词
石胆酸
PPARΑ
MIR-21
MRNA稳定性
lithocholic acid
peroxisome proliferator-activated receptor-alpha
miR-21
mRNA stability