[石胆酸通过上调肝癌HepG2细胞中miR-21的表达降低核受体PPAR的mRNA稳定性]
[Lithocholic acid decreases mRNA stability of nuclear receptor PPAR by upregulating miR-21 expression in hepatoma HepG2 cells].
作者信息
Pang B, Huang N, Huang X, Li X, Xiong W, Kong B, Yao Y
机构信息
School of Life Science, Guangzhou University, Guangzhou 510000, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Dec 20;43(12):2086-2094. doi: 10.12122/j.issn.1673-4254.2023.12.13.
OBJECTIVE
To investigate the regulatory effects of lithocholic acid (LCA) on nuclear receptor peroxisome proliferatoractivated receptor-alpha (PPAR) mRNA stability at the post-transcriptional level.
METHODS
PPAR 3'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 RTqPCR were used to detect the activated signaling pathway and the expression levels of its downstream transcription factors in LCA-treated cells. The changes in PPAR protein expression level were detected in the cells following overexpression of the transcription factors.
RESULTS
Treatment with 100 μmol/L LCA significantly reduced luciferase activity of PPAR 3'UTR1 and 3'UTR2 in HepG2 cells by more than 50% (<0.01) and induced significant upregulation of miR-21 and miR-22, especially the former (by 2.35 folds, <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 100 μmol/L LCA upregulated the expression of transcription factor early growth response 1 (EGR1) by 5.83 folds (<0.01). Transient overexpression of EGR1 significantly decreased cellular PPAR protein levels (<0.05).
CONCLUSION
LCA reduces PPAR mRNA stability and thus decreases PPAR mRNA 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 PPAR mRNA.
目的
在转录后水平研究石胆酸（LCA）对核受体过氧化物酶体增殖物激活受体α（PPAR）mRNA稳定性的调控作用。
方法
构建PPAR 3'UTR荧光素酶报告基因载体并转染至HepG2细胞，观察LCA处理后细胞荧光素酶活性的变化。采用生物信息学预测和miRNA PCR芯片技术鉴定LCA诱导的差异表达miRNA及其在3'UTR上的潜在结合位点。对结合位点（Mut1、Mut2和Mut1 + Mut2）进行突变，以比较LCA处理后细胞荧光素酶活性的变化。采用蛋白质免疫印迹法（Western blotting）和实时定量逆转录聚合酶链反应（RT-qPCR）检测LCA处理细胞中激活的信号通路及其下游转录因子的表达水平。在转录因子过表达后检测细胞中PPAR蛋白表达水平的变化。
结果
100 μmol/L LCA处理显著降低HepG2细胞中PPAR 3'UTR1和3'UTR2的荧光素酶活性超过50%（<0.01），并诱导miR-21和miR-22显著上调，尤其是前者（上调2.35倍，<0.05）。对3'UTR1中两个预测的miR-21结合位点进行突变，构建Mut1、Mut2和Mut1 + Mut2报告基因载体。LCA处理使3'UTR1荧光素酶活性下调51%，而Mut1、Mut2和Mut1 + Mut2分别下调37%、39%和13%。LCA导致ERK1/2磷酸化并激活ERK1/2信号通路，100 μmol/L LCA处理使转录因子早期生长反应因子1（EGR1）的表达上调5.83倍（<0.01）。瞬时过表达EGR1显著降低细胞中PPAR蛋白水平（<0.05）。
结论
LCA通过激活ERK1/2信号通路并上调EGR1和靶向PPAR mRNA 3'UTR调控区域的miR-21，降低PPAR mRNA稳定性，从而降低肝细胞中PPAR mRNA和蛋白表达。
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