miR-132-3p 通过靶向调控 MAPK1 表达对过量氟暴露致大鼠脑及 SH-SY5Y 细胞 MAPK 通路变化的调控作用
Regulating effect of miR-132-3p on the changes of MAPK pathway in rat brains and SH-SY5Y cells exposed to excessive fluoride by targeting expression of MAPK1.
机构信息
Department of Pathology at the Affiliated Hospital of Guizhou Medical University, Guiyang 550004, PR China.
Key Laboratory of Endemic and Ethnic Diseases (Guizhou Medical University) of the Ministry of Education and Provincial Key Laboratory of Medical Molecular Biology, Guiyang 550004, PR China.
出版信息
Ecotoxicol Environ Saf. 2024 Jul 1;279:116467. doi: 10.1016/j.ecoenv.2024.116467. Epub 2024 May 17.
BACKGROUND
Although the changes of mitogen-activated protein kinase (MAPK) pathway in the central nervous system (CNS) induced by excessive fluoride has been confirmed by our previous findings, the underlying mechanism(s) of the action remains unclear. Here, we investigate the possibility that microRNAs (miRNAs) are involved in the aspect.
METHODS
As a model of chronic fluorosis, SD rats received different concentrations of fluoride in their drinking water for 3 or 6 months and SH-SY5Y cells were exposed to fluoride. Literature reviews and bioinformatics analyses were used to predict and real-time PCR to measure the expression of 12 miRNAs; an algorithm-based approach was applied to identify multiply potential target-genes and pathways; the dual-luciferase reporter system to detect the association of miR-132-3p with MAPK1; and fluorescence in situ hybridization to detect miR-132-3p localization. The miR-132-3p inhibitor or mimics or MAPK1 silencing RNA were transfected into cultured cells. Expression of protein components of the MAPK pathway was assessed by immunofluorescence or Western blotting.
RESULTS
In the rat hippocampus exposed with high fluoride, ten miRNAs were down-regulated and two up-regulated. Among these, miR-132-3p expression was down-regulated to the greatest extent and MAPK1 level (selected from the 220 genes predicted) was corelated with the alteration of miR-132-3p. Furthermore, miR-132-3p level was declined, whereas the protein levels MAPK pathway components were increased in the rat brains and SH-SY5Y cells exposed to high fluoride. MiR-132-3p up-regulated MAPK1 by binding directly to its 3'-untranslated region. Obviously, miR-132-3p mimics or MAPK1 silencing RNA attenuated the elevated expressions of the proteins components of the MAPK pathway induced by fluorosis in SH-SY5Y cells, whereas an inhibitor of miR-132-3p just played the opposite effect.
CONCLUSION
MiR-132-3p appears to modulate the changes of MAPK signaling pathway in the CNS associated with chronic fluorosis.
背景
虽然我们之前的研究已经证实，中枢神经系统（CNS）中过量氟化物诱导的丝裂原活化蛋白激酶（MAPK）通路变化，但作用机制尚不清楚。在这里，我们研究了 microRNAs（miRNAs）是否参与其中的可能性。
方法
以慢性氟中毒模型为例，SD 大鼠饮用不同浓度的氟水 3 或 6 个月，SH-SY5Y 细胞暴露于氟化物中。通过文献回顾和生物信息学分析预测和实时 PCR 测量 12 种 miRNAs 的表达；应用基于算法的方法识别多个潜在靶基因和途径；双荧光素酶报告系统检测 miR-132-3p 与 MAPK1 的结合；荧光原位杂交检测 miR-132-3p 的定位。将 miR-132-3p 抑制剂或模拟物或 MAPK1 沉默 RNA 转染到培养细胞中。通过免疫荧光或 Western blot 检测 MAPK 通路蛋白成分的表达。
结果
在暴露于高氟的大鼠海马体中，有 10 种 miRNA 下调，2 种上调。其中，miR-132-3p 表达下调最明显，MAPK1 水平（从预测的 220 个基因中选择）与 miR-132-3p 的变化相关。此外，在暴露于高氟的大鼠脑组织和 SH-SY5Y 细胞中，miR-132-3p 水平下降，而 MAPK 通路成分的蛋白水平升高。miR-132-3p 通过直接结合其 3'-非翻译区上调 MAPK1。显然，miR-132-3p 模拟物或 MAPK1 沉默 RNA 可减弱氟中毒诱导的 SH-SY5Y 细胞中 MAPK 通路蛋白成分的升高表达，而 miR-132-3p 抑制剂则起到相反的作用。
结论
miR-132-3p 似乎调节与慢性氟中毒相关的中枢神经系统中 MAPK 信号通路的变化。
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