敲除PAMM基因对小鼠白色和棕色脂肪组织基因表达的影响

Impact of PAMM gene knockout on gene expression in white and brown adipose tissues of mice

目的:巨噬细胞集落刺激因子诱导单核细胞活化的类过氧化物酶2(peroxiredoxin-like 2 activated in M-CSF stimulated monocytes,PAMM)是一种具有抗炎、抗氧化功能的蛋白,在脂肪组织中高表达,本文拟利用基因敲除小鼠,探究PAMM对白色和棕色脂肪组织形态及基因表达等的影响,以期为其功能研究提供新思路。方法:利用CRISPR-Cas9等技术构建PAMM基因敲除(knockout,KO)小鼠,用野生型(wild-type,WT)小鼠作为对照。收集腹股沟皮下白色脂肪组织(inguinal adipose tissue,IAT)和背部棕色脂肪组织(brown adipose tissue,BAT),通过HE染色观察组织形态,利用RNA-seq和RT-qPCR检测相关基因的表达。结果:通过基因组PCR及测序,证明基因敲除小鼠构建正确。敲除PAMM后IAT和BAT形态无明显变化,但E2F靶点、未折叠蛋白反应等基因集在IAT或BAT中富集。比较WT和KO小鼠组织,发现33个基因的变化趋势在IAT和BAT中保持一致;而仅在IAT中出现变化的基因,分布在胞外、胞膜等部位,参与免疫和趋化反应等,与钙信号通路等相关;仅在BAT中出现变化的基因,分布在胞外基质、胞核等部位,参与葡萄糖稳态、生物节律等过程。RT-qPCR检测钙通路及生物节律相关基因,变化趋势与测序结果类似。本文还观察了敲除PAMM对IAT和BAT之间差异表达基因的影响,发现WT和KO小鼠BAT/IAT的差异基因和通路大多相同,但也有部分基因和通路发生了变化。结论:PAMM敲除对IAT和BAT的形态结构无明显影响,但显著影响了二者的基因表达及相关信号通路,还可能影响IAT和BAT之间的相互作用。

AIM:The protein peroxiredoxin-like 2 activated in M-CSF stimulated monocytes(PAMM)possesses anti-inflammatory and antioxidant attributes and exhibits high expression in adipose tissue.To contribute new perspectives to the functional research on PAMM,the objective of this study is to examine the influence of PAMM on the morphology and gene expression profiles of white and brown adipose tissues by using mice with PAMM gene knockout.METHODS:Homozygous knockout(KO)mice for the PAMM gene were modeled using techniques such as CRISPRCas9,with wild-type(WT)mice serving as controls.Subsequently,subcutaneous inguinal adipose tissue(IAT)and dorsal brown adipose tissue(BAT)were procured,and tissue morphology was assessed through hematoxylin and eosin(HE)staining.Gene expression patterns were comprehensively analyzed using RNA sequencing(RNA-seq)and further confirmed with quantitative reverse transcription-polymerase chain reaction(RT-qPCR).RESULTS:Genomic PCR and the product sequencing were used to validate gene knockout in mice.Notably,in PAMM knockout mice,there were no significant changes in the morphology of subcutaneous IAT and dorsal BAT.However,enrichment of gene sets associated with E2F targets and unfolded protein response was evident in either IAT or BAT in KO mice.A comparative analysis between WT and KO tissues revealed 33 genes exhibiting consistent changes in both IAT and BAT.Genes manifesting changes exclusively in IAT were predominantly located in extracellular and plasma membrane domains,contributing to immune and chemotactic responses,as well as calcium signaling pathways.Conversely,genes displaying changes solely in BAT were primarily distributed in extracellular matrix and nucleus regions,involved in glucose homeostasis and biological rhythms.RT-qPCR analysis of calcium signaling and biological rhythm-related genes corroborated trends observed in RNA-seq.The influence of PAMM knockout on differentially expressed genes between IAT and BAT was also evaluated in this study.While the majority of differential genes and pathways of BAT/IAT demonstrated consistency between WT and KO mice,noteworthy expression changes were observed in specific genes or pathways.CONCLUSION:PAMM knockout demonstrates no notable influence on the morphological characteristics of IAT and BAT.However,it exerts a significant effect on gene expression profiles and associated signaling pathways in both tissues,further indicating a potential impact on the crosstalk between IAT and BAT.