Circular RNA Expression Profiling In The Hypothalamus Of Heat-acclimated Rats

Background: Heat acclimation（HA）is an adaptive process that is attained in the organism by repeated exposure to hyperthermia.At present,the physiological effect of HA has been investigated indepth,but its molecular mechanism has not yet been fully elucidated.The lack of research on HA molecular mechanism and its regulatory network has severely restricted the prevention and treatment of thermal injury.Circular RNAs（circRNAs）are a subclass of non-coding RNAs.They play crucial roles in diverse biological processes and are potential biomarkers in diverse diseases and cancers.However,changes and roles of circRNAs related to HA are not fully known.Differentially expressed circRNAs and their regulated downstream miRNAs and target genes,their molecular mechanisms and regulatory networks were initially investigated in this study.It will provide a molecular basis research gist of the monitoring and accurate evaluation of HA,and the development of measures related to high temperature promotion HA.Methods:（1）Animals and experimental conditions: SPF male SD rats were randomly divided into HA group and control group.HA group rats were subjected to an environmental temperature of 35 ± 1°C and a relative humidity of 55% ± 5% for 2 h daily for 30 days to acquire HA.To avoid the influence of diurnal cycling,all experiments were started at approximately the same time each day（between 2:30 and4:30 PM）.（2）Microarray assays by using arraystar rat circRNA arrays（8x15K,Arraystar）of circRNA were performed to identify circRNA expression signatures in the hypothalamus of HA rats.（3）Differential circRNAs and their regulated downstream miRNAs and mRNAs were quantitatively validated using real-time quantitative polymerase chain reaction（RT-qPCR）.The expression of MFAP4 protein and HIF-1α protein were determined using enzyme-linked immunosorbent assay（ELISA）.Results:（1）A total of 13860 circRNA targets were detected in three pairs of rathypothalamus tissue samples.53 circRNAs were differentially expressed in the HA group compared with the C group.28 of these circRNAs were upregulated and 25 were downregulated（Fold change ≥1.5,P < 0.05）;（2）In addition,GO and KEGG pathway enrichment analysis of the parental genes was proved to be closely related to HA formation,for example the Hippo signaling pathway,the PI3K-Akt signaling pathway,the FoxO signaling pathway,the Ras signaling pathway,the cGMP-PKG signaling pathway,the MAPK signaling pathway,the Phospholipase dsignaling pathwaya nd the mTOR signaling pathway;（3）A total of 10 circRNAs,4 miRNAs and17 mRNAs were further verified by using RT-qPCR.The results showed the expression of rno_circRNA₀02960,rno_circRNA₀14301 and rno_circRNA₀11190 were upregulated 3.34-fold,1.41-fold,1.73-fold respectively（P< 0.05）,whereas expressions of rno_circRNA₀10393 was downregulated0.74-fold（P < 0.05）.Among the 4 miRNAs,rno-miR-23a-5p,rno-miR-3575 and rno-miR-3561-3p were down-regulated 0.65-fold,0.39-fold and 0.35-fold respectively（P < 0.05）,while rno-miR-20b-3p was up-regulated 1.42 fold（P < 0.05）.Among the 17 mRNAs,Dusp7,Hnrnpa3,Lppr4,Hif-1α,Sec31 a,Zbtb20,Fabp3 and Lin28 b were up-regulated 1.31-fold,1.38-fold,1.44-fold,1.56-fold,2.08-fold,2.01-fold,1.61-fold,1.54-fold and 3.34-fold respectively（P < 0.05）,whereas expressions of Mfap4 were downregulated 0.67-fold（P < 0.05）.（4）In this study,the HIF-1α protein was up-regulated,and the MFAP4 protein was down-regulated in the hypothalamus of HA rats were been proven.The body’s ability to resist oxidative stress and lipid metabolism may improve,and maintain the normal physiological function of the body in HA.Conclusions:（1）Study has shown that many circRNAs regulate expressions of genes that interact with each other to exert their functions during HA.（2）Bioinformatics analysis was used to perform functional analysis of differentially expressed circRNAs.The results jointly suggest that circRNA may play an important role in HA through different mechanisms.（3）Therefore,according to the circRNA-miRNA-mRNA bioinformatics analysis of regulatory networks,four pathways may promote HA formation: thatrno_circRNA₀14301-vs-rno-miR-3575-vs-Hif-1α in the hypoxia response pathway;rno_circRNA₀14301-vs-rno-miR-3575-vs-Lppr4,rno_circRNA₀10393-vs-rno-miR-20b-3p-vs-Mfap4 in the lipid/lipoprotein metabolic pathways;rno_circRNA₀11190 may affect the expression of Fabp3 by regulating rno-mir-667-5p in regulate the lipid metabolism pathway;Preliminary experimental results suggest that circRNA may have important research value in HA promote the formation.The next step still has yet to be verified by in vitro and in vivo experiments.