Body temperature-dependent microRNA expression analysis in rats: rno-miR-374-5p regulates apoptosis in skeletal muscle cells via Mex3B under hypothermia

Abstract

Forensic diagnosis of fatal hypothermia is considered difficult because there are no specific findings. Accordingly, exploration of novel fatal hypothermia-specific findings is important. To elucidate the molecular mechanism of homeostasis in hypothermia and identify novel molecular markers to inform the diagnosis of fatal hypothermia, we focused on microRNA expression in skeletal muscle, which plays a role in cold-induced thermogenesis in mammals. We generated rat models of mild, moderate, and severe hypothermia, and performed body temperature-dependent microRNA expression analysis of the iliopsoas muscle using microarray and quantitative real-time PCR (qRT-PCR). The results show that rno-miR-374-5p expression was significantly induced only by severe hypothermia. Luciferase reporter assay and qRT-PCR results indicated that Mex3B expression was regulated by rno-miR-374-5p and decreased with decreasing body temperature. Gene ontology analysis indicated the involvement of Mex3B in positive regulation of GTPase activity. siRNA analysis showed that Mex3B directly or indirectly regulated Kras expression in vitro, and significantly changed the expression of apoptosis-related genes and proteins. Collectively, these results indicate that rno-miR-374-5p was activated by a decrease in body temperature, whereby it contributed to cell survival by suppressing Mex3B and activating or inactivating Kras. Thus, rno-miR-374-5p is a potential supporting marker for the diagnosis of fatal hypothermia.

Potential predictive value of circulating tumor DNA (ctDNA) mutations for the efficacy of immune checkpoint inhibitors in advanced triple-negative breast cancer

“…Additionally, to explore the linkage among these 12 mutated genes, we utilized the KEGG database to examine the signaling pathways in which these genes are located. We found that several of the mutant genes are implicated in numerous important signaling pathways, both upstream and downstream, including the PI3K-AKT, MAPK, Ras, and JAK-STAT signaling pathways ( Christov et al, 2011 ; Hernandez-Varas et al, 2011 ; Clinkenbeard et al, 2016 ; Wang et al, 2017 ; Luo et al, 2018 ; Wang et al, 2018 ; Wu et al, 2019 ; Zhang et al, 2019 ; Chang et al, 2020 ; Schafer et al, 2020 ; Umehara et al, 2020 ; Ho and Bergwitz, 2021 ) ( Figure 5 ). These signaling pathways are involved in the regulation of cancer-related phenotypes, including cell cycle progression, cell survival, cell migration, signal transduction, and more.…”

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“…It is involved in the precursor mRNA shearing and RNA processing and is associated with the action of cell cycle protein-dependent kinases ( 46 ). Additionally, CCNL1 may contribute to cell survival during cellular stress, such as cold stimulation ( 47 ). CCNT2 is a cyclin involved in the cell cycle and RNA transcription ( 48 ).…”

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“…Some of these studies shed light on our understanding of basic molecular and cellular pathways that underlie the beneficial roles of posttraumatic hypothermia, yet many questions still need to be answered, including the roles of posttranscriptional regulation. Both cell cultures and animal model studies have demonstrated the effects of many miRNA species under hypothermic versus normothermic conditions [ 150 , 151 ]. For example, a study of the role of temperature change in dealing with traumatic brain injury (TBI) showed that lower levels of miR-874 under hypothermic conditions could promote cell function by upregulating the levels of essential proteins for normal cell function (e.g., cytosolic or membrane proteins), suggesting role of cooling temperatures in inducing miRNA expression during TBI [ 150 ].…”

“…elegans showed elevated levels of miR-21 in kidney, muscle and spleen and this also correlated with apaf1 , casp3 and casp7 regulation, suggesting a similar role in mediating an anti-apoptosis response under anoxia exposure [ 162 , 163 ]. Another study using a hypothermic rat model showed activation of miR-374-5p in skeletal muscle in response to hypothermia (12 °C), and inducing suppression of Mex3B (muscle excess 3) that ultimately affects genes involved in the apoptosis cascade [ 151 ]. Overall, these studies highlight the roles of temperature and stress specific miRNAs in activating different regulatory pathways and mediating positive metabolic consequences similar to the effects of therapeutic hypothermia after traumatic injury and may provide a guide to comprehend temperature/stress specific miRNA regulation involved in these basic cellular mechanisms.…”