Analysis of potential functional significance of microRNA‑3613‑3p in human umbilical vein endothelial cells affected by heat stress

Abstract

Dysregulation of microRNA-3613-3p (miR-3613-3p) was previously reported in endothelial cells (ECs) during heat stress. The aim of the present study was to investigate the precise role of miR-3613-3p in heat stress. In the present study, potential gene targets of miR-3613-3p in heat-treated ECs were assessed, and the potential effects of miR-3613-3p were determined using Gene Ontology enrichment analysis. Kyoto Encyclopedia of Genes and Genomes pathway analysis was used to identify signaling pathways that may be affected by miR-3613-3p in heat-treated cells. Reverse transcription-quantitative PCR, western blotting and annexin V-FITC/propidium iodide staining were performed to detect miRNA expression, protein expression and apoptosis, respectively. Luciferase gene reporter assay was performed to evaluate the association between miR-3613-3p and mitogen-activated protein kinase kinase kinase 2 (MAP3K2). Bioinformatics analysis revealed 865 potential gene targets for miR-3613-3p and a series of functions and pathways in heat-treated ECs. ‘Negative regulation of apoptotic process’ was identified as a potential function of miR-3613-3p. In addition, functional analysis confirmed the downregulated expression levels of miR-3613-3p in ECs during heat stress, which was accompanied by an increase in apoptosis; restoration of miR-3613-3p expression inhibited apoptosis. MAP3K2 protein was demonstrated to be upregulated in heat-treated ECs, and overexpression of miR-3613-3p reduced MAP3K2 expression levels. Additionally, MAP3K2 was targeted by miR-3613-3p. These results indicated that miR-3613-3p may have complicated roles in ECs under heat stress. miR-3613-3p may serve an important role in the apoptosis of heat-treated ECs, and this effect may be partly achieved by targeting MAP3K2.

miR‑3613‑3p/MAP3K2/p38/caspase‑3 pathway regulates the heat‑stress‑induced apoptosis of endothelial cells

“…Our previous miRNA expression profiling data in HUVECs indicated that miR-3613-3p may be a HS-related miRNA ( 12 ). Furthermore, using an in vivo approach, our previous study revealed a regulatory relationship between miR-3613-3p and MAP3K2 that mediated the apoptosis of HS-treated HUVECs ( 19 ).…”

“…Protein extraction and western blot analysis. Protein extraction, measurement of total protein concentration and western blot analysis were performed as described previously (19). The primary antibodies used were as follows: Mouse anti-human MAP3K2 (cat.…”

“…Our previous study demonstrated that miR-3613-3p was suppressed in HS-treated human umbilical vein endothelial cells (HUVECs) ( 19 ). In our preliminary study, it was revealed that a decrease in miR-3613-3p might promote cell apoptosis by negative post-transcriptional regulation of the target gene MAPK kinase kinase 2 (MAP3K2) ( 19 ), a component of the MAPK pathway. However, whether the miR-3613-3p/MAP3K2 axis, along with downstream signalling molecules, contributes to HS-induced apoptosis remains unclear.…”