In the current study, we found that the M2 murine BMDM proportion was significantly decreased in the Hypoxia process (P < 0.05). After transfection with lncRNA260 siRNA, the M2 murine BMDM proportion was significantly increased (P < 0.05). Meanwhile, the proteins expression of the Arg 1, p-AKT, PI3KCG, IL28RAV1 changed in the same way to the M2 murine BMDM proportion. Conversely, the IL28RAV2 protein expression presented the changes in the opposite direction in the murine BMDM hypoxia process. It was indicated that lncRNA260 siRNA could promote the M2 polarization of the hypoxia murine BMDM by reducing the IL28RAV2 alternative splicing variant, which might be related to the activation of the JAK-STAT and PI3K/AKT signaling pathways.
The degree of inflammation after AMI is positively correlated with the size of myocardial infarction, but the research on blocking inflammation has not obtained ideal results [15–22]. It was indicated that moderate suppression of inflammatory storm after AMI could reduce myocardial injury and improve prognosis, while blocking the inflammatory process was not conducive to myocardial repair, but increased the risk of pathological cardiac remodeling and cardiac rupture. The latest evidence for anti-inflammatory therapy after AMI is available. After PCI treatment of AMI, anti-inflammatory treatment with colchicine could reduce infarct size and improve prognosis . Intravenous application of metoprolol after AMI could reduce the inflammatory response and limit the infarct size by reducing neutrophil tissue infiltration and its interaction with platelets in myocardial infarction area .
Peripheral blood mononuclear macrophages are recruited into damaged myocardium after AMI. This process was critical for cardiac repair because they could adopt pro-inflammatory or repair phenotypes to regulate inflammatory and repair responses, respectively [25, 26]. At first, M1 type macrophage was dominant, and reached the peak on 3–4 days after AMI, mainly producing inflammatory cytokines such as TNF-α, IL-1β, IL-6, chemokines such as CCR2, CXCL1, IL-8, and so on, thus aggravating the degree of inflammation. Then entering the fiber proliferation stage, Arginase 1, IL-10, VEGF and TGF-β1were mainly produced, and necrotic cells were cleared through cell burial [27, 28], inflammation was inhibited, neovascularization was promoted, and damaged tissue repair was promoted. The peak value was reached on 6–7 days after AMI. Finally, cardiac remodeling, or scar formation, occurs, and the necrotic areas of the myocardium are replaced by fibrous scar tissue formed by crosslinked fibers. The early stage of inflammatory response after AMI is a critical time for cardiac remodeling. It is very important to find genes or drugs that can promote the Mφ phenotype from pro-inflammatory M1 to anti-inflammatory M2. Macrophages can promote the transformation from inflammatory M1 type to anti-inflammatory M2 type by activating PI3K/AKT signaling pathway [29–30]. In our previous study, we found that down-regulation of lncRNA260 could ameliorate hypoxic cardiomyocytes injury by regulating IL28RA through the activation of PI3K/AKT signaling pathways. It was suggested that lncRNA260 siRNA could promote the macrophages toward M2 polarization by regulating IL28RA.
Studies have shown that IL28RA has a variety of alternative spliceosomes with different functions . IL28RAVl is a normal functioning CRF2-12. IL28RAV2 lacks 29 amino acids in the intracellular region and can bind to type III interferon, but it loses the signal transduction function. IL28RAV2 can inhibit the activity of IFN-λ1, and is a negative regulator of type III interferon, which plays an anti-cell proliferation and promotes inflammatory response. In the current study, it was found that mouse macrophages also expressed two AS variants, namely, 59KD and 55KD respectively. After 24h hypoxic, the full length of 59KD IL28RAVl was significantly reduced, and the expression of 55KD IL28RAV2 was significantly increased. It is speculated to be related to IL28RA mRNA alernative splicing caused by hypoxia, which inhibits JAK-STAT and PI3K/AKT signaling pathways, promotes polarization of macrophages to M1, enhances inflammatory response, significantly increases apoptosis and necrosis of myocardial cells, and increases the risk of myocardial fibrosis and heart rupture. After lncRNA-260 siRNA intervention, the IL28RAV2 AS was significantly reduced and IL28RAV1 was significantly increased compared with that of the hypoxia group, which promoted the activation of JAK-STAT and PI3K/AKT signaling pathways, caused the M2 polarization of macrophages, and reduced the inflammatory response. IL10RB is a common receptor ligand of IL28RA and IL10RA. During AMI, the expression of IL28RAV2 is up-regulated in macrophages, resulting in a competitive increase in the heterodimer formed by IL10RB and IL28RAV2, while the binding of IL10RA receptor is reduced. In addition, when hypoxia occurs, the expression of IL10RA is down-regulated due to the inhibition of type III interferon signaling pathway, which further reduces the activation of IL10/STAT3/IL4RA/STAT6, decreases M2 macrophages, further reduces the secretion of IL10, and aggravates inflammation.
CatRAPID omics is a server for large-scale calculations of protein-RNA interactions . It was showed that lncRNA260 could bind to helicase-like transcription factor (HLTF), which has helicase and ATPase activity, and could bind to IL28RA promoter by using the catRAPID omics assay. HLTF could cause the chromatin structure around IL28RA gene change to promote the transcription of IL28RA gene. Therefore, it is speculated that lncRNA260 can promote the transcription of IL28RA gene through trans action by recruiting HLTF to the promoter site of IL28RA. These results suggest that lncRNA260 can regulate IL28RA gene and participate in the signal transduction process of AMI through JAK-STAT and PI3K/AKT signaling pathways.
The catRAPID omics predicted that lncRNA260 also competitively could bind to the SF3B2, SF3B3 splicing complex with IL28RA. The ESE finder predict that lncRNA260 also has the exon splicing enhancer (ESE) sequence of the SRSF protein recognition site of the splicing enhancer, which competitively binds to the SRSF protein with IL28RA (http://krainer01.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi?process=home) [32, 33]. Thus, it affected the recognition and splicing of the 7th exon of IL28RA mRNA by the splicing body, resulting in partial loss of the 7th exon, and increased the IL28RAV2 AS variant.
LncRNA260 is distributed in both nucleus and cytoplasm. According to the bioinformatics prediction of miRDB (http://mirdb.org/cgi-bin/custom.cgi) and Targetscan databases (http://www.targetscan.org/mamm_31/), both of lncRNA260 and IL28RAV2 mRNA can bind to miR-3622. LncRNA260 acts as competing endogenous RNAs (ceRNA). Hence, the degradation of IL28RAV2 gene by miR-3622 is weakened, and the expression of IL28RAV2 gene is further increased. In contrast to IL28RAV1, the function of this IL28RAV2 variant blocked JAK-STAT, PI3K/AKT signaling pathways, resulting in decreased M2-type polarization of macrophages and enhanced inflammatory response, resulting in myocardial cell damage and ventricular remodeling during AMI.
In a word, lncRNA260 siRNA promotes M2 macrophage polarization by reducing IL28RAV2 AS in the murine BMDM hypoxia process. Considering the current study is an in vitro experiment, this conclusion will be further confirmed in the animal experiments. This study will lay a foundation for future in vivo research.