脑损伤
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Figure 1｜Verification of M0, M1, and M2 macrophages by cell morphology and RNA and protein expressions.
To examine the expression of lnc_000048 in macrophages. we generated M0, M1, and M2 macrophages from THP-1 cells with different stimuli. M0 and M2 macrophages were mainly round and adherent with short pseudopodia. M1 macrophages were larger with extended long pseudopodia in an irregular shape (Figure 1A). We next performed qRT-PCR for the mRNA expression of secretory marker and cell surface marker genes (Huang et al., 2019; Munteanu et al., 2020; Ren et al., 2021), The mRNA expressions of TNF-α, IL-6, and IL-1β were significantly higher in M1 macrophages than in M0 macrophages (P < 0.05; Figure 1B), and the expressions of CD38 and CD80 mRNA were significantly higher in M1 macrophages than in M0 macrophages (P < 0.05; Figure 1C). The mRNA expressions of IL-10, CCL18, and CCL22 were significantly higher in M2 macrophages than in M0 macrophages (P < 0.05; Figure 1D), and the expression of CD163 mRNA was significantly higher in M2 macrophages than in M0 macrophages (P < 0.05; Figure 1E). Flow cytometry further showed that the proportion of cells expressing the M0 macrophage surface signature protein CD11b was increased (Figure 1F), and the proportion of M1 macrophage surface signature proteins CD38 and CD80 expression was also increased (Figure 1G). Together, these results confirmed successful macrophage polarization.
Figure 5｜Lnc_000048 binds to PKR in the cytoplasm of M1 macrophages.
We speculated that lnc_000048 may regulate the activation of the STAT1 pathway in macrophages by binding to RBPs. To test this hypothesis, we performed RNA pull-downs with lnc_000048 in THP-1 cells and identified all lnc_000048-interacting proteins by mass spectrometry. In parallel, the catRAPID database was used to identify candidate RBPs that may bind lnc_000048. An examination of the top 20 RBPs identified by mass spectrometry, along with the predictions made by catRAPID, led to the identification of PKR as a potential binding partner of lnc_000048 (Figure 5A and B). We then used RPISeq and catRAPID online databases to predict the binding ability of lnc_000048 to EIF2AK2, which is the gene encoding PKR. In RPISeq, both support vector machine (SVM) and random forest (RF) values were more significant than 0.5, indicating that lnc_000048 and PKR had a high possibility of binding (Figure 5C and D).
We next examined the subcellular localization of lnc_000048 and PKR by FISH and IF. Confocal microscopy revealed that lnc_000048 co-localized with PKR in the cytoplasm of M1 macrophages (Figure 5E). From the above results, lnc_000048 and PKR jointly contribute to the polarization of M1 macrophages.