Lnc＿000048 Targets PKR To Enhance JAK1/STAT1 Pathway Mediated M1-type Macrophage Polarization

Objective:Atherosclerosis can lead to Large Artery Atherosclerosis(LAA)cerebral infarction.The etiology of atherosclerosis is complex,and the exact molecular mechanism is still unclear.Macrophages are the core immune cells of atherosclerosis,and their number and phenotype influence the progression and regression of the disease.Long non-coding RNA(lnc RNA)can regulate macrophage phenotype through a variety of mechanisms.Doublestranded RNA-dependent protein kinase(PKR)is an immune-related RNA-binding protein(RBP).Previous studies have found that lnc RNA binds to PKR to form complexes that can further activate downstream pathways.In our previous study,lnc＿000048 was found to be associated with LAA progression and poor prognosis in patients,but the molecular regulation mechanism is still unclear.Therefore,our study aimed to explore the function of lnc＿000048 in macrophages and verify whether it enhances JAK1/STAT1pathway-mediated macrophage polarization by targeting PKR.Methods:Human monocytic cells(Tohoku Hospital Pediatrics-1,THP-1)were cultured in vitro.The cells were induced by Phorbol 12-myristate 13-acetate(PMA)for 24 hours and then cultured for another 24 hours without PMA to become quiescent macrophages(M0),further stimulated with Lipopolysaccharide(LPS)/ Interferon-gamma(IFN-γ)for 48 hours to construct classical activated macrophages(M1).The expression of marker genes(TNF-α,IL-6,IL-1β,CD38,CD80)m RNA and phenotypic proteins(CD11b,CD38,CD80)were verified by real-time fluorescence quantitative PCR(RT-q PCR)and flow cytometry,respectively.RT-q PCRwas used to detect the differential expression of lnc＿000048 in different macrophage models.The Sh-lnc＿000048,Sh-NC,Oe-lnc＿000048,Oe-NC were constructed by infection with lentiviruses.The effect of lnc＿000048 on macrophage polarization was verified by flow cytometry,western blot(WB)and RTq PCR.cat RAPID website prediction,RNA-pull down and mass spectrometry were used to identify and screen the binding protein PKR(another name is eukaryotic translation initiation factor 2-alpha kinase,2EIF2AK2)of lnc＿000048,and cat RAPID and RPIseq websites were used to predict the binding ability of lnc＿000048 to PKR.Subcellular localization of lnc＿000048 and PKR was detected using the lnc Locator online database,in-situ hybridization,and immunofluorescence.Sh-lnc＿000048-M1 group and Sh-NC-M1 group,Oe-lnc＿000048-M1 group and Oe-NC-M1 group,blank control group and JAK inhibitor group,DMSO control group and PKR inhibitor group,PKR inhibitor + Oelnc＿000048 group and PKR inhibitor + Oe-NC group were constructed.Protein expression levels of STAT1,p-STAT1,PKR,and p-PKR were detected using WB and the relative protein expression was analyzed using Image J.The content of CD38-tagged M1-type macrophages was analyzed by flow cytometry.RT-q PCR was used to detect differences in m RNA expression of M1-type macrophage-related polarization marker genes.?Results:1.The M0-type and M1-type macrophage models were successfully constructed by PMA and LPS/IFN-γ,and the expression of lnc＿000048 in M1-type macrophages was higher than that in M0-type macrophages(P < 0.05).2.Compared with M1-type macrophages in Sh-NC group,M1-type macrophages in Sh-lnc＿000048 group showed morphological changes,most of which were round or irregular adherent,with shorter pseudopodia;Moreover,Sh-lnc＿000048 could significantly inhibit the m RNA expression of M1-type macrophage marker genes TNF-α,IL-6,IL-1β,CD38 and CD80(P < 0.05);At the same time,Sh-lnc＿000048 also inhibited the expression of secretory markers TNF-α and L-1β proteins in M1-type macrophages(P< 0.05).After Oe-lnc＿000048,the expression of corresponding genes was increased(P <0.05);The results of flow cytometry showed that the number of CD38-labeled M1-type macrophages decreased after Sh-lnc＿000048 treatment.3.WB results showed that down-regulation of lnc＿000048 did not change the total protein expression of STAT1 in Sh-lnc＿000048-M1 group and Sh-NC-M1 group,but the expression of p-STAT1 protein decreased.On the contrary,p-STAT1 protein expression was relatively increased after upregulation of lnc＿000048.After adding JAK inhibitor,the expression of total STAT1 and p-STAT1 in M1-type macrophages did not change significantly(P > 0.05),indicating that inhibition of JAK activity did not affect the expression of total STAT1 and phosphorylated STAT1,indicating that lnc＿000048 could enhance the activation of JAK1/STAT1 pathway and mediate the M1-type macrophages polarization.4.Lnc＿000048 was predicted to be localized in the cytoplasm by the lnc Locator online database,and the binding protein of lnc＿000048 was identified by cat RAPID website prediction and RNA-pull down and mass spectrometry.The immune-related RNA-binding protein PKR was screened,and cat RAPID and RPIseq websites were used to predict the presence and binding ability of lnc＿000048 to PKR.In situ hybridization and immunofluorescence showed subcellular colocalization of lnc＿000048 and PKR in the cytoplasm of M1-type macrophages.5.Lnc＿000048 was found to act as the upstream of PKR to regulate PKR activity when lnc＿000048 was down-regulated or PKR inhibitor was added.Compared with the control group,p-STAT1 protein expression was decreased after adding PKR inhibitor C16.The results of RT-q PCR showed that the m RNA expression levels of the secretory markers TNF-α,IL-6 and IL-1β of M1 macrophages in the inhibitor group were significantly lower than those in the non-inhibitor group(P < 0.05).When lnc＿000048was overexpressed in the C16 group,WB results showed that the expression of p-STAT1 was increased compared with the control group(Oe-NC).RT-q PCR results showed that the m RNA expression of marker genes TNF-α,IL-6 and IL-1β was also increased.These results indicated that upregulation of lnc＿000048 could rescue the inhibitory effect of C16 on M1-type macrophage polarization,indicating that lnc＿000048 promoted PKR phosphorylation and combined with PKR to enhance the JAK1/STAT1 pathway mediated M1-type macrophages polarization.Conclusions:1.Lnc＿000048 was found to be mainly enriched in M1 macrophages and colocalized with PKR in the cytoplasm of M1 macrophages.2.Lnc＿000048 as PKR upstream,regulating the PKR phosphorylated activated and combined with PKR to enhance the JAK1/STAT1 pathway mediated M1-type macrophages polarization.