Microarray Analysis Of Long Non-coding RNA Expression Patterns In Polarized Monocyte-derived Macrophages

Objective:To test the expression profile of long non-coding RNA(1ncRNA)and mRNA in polarized monocyte-derived macrophages(MDMs)by using lncRNA expression microarray,and to explore lncRNA nuclear enriched abundant transcript 1(NEAT1)effect on MDMs polarization to M1 or M2 phenotype.Methods:Monocytes were isolated from health donors peripheral blood mononuclear cells(PBMC)using magnetic activated cell sorting(MACS)anti-CD14 microbead.The purified monocytes were cultured in RPMI 1640 medium contains 10% Human serum and 0.05% Glutamine for 7 days to generate MDMs.To induce MDMs polarization,CD68-positive cells were treated with 20 ng/ml IFN-? and 100 ng/ml of LPS to achieve M1 polarization,or with 20 ng/ml IL-4 to achieve M2 macrophage.The non-polarized MDMs were cultured without treatment(M0 phenotype).After 18 h,the supernatant was collected and adherent cells were harvested for further analysis.IL-6,IL-10,IL-12,TNF-?,CCL17 and CCL18 secretion induced by different condition were detected by Enzyme linked immunosorbent assay(ELISA);The expression level of mRNA of M1 markers(CXCL10,CXCL11 and Nos2)and M2 markers(CCL17,CCL18 and CCL22)were detected by quantitative real-time polymerase chain reaction(RT-PCR)respectively.Surface moleculars CD86 and CD206 were evaluated by flow cytometry to show whether M1/M2 macrophages were induced successfully.1.The technique of lncRNA microarray was used to test the lncRNA and mRNA expression profile in M1 macrophages,M2 macrophages and control macrophages.Bioinformatic analysis including gene ontology(GO)analysis,pathway(KEGG)analysis and network analysis was done for further investigation.RT-PCR was used to validate 6 lncRNA(ENST00000569328,ENST00000414554,ENST00000474886,CMPK2,NEAT1 and THRIL).2.20 ng/ml IFN-? and 100 ng/ml of LPS were used to induce M2 macrophages to M1 macrophages;20 ng/ml IL-4 was used to induce M1 macrophages to M2 macrophages.Molecular markers of M1/M2 macrophages were detected by RT-PCR and ELISA to show whether re-polarized macrophages were induced successfully.The expression of lncRNA NEAT1(nuclear enriched abundant transcript 1)in re-polarized macrophages were analyzed by RT-PCR.3.We designed and synthesized siRNA oligo for lncRNA NEAT1 and unrelated sequences at the same time.Then we transfected the siRNA oligo and NC oligo into MDMs by Lipo 2000,and the MDMs were induced to polarize to M1 by IFN-? and LPS,and to M2 by IL-4.IL-10 and IL-12 secretion induced by different condition were detected by ELISA;the mRNA expression level of CXCL10,CXCL11,CCL17 and CCL18 of M1/M2 phenotypes were detected by RT-PCR respectively.M1 macrophages were transfected with siRNAs to knock down lncRNA NEAT1,to investigate the biological effects of lncRNA NEAT1 on the phenotypes of macrophages.Results:1.The results showed that with the IFN-? and LPS induction,the surface levels of CD86 was significantly increased,the mRNA expressions of M1 markers(NOS2,CXCL10 and CXCL11)were significantly increased,and the resulting proinflmmatory cytokine TNF-?,IL-6 and IL-12 were also significantly enhanced.On the other hand,IL-4 induction in MDMs up-regulated surface levels of CD206 and the mRNA expression levels of M2 molecular markers,such as CCL17,CCL18 and CCL22,and showed increased IL-10,CCL17 and CCL18 production,suggesting that,M1/M2 macrophages were induced successfully.2.To identify the most significant candidates,lncRNA with at least two fold expression changes were selected.Under the criteria,5820 lncRNA were up-regulated and 3523 lncRNA were down-regulated in the M1 group compared with the primary M0 group.2621 lncRNA were increased and 1971 lncRNA were decreased in the M2 group compared with the M0 group.4673 lncRNA were overexpressed and 5807 lncRNA were downexpressed in the M2 group compared with the M1 group.From the mRNA analysis,3387 mRNAs were up-regulated and 2516 mRNAs were down-regulated in the M1 group compared with the M0 group.1121 mRNAs were increased and 2001 mRNAs were decreased in the M2 group compared with the M0 group.2528 mRNAs were overexpressed and 4534 mRNAs were downexpressed in the M2 group compared with the M1 group.3.In this study,GO analysis was performed to determine the gene and gene product attributes in biological pocesses,cellular components and molecular functions.GO analysis indicated that the identified mRNA were mainly related to the immune response,immune system process,defense response,innate immune response,chemokine activity,cytokine activity,extracellular space and plasma membrane.Pathway analysis indicated that the deregulated mRNAs between M2 and M1 group were mainly involved in cytokine-cytokine receptor interaction,TNF signaling pathway,Chemokine signaling pathway,NF-kappa B signaling pathway,NOD-like receptor signaling pathway,MAPK signaling pathway,fatty acid metabolism,PPAR signaling pathway,and pathways in cancer.RT-PCR results were generally consistent with the microarray data.4.M1 macrophage was able to redifferentiate to M2-like macrophage while M1 macrophage could redifferentiate to M2-like macrophage when induced conditions in vitro environment changed.Furthermore,we found that lncRNA NEAT1 is expressed at a higher level in M1 macrophages than in M2 macrophage.lncRNA NEAT1 expression was decreased when M1 converted to M2 whereas increased when M2 converted to M1.5.Compared to the control group,lncRNA NEAT1 inhibited the increase of IL-12 secretion,CXCL10 and CXCL11 mRNA expression level in macrophages induced by IFN-? and LPS.lncRNA NEAT1 promotes the increase of IL-10 secretion,CCL17 and CCL18 mRNA expression level in macrophages induced by IL-4.Compared to the control group,lncRNA NEAT1 inhibited the increase of IL-12 secretion,promotes the increase of IL-10 secretion in M1 macrophages.Conclusions:1.These data suggested that IFN-? and LPS successfully induced the alteration of MDMs to M1-polarized macrophages;IL-4 successfully induced the alteration of MDMs to M2-polarized macrophages.2.These data show a significantly altered lncRNA and mRNA expression profile in macrophage exposure to different activating conditions.GO and KEGG analysis revealed that deregulated mRNA may play important roles in regulating macrophage polarization.Some of the differentially expressed lncRNA were validated by RT-PCR to promote the credibility of results.3.lncRNA NEAT1 is expressed at a higher level in M1 than in M2.Moreover,we found that lncRNA NEAT1 may plays an important role in regulating macrophage polarization,it could promote M1 macrophage polarization,and inhibit M2 macrophage polarization.