The Role And Mechanism Of LncRNA GAS5 In The Polarization Of Microglia

Objective: Multiple sclerosis( MS) is an inflammatory demyelinating disease in the central nervous system, which affects the white matter majorly. The clinical manifestations of MS are quite complicated and multiple systems suffer systemic neurological disorders. In the early stage of the disease, neurological symptoms appear in a relapsing-remitting pattern and can be controlled by drugs, but in the late stage they develop into a secondary progressive course without remission. Currently, no effective treatment is available for progressive MS, and new therapeutic strategies and targets are greatly needed.In the current study and treatment of MS, the regulation of inflammation in the central nervous system has been a key point in controlling the occurrence and development of MS. Recent studies have showed that MS is not only an autoimmune disease but also a neurodegenerative disorder. The inflammation process reacts with the degeneration process thus promotes the exacerbation of MS. As the major innate immune cells in the central nervous system as well as the bridge linking innate immunity and acquired immunity, microglia not only affect the inflammatory process of MS but also play an important role in neuronal degeneration. Activated microglia are classified into two functional subtypes: one promotes the secretion of inflammatory cytokines and toxic substances, thus plays the pro-inflammatory and foreign matter and debris cleaning role;and the other secrets anti-inflammatory cytokines and neuroprotective substances, thus plays the anti-inflammatory and repair promoting role. The two functional subtypes are called M1/classic and M2/alternative polarization respectively. It is important in regulating the function of microglia to clarify the mechanism of M1 and M2 polarization.Currently, the understanding of M2 polarization is more urgent to be developed since less knowledge is available.Long noncoding RNAs(lncRNAs) are a class of RNAs greater than 200 bp in length with no protein-coding capacity. With the development of microarray and RNA-sequencing technology in recent years, lncRNAs were found not only widely transcribed, but also exhibited changes in expression in almost all biological processes.More and more studies relating lnc RNAs discovered many functional lnc RNAs.According to the in-depth studies of the function of lncRNAs, they were characterized ashighly sensitive to environmental changes, expressed widely and harbored diverse action modes, which make lncRNAs an important player in epigenetic regulation. In the central nervous system, lnc RNAs are not only expressed in all types of brain cells, but the proportion of expressed lncRNAs in all discovered lnc RNAs is probably highest compared with other systems. This makes it possible that lnc RNAs may play a more important role in the central nervous system. However, the role of lncRNAs in microglia function and the pathogenesis of MS is unknown and arouses our interests.Methods: This study was designed to elucidate the expression profile of lnc RNAs in M2 polarized microglia and the quiescent control. By bioinformatic analysis of comparative expression pattern and co-expression network, we wanted to select some potential functional ones in the regulation of microglia M2 polarization. Then we planed to combine the means of molecular biology, cell biology, morphology and praxiology and other to elucidate the role of the selected lncRNA in microglia M2 polarization and the detailed molecular mechanisms. We wish our work shed some light on the role of lnc RNAs in microglia polarization.Results: Through microarray screening, we found 120 lnc RNAs were differentially expressed between M2 polarized microglia and the quiescent control. Among them, the lnc RNA growth arrest-specific 5( GAS5) was significantly down-regulated in IL-4stimulated M2 microglia. Overexpressing or interfering GAS5 in primary cultured microglia in vitro showed that GAS5 inhibited the expression of M2 markers like Ym-1,Fizz-1, CD206 and IGF-1, while increased the secretion of M1 inflammatory cytokines like TNF-α and IL-1β. The conditioned medium from GAS5-modified microglia significantly affected the apoptosis and differentiation of oligodendrocyte precursor cells(OPCs) and the neurite growth of neurons. In vivo, the transplantation of GAS5-modified microglia influenced the severity of experimental autoimmune encephalomyelitis(EAE)and transplantation of GAS5-interfered microglia promoted the remyelination in lysophosphatidylcholine( LPC) induced demyelination model. Our mechanism work by RNA-IP, RNA-pull down and ChIP showed that GAS5 suppressed the transcription of IRF4 which is pivotal in M2 polarization by binding with PRC2. Besides, we found that GAS5 expressed more in microglia of aged mice. In the brain slices of MS patients,GAS5 expressed more in amoeboid-shaped microglia. Human GAS5 showed a similar role on microglia polarization. These results indicate that lncRNAs participate in the regulation of microglia polarization and GAS5 plays an important role on microglia M2 polarization. Our work also partially illuminate the reason for the obstacle of microglia M2 polarization in aged mice.Conclusions: According to this study, we get the following conclusions:1. Lnc RNAs participate in the process of microglia polarization and lnc RNA GAS5 inhibits microglia M2 polarization as well as promotes microglia polarizing to M1 type;2. The transplantation of GAS5-modified microglia influenced the severity of EAE and the remyelination of LPC model, indicating a functional role in vivo;3. GAS5 inhibits microglia M2 polarization by binding with PRC2 thus suppressing the transcription of IRF4.To our knowledge, this is the first work showing the function of a lnc RNA in microglia. This is also the first work showing how a lnc RNA participates in the pathogenesis of EAE and MS by affecting the immune system.