Role And Underlying Mechanism Of Long Non-coding RNA Snhg1 In Memory CD8~+T Cell Differentiation

BackgroundInfectious diseases have always been a major threat to human health.In recent years,in addition to traditional infectious diseases such as viral hepatitis,tuberculosis,and acquired immunodeficiency syndrome,which are still wreaking havoc on public health,some new infectious diseases include Severe Acute Respiratory Syndromes,Ebola hemorrhagic fever,Middle East respiratory syndrome,and COVID-19 have emerged one after another,seriously threatening the health of all human beings.One of the most effective measures of preventing and controlling infectious diseases is vaccination.However,many infectious diseases such as AIDS,tuberculosis and COVID-19 still lack effective vaccines,and the research and development of vaccines against infectious diseases still has a long way to go.The principle of vaccine protection is to induce the human body to produce immunological memory.Immunological memory means that the body will remember the encountered antigen,and will produce a faster and stronger immune response when re-encounters the same antigen.Immunological memory can be maintained in the body for a long time and provide long-term protection.The basis of immunological memory is to form memory cells and maintain them in the body for a long time,and how to generate more endurable memory cells is the core scientific issue for vaccine development.CD8⁺T cells are of vital importance for controlling infection of virus and intracellular pathogens.During acute viral infection,naive CD8⁺T cells are activated and proliferate rapidly after exposure to antigens,resulting in a large number of effector CD8⁺T cells.Effector CD8⁺T cells have cytotoxic effects,they can directly dissolve infected cells by secreting perforin,or mediate apoptosis of target cells through granzyme and FAS-FASL pathway.After the elimination of pathogens,most of the effector CD8⁺T cells gradually die via apoptosis,but a small percentage（～5-10%）survive and further differentiate into long-lived memory CD8⁺T cells,which can provide enhanced protection against previously encountered pathogens.The differentiation and fate of memory CD8⁺T cells are regulated by many factors,including T cell receptor（TCR）signals,surface markers,transcription factors,epigenetic regulation,cellular metabolic state and the cytokine status in the microenvironment.However,the current researchs on memory CD8⁺T cell differentiation mainly focus on transcriptional regulation,and the roles of long non-coding RNAs（LncRNAs）s in this process are not yet clear.LncRNAs were originally considered to be"noise"produced by gene transcription and ignored by people.However,with the development of high-throughput sequencing technology in recent years,people are increasingly aware of the importance of lncRNAs in regulating gene expression.Studies have shown that lncRNAs can be widely involved in various physiological or pathological processes,such as hematopoiesis,nervous system development,tumors and infections.In the immune system,lncRNAs has also been reported to affect the development and function of various immune cells such as dendritic cells,neutrophils,and macrophages.However,the roles of lncRNAs in CD8⁺T cells are not clear,especially in the formation and function of memory CD8⁺T cell during acute viral infection.Combined with the research progress of memory CD8⁺T cells and lncRNAs,we speculate that lncRNA may play an important role in the formation and function of memory CD8⁺T cells.Methods and results1.To explore the roles of lncRNAs in the development of memory CD8⁺T cells and to find the lncRNA that may play a key role in this process,we first used the lymphocytic choriomeningitis virus（LCMV）Armstrong strain to establish an acute viral infection mouse model,and through RNA-sequencing,we obtained the expression pattern of lncRNAs in naive,effector and memory CD8⁺T cells respectively.Further,through biological analysis and quantitative real-time PCR,we identified that lncRNA Snhg1 may play an important role in the development of memory CD8⁺T cells.Next,we used short hairpin RNA（sh RNA）to knock down the expression of Snhg1 in CD8⁺T cells,and found that the formation of memory precursor（MP）and central memory T cells(T_CM)were significantly impaired after Snhg1 knockdown.2.Lcn RNAs usually need to interact with proteins to perform its biological functions.To explore the protein that interacts with Snhg1,we performed experiments such as RNA pull down,mass spectrometry and RNA-binding protein immunoprecipitation（RIP）,and found the protein that interacted with Snhg1-Vps13D,a kind of vesicle transport protein.Further,through knocking down the expression of Vps13D in CD8⁺T cells with sh RNA,we found that the formation of MP cells and T_CM cells were also impaired after Vps13D knockdown,suggesting that Snhg1 regulates the differentiation of memory CD8⁺T cells via interacting with Vps13D.3.To explore the mechanism of Snhg1 and Vps13D regulating memory CD8⁺T cell differentiation,we performed RNA sequencing and analyzed the changes in gene expression profiles after Snhg1 or Vps13D knockdown,and found that the genes regulated by Snhg1 or Vps13D were mainly related to membrane receptor-associated immune process.Subsequently,we detected that the surface expression of IL-7Rα（CD127）was significantly down-regulated after Snhg1 or Vps13D knockdown through flow cytometry.Further,through experiments such as immunofluorescence staining,immunoprecipitation（IP）and mass spectrometry,we found that the down-regulation of surface IL-7Rαexpression after Snhg1/Vps13D knockdown was due to Snhg1/Vps13D affecting the trafficking of IL-7Rαfrom ER-Golgi to cell membrane.Finally,through chromatin immunoprecipitation,flow cytometry and TCF-1overexpression experiment,we demonstrated that Snhg1-Vps13D regulates the differentiation of memory CD8⁺T cells through IL-7-IL-7R-STAT3-TCF-1 axis.Conclusions1.LncRNA Snhg1 promotes the differentiation of virus-specific memory CD8⁺T cells during acute viral infection;2.Snhg1 regulates the differentiation of memory CD8⁺T cells by interacting with Vps13D;3.Snhg1-Vps13D regulates the differentiation of memory CD8⁺T cells through IL-7-IL-7R-STAT3-TCF-1 axis.