Rapamycin Can Inhibit The Increase Of Type Ⅰ Interferon Caused By Down-regulation Of SAMHD1 In THP-1 Cells

Background and Objiective Aicardi-Goutieres syndrome(Aicardi-Goutieres syndrome,AGS)is a neurodamaging disease classified in the category of neurological diseases.The early symptoms of AGS patients are similar to congenital viral infections.The main symptoms of patients are brain damage to varying degrees and an abnormal increase in the expression of interferon in the body.Studies have found that AGS is a genetic disease related to single-gene inheritance.Mutations in any of the seven genes,including TREX1,RNASH2 A,RNASH2B,RNASH2 C,SAMHD1,ADAR1,and IFIHI,can cause AGS.SAMHD1(Sterile alpha motif domain and histidine/aspartic acid domain-containing protein1)is a deoxynucleoside triphosphate hydrolase containing SAM and HD domains,which can degrade intracellular deoxynucleoside triphosphates(d NTPs)Create an environment lacking d NTPs to inhibit virus replication.It has been found that the inability of SAMHD1 mutants to inhibit LINE-1 retrotransposition can cause LINE-1 DNA to accumulate in the cytoplasm.The accumulated DNA is recognized by c GAS and induces type I interferon overexpression can lead to AGS.In addition,the SAMHD1 mutation can block DNA replication and repair,promote the accumulation of single-stranded DNA fragments into the cytoplasm,and induce the expression of type I interferon through the c GAS-STING-IRF3 pathway,thereby causing AGS.However,the mechanism of how the SAMHD1 mutation causes AGS is still not fully understood.Cells can use autophagy to remove damaged DNA from the nucleus of the cytoplasm.If the autophagy function is normal,it can effectively eliminate cytoplasmic DNA and will not induce the expression of type I interferon.Therefore,the SAMHD1 mutation may inhibit or weaken the autophagy function in the cells of AGS patients.The purpose of this study is to use THP-1 cell line to preliminarily clarify the role of autophagy in AGS caused by SAMHD1 mutation.Methods(1)Does down-regulation of SAMHD1 lead to cytoplasmic DNA aggregation and induce type I interferon expression a.Use lentivirus with SAMH1 shRNA and control shRNA to infect THP-1 cells,and then pass puromycin selection to obtain THP-1-SAMHD1 shRNA cells,and negative control THP-1-Control shRNA cells.Detect the expression of SAMHD1 in the control and experimental groups by western blot.b.Western blot to detect the expression of SAMHD1 in THP-1-SAMHD1 shRNA and THP-1-control shRNA cells.c.Immunofluorescence to detect the expression of DNA damage marker molecules 53BP1 and γH2AX in THP-1-SAMHD1 shRNA and THP-1-control shRNA cells,and cytoplasmic DNA aggregation.d.qRT-PCR detection of THP-1-SAMHD1 shRNA and THP-1-control shRNA cell interferon α,β and interferon stimulation gene ISG15 and MX1 expression.(2)Whether down-regulation of SAMHD1 activates PI3K/AKT/m TOR and prevents autophagy a.Western blot to detect the expression of PI3 K,AKT,m TOR and phosphorylated AKT and m TOR in THP-1-SAMHD1 shRNA and THP-1-control shRNA cells.b.Western blot to detect the expression of autophagy-related proteins LC3,Beclin-1 and ATG-5 in THP-1-SAMHD1 shRNA and THP-1-control shRNA cells.(3)Does autophagy inducer rapamycin down-regulate the increase in interferon expression caused by SAMHD1 a.Find out the best time and dose of rapamycin to treat THP-1 cells..b.Western blot detection of LC3 expression in THP-1-SAMHD1 shRNA and THP-1-ControlshRNA cells treated with rapamycin.Test whether rapamycin blocks the increase in interferon expression caused by down-regulation of SAMHD1.c.qRT-PCR detects whether THP-1-SAMHD1 shRNA and THP-1-ControlshRNA cells are treated by rapamycin to block the increase in interferon expression caused by down-regulation of SAMHD1.(4)Does rapamycin treatment reduce SAMHD1 down-regulation and cause cytoplasmic DNA aggregation a.Detection of DNA damage and cytoplasmic DNA accumulation changes after rapamycin treatment of THP-1-SAMHD1 shRNA and THP-1-ControlshRNA cells by immunofluorescence.Results(1)The results of western blotting showed that the SAMHD1 gene was knocked down in THP-1-SAMHD1 shRNA cells.(2) DNA damage and cytoplasmic ds DNA aggregation were more obvious in THP-1 cells knocked down by SAMHD1 by immunofluorescence.(3)Down-regulation of SAMHD1 in THP-1 cells will increase the expression of type I interferon and interferon-stimulating genes in the cells.(4)The expression of phosphorylated AKT and m TOR increased in THP-1 cells down-regulated by SAMHD1.(5)The ratio of LC3II/LC3 I and the expression of autophagy-related proteins ATG-5 and Beclin-1 decreased in THP-1 cells down-regulated by SAMHD1.(6)Compared with THP-1 control shRNA cells,rapamycin is more difficult to induce autophagy in THP-1-SAMHD1 shRNA.(7)The results of qRT-PCR showed that interferon and interferon-induced gene expression in THP-1-SAMHD1 shRNA cells decreased after adding rapamycin.(8)Immunofluorescence results showed that the DNA damage and cytoplasmic DNA accumulation in THP-1 cells down-regulated by SAMHD1 were reduced after rapamycin treatment.Conclusion Down-regulation of SAMHD1 expression in THP-1 cells causes DNA damage,cytoplasmic DNA aggregation and increased interferon expression,while rapamycin can reduce DNA damage,cytoplasmic DNA aggregation and interferon caused by SAMHD1 knockdown in THP-1 cells Increased expression.