The Study Of The Mechanism By Which SPG6 Regulates Acute Myeloid Leukemia Development

Objective:To investigate the mechanism of occurrence and development of SPG6(Spastic Paraplegia 6)in acute myeloid leukemia(AML).Methods:Firstly,three AML patient databases(GSE10358,TCGA,GSE6891)were analyzed to find the relationship between SPG6 expression and survival rate of AML patients.Relative expression of SPG6 was analyzed in different types of leukemia cells.Two pairs of interference sequences were designed for the SPG6 mRN A sequence and the lentiviral vector was constructed.Lentivirus containing SPG6 shRNA were ackaged in HEK293T cells.AML cells NB4 and MV4-11 were infected by the SPG6 lentivirus to knockdown expression of SPG6.Then,GFP-positive AML cells were sorted by flow cytometry.The the growth of sorted cells was detected by cell counting method,apoptosis of sorted cells was detected by flow cytometry.Expression and phosphorylation of BMPR2,Smad,caspase-3 was detected by western blot;transcription of Bcl-2 and Bcl-xl was detected by RT-PCR.Results:Firstly,we analyzed the database of three AML patients and found that there was a significant negative correlation between the expression level of SPG6 and the survival time of AML patients,and found that the expression of SPG6 in various types of AML was higher than that of normal bone marrow cells.Two lentiviral vectors targeting SPG6 mRNA were constructed.Lentiviruses targeting SPG6 mRNA were successfully packaged in 293T cells using the lentivirus three-plasmid packaging system.MV4-11 and NB4 cells were infected with SPG6 shRNA lentivirus,the infection efficiency can reach 30%-50%.After verification,the knockout effect of SPG6 shRNA-1 and SPG6 shRNA-2 was found to be between 50%and 80%.Knockdown expression of SPG6 resulted in decreased cell growth and elevated apoptosis of MV4-11 and NB4 cells.Notably,the SPG6 deficiency resulted in higher BMPR2 expression indicating that BMPR2 signaling contributes to AML pathogenesis.Furthermore,SPG6 deficiency promoted phosphorylation of Smadl/5/9 and decreased transcription of Bcl-2 and Bcl-xl.Conclusion:1.SPG6 supports development of acute myeloid leukemia;2.SPG6 promotes growth and inhibits apoptosis of AML cells;3.SPG6 inhibits expression of BMPR2;4.SPG6 suppresses Smad signaling activation and inhibits transcription of Bcl-2 and Bcl-xl.