Effect Of Lentivirus-mediated SNX17 On Expression Of Acetylcholine Receptor Clustering-related Proteins In C2C12 Myotubes

BackgroundSorting nexin 17(SNX17),a member of the sorting nexin family,which contains a characteristic conserved phox homology(PX)domain that binds specific phosphoinositides,and FERM domains that participate in the intracellular trafficking of membrane proteins.SNX17,specifically through its FERM domain,recognizes an NPxY motif in the cytoplasmic domains of LDLR family,participates in the recycling of the LDLR family regulates the cell surface levels and mediates the recycling and degradation.Lipoprotein receptor-related protein 4(LRP4)is a single-pass transmembrane protein that belongs to the LDL receptor family,and has a small intracellular domain that contains a NPXY motif C-terminus.As a functional receptor for the nerve-derived agrin,LRP4 forms a complex with MuSK at the NMJ,where it plays a critical role in agrin-stimulated MuSK activation and AChR clustering in muscle cells.Recent advances are exciting that anti-LRP4 antibodies have been detected in myasthenia gravis patients.C2C12 myoblast is a model for in vitro culture and differentiation of skeletal muscular cells and identification of multinuclear contractile myotube cells,used for the study of myasthenia gravis.The study of C2C12 myoblast differentiation and the expression of related receptors is very important for studying the function and repair of neuromuscular junctions.Early co-immunoprecipitation verified that SNX17 can directly bind to the intracellular domain of LRP4.And it was speculated that:when the LRP4 internalization,the NPxY motif of LRP4 cytoplasmic binds to the FERM domain of SNX17,the PX domain is combined with phosphatidylinositol on the membrane of the early endosome.SNX17 mediates LRP4 into the early endosome,escape the lysosomal degradation pathway,and regulates the cell surface levels and mediates the recycling and degradation,improves efficient reuse of acetylcholine receptor clustering-related proteins.ObjectIn order to verify the hypothesis,this topic uses C2C12 myoblast as the research object,using lenti virus transfection technology to explore the effect of lentivirus-mediated SNX17 silencing and overexpression on the expression of acetylcholine receptor clustering-related proteins in C2C12 myotubes.Methods1.C2C12 myoblast culture and identification:C2C12 myoblast were cultured in complete medium,induced differentiation with 2%horse serum,and incubated with 10 ng/mL Agrin for 16 h,followed by fluorescently labeled ?-BTX staining.2.Silent C2C12 cell SNX17:design and screen SNX17 shRNA,construct SNX17 lentiviral silencing plasmid,transfect 293T cells,and detect the expression level of SNX17 by western blot;lentiviral shRNA lentiviral silencing plasmid and two lentiviral packaging plasmids co-transform Transfected 293T cells for lentiviral packaging,fluorescent counting method for lentiviral particle titer detection;infected C2C12 cells;qPCR and western blot to verify the shRNA target sequence of SNX17.3.Overexpression of C2C12 cell SNX17:construction of SNX17 lentiviral overexpression plasmid,lentiviral packaging and titer detection,infection of C2C12 cells,western blot to verify SNX17 overexpression.4.Western blot to detect the expression of acetylcholine receptor clustering-related proteins LRP4 and MuSK after silencing and overexpression of C2C12cells SNX17.Results1.C2C12 myoblast can be induced to differentiate into myotubes by DMEM medium with 2%horse serum.After Agrin stimulated myotubes for 16 hours,the red fluorescence on the surface of myotubes can be seen,and the number and number of red fluorescent clusters and the length was significantly higher than the group without Agrin stimulation,and the difference was statistically significant(P<0.05).2.The four SNX17 shRNA and shRNA-NC plasmids were successfully constructed,and sequencing was in line with expectations.The efficiency of plasmid transfection of 293T cells was higher than 70%.The SNX17 shRNA-2 group had a silencing efficiency of about 30%for SNX17 protein levels.SNX17 shRNA and shRNA-NC lentiviral particle titers were successfully packaged,and the myotube infection rate was higher than 60%.The results showed that the SNX17 shRNA-2 group had the highest silencing efficiency for SNX17 at the gene and protein levels,which were about 46%and 50%,respectively,and the differences were statistically significant(P<0.05).3.The plasmids of SNX17 OE and SNX17 OE-con group(control group)were successfully constructed and the sequencing results were in line with expectations.The lentiviral particle titers of SNX17 OE and SNX17 OE-con group were successfully packaged,and the myotube infection rate was higher than 60%.It showed that the overexpression level of SNX17 protein was higher than that of the control group,and the difference was statistically significant(P<0.05).4.The expression of LRP4 in the SNX17 silencing group was not significantly different from that of the shRNA-NC group(negative control group)(P>0.05).The expression level of MuSK was significantly lower than that of the shRNA-NC group(negative control group).The difference was statistically significant(P<0.05).5.The expression level of LRP4 in the SNX17 overexpression group was significantly higher than that in the SNX17 OE-con group(control group),and the difference was statistically significant(P<0.05).Significantly increased,the difference was statistically significant(P<0.05).Conclusions1.C2C12 myoblast can be induced into C2C12 myotubes by 2%horse serum.Myotubes can accumulate nAChR under the stimulation of Agrin.2.Silencing with C2C12 myotube SNX17,LRP4 protein expression decreased,MuSK protein expression levels were significantly reduced;C2C12 myotube SNX17 overexpression,LRP4 protein and MuSK protein expression levels were significantly increased.This indicates that SXN17 has the potential to return LRP4 to the cell membrane for recycling.