The Preliminary Mechanisms Study Of RTN3Involved In Autophagy And Influence Cholesterol Efflux

Objective:In this study, HepG2was used as a model to investigate the relationship between RTN3and autophagy. This study was designed to explore whether RTN3is involved in cellular lipid metabolism and cholesterol efflux, and to do a preliminary study of its possible mechanism.Methods:(1) Protein levels of RTN3and LC3B under the condition of cell autophagy induced by nutritional deprivation was detected by western blot. Higher and lower expression of RTN3than normal cells was induced by transfecting with plasmids, and then RTN3, Beclinl and LC3B was detected by western blot. Whether RTN3is invoved in the formation of autophagic vacuole was detected by laser scanning confocal microscopy.(2) The interaction between RTN3and Beclinl, Bcl-2was detected by co-immunoprecipitation. Whether colocalization exists between RTN3and Beclinl was detected by laser scanning-confocal microseopy.(3) Differentiation expression of RTN3caused by interventions of HDL and statin was detected by WB, then explore whether RTN3is related to lipid metabolism regulated by HDL and statin. (4) Red Oil staining and cholesterol efflux assay are employed to test Whether overexpression and knockdown of RTN3affects intracellular lipid metabolism and cholesterol efflux.(5) In order to explore the mechanism of RTN3in cholesterol efflux, expression of ABCA1was detected by WB under the overexperssion and knockdown of RTN3, and colocalization of ABCA1and RTN3was analyzed by laser scanning confocal microscopy in cell treated with HDL or statin.Results:(1) The expression levels of RTN3and LC3B were upregulated in autophagy induced by nutritional deprivation. There was significant change in expression levels of autophagy protein LC3B under overexpression of RTN3. Besides, LC3B and RTN3displayed a colocalization in autophagy induced nutritional deprivation.(2) Co-immunoprecipitation and confocal experiment showed that RTN3and Beclinl may have a direct interaction.(3) Protein levels of LC3B and RTN3were decreased with increasing concentration of HDL, In contrast with treat statin.(4) Red oil staining showed that overexperssion of RTN3led to decrease of lipid droplets, while knockdown of RTN3led to increase of lipid droplets. Without treatment of HDL, overexpression of RTN3group led to higher cholesterol efflux than knockdown of RTN3group and control group. However, cholesterol effluxes in both over and low expression of RTN3groups were suppressed under high concentrations of HDL..(5) The expression of ABCA1was down regulated in both overexpression and knockdown of RTN3groups, and confocal assay displayed that ABCA1and RTN3was located in the intracellular membrane vesicles.Conclusion:(1) the autophagic level in HepG2was affected by the interaction of RTN3and Beclinl.(2) RTN3was involed in lipid metabolism through autophagy.(3) the lipid droplets accumulation and cholesterol efflux was affected by RTN3.