KIF16B可能参与LDLR在体外培养的肝细胞膜上的分布

KIF16B may participate in the distribution of LDLR on the cell membrane

目的探究驱动蛋白超家族成员16B(KIF16B)在低密度脂蛋白受体诱导降解蛋白(IDOL)调节肝细胞摄取低密度脂蛋白胆固醇(LDLC)中的作用。方法干扰/过表达IDOL(RNAi/OE-IDOL)的慢病毒感染HepG2和LO2细胞,通过倒置荧光显微镜来观测病毒感染效果;油红O染色观察细胞内脂质蓄积水平;DiI荧光染料标记的LDL(DiI-LDL)摄取实验检测肝细胞对LDLC的摄取能力,流式细胞术检测肝细胞表面低密度脂蛋白受体(LDLR)丰度;Western blot检测IDOL、KIF16B及LDLR蛋白的表达变化,并免疫共沉淀进一步检测蛋白之间的相互作用。结果两种感染慢病毒的肝细胞内均显示绿色荧光,提示RNAi/OE-IDOL的慢病毒已感染HepG2和LO2细胞;与无慢病毒感染的Control组比较,OE-IDOL组的细胞内脂质含量显著减少(P<0.05),LDLC的摄取显著减弱(P<0.05),且肝细胞表面LDLR丰度显著降低(P<0.01);RNAi-IDOL组的细胞内脂滴含量显著增加(P<0.01),LDLC摄取增强,且肝细胞表面LDLR丰度显著增高(P<0.01);与慢病毒载体的Control组比较,RNAi-IDOL组LDLR蛋白和KIF16B蛋白的表达升高(P<0.01),且KIF16B与LDLR的相互作用增强;OE-IDOL组LDLR蛋白和KIF16B蛋白的表达降低(P<0.05),LDLR与KIF16B相互作用随之减弱。结论IDOL调节的肝细胞摄取LDLC的过程可能与KIF16B跟LDLR之间存在相互作用有关。

Aim To confirm the role of kinesin superfamily member 16B(KIF16B)in the process of low density lipoprotein cholesterol(LDLC)uptake of hepatocyte which is regulated by the inducible degradation of low density lipoprotein receptor(IDOL).Methods The intracellular fluorescence intensity was observed by the inverted fluorescence microscope.The intracellular lipid content was measured by oil red O staining,and the LDLC uptake was detected by DiI-LDL uptake experiment.The low density lipoprotein receptor(LDLR)abundances on the cell surface of hepatocytes were assayed by immune flow cytometry.The protein expression of IDOL,KIF16B and LDLR was detected by Western blot,and the interaction between LDLR and KIF16B protein was carried out by co-immunoprecipitation.Results Compared with white light view,the observed green fluorescence results showed that both HepG2 and LO2 cells were infected by the RNA-interference or overexpression IDOL(RNAi/OE-IDOL)lentivirus.Compared with the non-lentivirus infected control group,both the intracellular lipid and the ability of the LDLC uptake were significantly decreased in the OEIDOL group(P<0.05),and also decreased in the abundances of LDLR on the surface of hepatocytes(P<0.01);and vice versa,the contrary results of these three experiments were observed in the RNAi-IDOL group(P<0.01),which indicated that overexpression IDOL would reduce the LDLC uptake of hepatocytes.Compared with the RNAi/OE-IDOL control group,the expression of LDLR and KIF16B protein was increased in the RNAi-IDOL group(P<0.01),and the interaction between KIF16B and LDLR was enhanced(P<0.01).While in the overexpression IDOL of HepG2 and LO2 cells,the expression of LDLR and KIF16B protein was decreased(P<0.05),meanwhile the interaction between LDLR and KIF16B was correspondingly weakened.Conclusion The interaction between KIF16B and LDLR possibly affects the process of that IDOL regulates LDLC uptake of hepatocytes.