| Cardiovascular disease(CVD)is the most serious diseases,which threaten human health,in the world.It is responsible for deaths in one-third of the global disease deaths each year.Atherosclerotic cardiovascular disease(ASCVD)is the main form of cardiovascular disease.It is a chronic disease of lipid deposition,vascular inflammation and plaque formation.Elevation of low-density lipoprotein cholesterol(LDL-C)is one of the key risk factors for atherosclerosis,so reducing the level of LDL-C in plasma is considered to be crucial factor in the treatment of ASCVD.It is also the cornerstone of ASCVD primary and secondary prevention.LDL-C is mainly eliminated by endocytosis mediated by low-density lipoprotein receptor(LDLR)on the surface of hepatocytes.Inducible degrader of LDLR(IDOL)is a post-transcriptional regulator of LDLR.It is an E3 ubiquitin ligase that can cause ubiquitination of the C-terminal cytoplasmic tail domain of LDLR,thereby targeting it for degradation by the lysosomal pathway.In addition,LDLR can also be recycled to the cell membrane through the copper metabolism MURR1domain-containing(COMMD1)-mediated endosome sorting method,which requires kinesins to participate in transportation and provide energy.Kinesin is a protein involved in the transport of organelles and vesicles.Kinesin super family 3(KIF3)is a superfamily of microtubule kinesins,which can catalyze the hydrolysis of adenosine triphosphate(ATP)and release it.Then the chemical energy is converted into mechanical energy,causing it to move continuously along the microtubules.Kinesin super family 16B(KIF16B)is unique among kinesins because it has a C-terminal PX domain.This domain can interact with the early endosome through phosphatidylinositol 3-phosphate(PI3P),regulated the positive movement of the early endosomes or related proteins.Therefore,this article speculates that KIF16 B may be involved in the regulation of the endocytic cycle of LDLR.Furthermore,Overexpression or RNA-interference IDOL(OE/RNAi-IDOL)lentivirals were used to infect two hepatocytes to explored the possible interaction between LDLR,IDOL and KIF16 B,in order to provide a new theoretical basis for cardiovascular disease drug research.Aim: To explore whether kinesin superfamily member 16B(KIF16B)is involved in the recirculation process of LDLR on the surface of liver cells.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 LDL-C uptake was detected by Di I-LDL uptake experiment.The LDLR abundances on the cell surface of hepatocytes were assayed by immune flow cytometry.The protein expression of IDOL,KIF16 B and LDLR was detected by Western Blot,and the interaction between LDLR and KIF16 B protein was carried out by co-immunoprecipitation.Result:Compared with white light view,the observed green fluorescence results showed that both HepG2 and LO2 cells were infected by the overexpression or RNA-interference IDOL(OE/RNAi-IDOL)lentivirus.Compared with the non-lentivirus infected control group,in the OE-IDOL group,both the intracellular lipid and the ability of the LDL-C uptake were significantly decreased(P<0.01,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 indicates that overexpression IDOL would reduce the LDL-C uptake of hepatocytes.Compared with the RNAi/OE-IDOL control group,the expression of LDLR and KIF16 B protein was increased in the RNAi-IDOL group(P<0.01),and the interaction between KIF16 B and LDLR was enhanced(P<0.01).While in the overexpression IDOL of HepG2 and LO2 cells,the expression of LDLR and KIF16 B protein was decreased(P<0.01;P<0.05),meanwhile the interaction between LDLR and KIF16 B was correspondingly weakened.Conclusion: KIF16B may be involved in the distribution of LDLR on the membrane of hepatocytes that cultured in vitro. |
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