Abstract:Phagocytosis of lipoprotein particles mediated by low density lipoprotein receptors on the surface of hepatocytes is the main pathway for the body to clear circulating low density lipoprotein, and plays a pivotal role in maintaining the homeostasis of cholesterol metabolism in the body. This article briefly reviewed the different links of low density lipoprotein receptor trafficking, including endocytosis, sorting, degradation or recycling channels of receptor molecules, and the main regulatory factors of each link, and summarized the latest advances in related fields. At present, the development of drugs indirectly targeting low density lipoprotein receptor transcriptional regulation (statins) and drugs targeting low density lipoprotein receptor degradation (proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors) have achieved tremendous success. However, our understanding of the molecular mechanisms regulating low density lipoprotein receptor trafficking remains to be incomplete, especially the intracellular recycling processes and the related signaling mechanisms need more research. It is anticipated that further in-depth research in these areas may foster the discovery of new targets for the treatment of hypercholesterolemia in the future.

Abstract:The occurrence and development of atherosclerotic cardiovascular diseases is closely related to abnormally elevated plasma low density lipoprotein cholesterol (LDLC) level. Low density lipoprotein receptor (LDLR) plays a central role in the maintenance of cholesterol homeostasis by mediating the endocytotic clearance of LDLC, and the abundance of LDLR on the surface of the cell membrane is closely related to the expression level and recirculation of LDLR. Recent studies have found that RING-E3 ubiquitin ligase can regulate LDLR levels through a dual mechanism:on the one hand, it directly ubiquitinates and modifies LDLR to promote its degradation via the endosome-lysosome pathway; on the other hand, it reduces LDLR synthesis through activation of the liver X receptor (LXR) pathway or inhibition of the nuclear translocation of sterol regulatory element-binding protein (SREBP). Together, these two mechanisms lead to a decrease in cell membrane LDLR abundance, impairing cholesterol metabolic homeostasis and exacerbating LDLC accumulation. Therefore, targeted inhibition of RING-E3 ubiquitin ligase activity may be a novel strategy to regulate LDLR expression, reduce plasma LDLC levels, and combat cardiovascular disease. This article reviews the mechanism of action of RING-E3 ubiquitin ligase in regulating LDLR and its related research progress.

Abstract:Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory serine protease, which is mainly expressed in hepatocytes. PCSK9 binds to low density lipoprotein receptor (LDLR) forming PCSK9-LDLR complex, and leads to LDLR degradation in lysosomes, which finally results in the reduction of cell membrane LDLR protein level and the elevation of plasma low density lipoprotein cholesterol (LDLC) level. PCSK9 inhibitors have become a hot spot in the research of hypercholesteremia and atherosclerotic cardiovascular disease (ASCVD) drugs discovery. There are three globally marketed PCSK9 inhibitors including evolocumab, alirocumab and PCSK9 siRNA drug inclisirian (Leqvio). This article reviewed the structure, functions and inhibitors of PCSK9.

Abstract:Aim To explore the expression of proprotein convertase subtilisin kexin 9 (PCSK9) in polycystic ovary syndrome (PCOS) ovarian granulosa cells, and the relationship between PCSK9 expression and the lipid status of PCOS ovarian tissue and testosterone-treated KGN cells. Methods A rat model of PCOS was constructed by letrozole combined with high-fat diet. Oil red O staining was used to observe the lipid distribution in the ovarian tissue of PCOS rats. Transcriptome data from PCOS ovarian tissue and normal ovarian tissue was analyzed by bioinformatics, and the differentially expressed genes involved in lipid metabolism pathway were screened. Western blot and immunohistochemical techniques were used to detect the protein expression of PCSK9 in PCOS rat tissues. Immunofluorescence, qRT-PCR and Western blot techniques were used to detect the expression differences of PCSK9 and low density lipoprotein receptor (LDLR) in KGN cells treated with different concentrations of testosterone. Immunofluorescence microscope was used to observe the ability of KGN cells uptake Dil-LDL. Results The results of Oil Red O staining showed that the distribution of lipid droplets in granular cells of PCOS ovarian tissue was significantly less than that of normal ovarian tissue. The results of bioinformatics analysis showed that the up-regulated expression of PCSK9 in the ovarian tissue of PCOS rats was mainly involved in the cholesterol metabolism pathway. The results of immunohistochemistry and Western blot showed that the PCSK9 protein was mainly localized in granulosa cells and its expression increased in the granulosa cells of PCOS rats.The results of immunofluorescence, qRT-PCR and Western blot showed that PCSK9 expression was up-regulated with the increased concentration of testosterone, and LDLR expression was decreased with the increased concentration of testosterone in KGN cells. Testosterone (100 nmol/L) could reduce the uptake of Dil-LDL by KGN cells. Conclusion PCSK9 was highly expressed in PCOS ovarian granulosa cells, which could reduce the expression of LDLR protein and lead to insufficient lipid uptake of granulosa cells.

Abstract:Aim To explore the regulatory effect of nicotinamide mononucleotide (NMN) on cholesterol in Huh7 cells and its molecular mechanism. Methods Huh7 cells were treated with different concentrations of NMN (0,2.5,5, 0,0, 200 μmol/L) for 24 hours, and the cell viability was detected by cell counting kit-8 (CCK-8). The lipid accumulation in cells was observed by oil red O staining. The uptake ability of cells to DiI-LDL was observed by immunofluorescence microscope. Quantitative real-time PCR (qRT-PCR) and Western blot were used to detect the expressions of mRNA and protein of intracellular hepatocyte nuclear factor 1α (HNF1α), proprotein convertase subtilisin kexin 9 (PCSK9), and low density lipoprotein receptor (LDLR). Results CCK-8 experiment showed that different concentrations of NMN had no significant effect on cell viability. Oil red O staining showed that the number of orange-red lipid droplets in the cells increased with the increase of NMN concentration after the addition of LDL. Immunofluorescence microscope observation results showed that the red fluorescence around the nucleus increased with the increase of NMN concentration. Western blot and qRT-PCR results showed that 100 and 200 μmol/L NMN significantly reduced the expressions of PCSK9 and HNF1α mRNA and protein, and significantly increased the expressions of LDLR mRNA and protein in Huh7 cells (P＜0.01). Conclusion NMN may be involved in regulating the cholesterol metabolism of Huh7 cells by mediating the HNF1α/PCSK9/LDLR signaling pathway, and enhance the uptake capacity of LDL by liver cells.

Abstract: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 OE-IDOL 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.

Abstract:Aim To explore the effect of Curcumin on that inducible degrader of the low density lipoprotein receptor (IDOL) regulating the uptake of low density lipoprotein cholesterol (LDLC) of hepatocytes. Methods HepG2 and LO2 cells (two kinds of hepatocytes) were infected with the constructed overexpression or RNA-interference IDOL (OE/RNAi-IDOL) lentivirus. The efficiency of the lentiviral infection experiment was evaluated by fluorescence microscopy. The expression of IDOL and low density lipoprotein receptor (LDLR) proteins was detected by Western blot. After HepG2 and LO2 cells were treated with Curcumin for 24 hours, intracellular lipid droplets were determined by red oil O staining; cholesterol content was detected by using cholesterol testing kits; the uptake of LDLC by hepatocytes was detected by DiI-LDL uptake experiment; LDLR abundances of hepatocytes surfaces were determined by immune flow cytometry. Results Compared with white light view, the phenomenon of the green fluorescence was observed both in HepG2 and LO2 cells infected by OE-IDOL and RNAi-IDOL lentivirus; Western blot results showed that both in HepG2 and LO2 cells infected by RNAi-IDOL-2 lentivirus, IDOL protein expression were decreased, while LDLR expression was increased (P＜0.01); on the contrary, in HepG2 and LO2 cells infected by OE-IDOL lentivirus, IDOL protein expression was increased, while LDLR expression was decreased, the above results indicated that both HepG2 and LO2 cells infected by OE/RNAi-IDOL lentivirus had been acquired. Compared with the control group of HepG2 and LO2 cells without any treatment, after 25 μmol/L Curcumin treatment for 24 hours in the OE/RNAi-IDOL lentivirus infected cells, both the intracellular lipid droplet content and relative cholesterol content were increased in the treatment group (P＜0.01), meanwhile the uptake LDLC ability and cell surface LDLR abundances of hepatocytes were also enhanced (P＜0.01), the same trend was also observed in the results of Rosuvastatin treatment group. Conclusion The levels of IDOL protein in liver cells were down-regulated by Curcumin, which further promotes the uptake of LDLC of liver cells.

Abstract:Aim To observe whether the effect of curcumin on lipid uptake of human hepatoma cell line HepG2 is related to kinesin family member 16B (KIF16B), and to explore the lipid-lowering mechanism of curcumin. Methods (1)The HepG2 cells cultured in vitro were divided into control group (curcumin concentration was 0) and 0,0, 40 μmol/L curcumin treatment groups, and CCK8 method was used to detect cell viability to determine the appropriate concentration of curcumin. (2)The HepG2 cells cultured in vitro were divided into blank control group, negative control group, rosuvastatin (positive drug) group and curcumin group. Cholesterol detection kit was used to detect the content of cholesterol in HepG2 cells; The uptake of DiI-labeled low density lipoprotein (DiI-LDL) was observed by fluorescence microscope; The protein expressions of KIF16B and low density lipoprotein receptor (LDLR) were detected by Western blot; The fluorescence co-localization of KIF16B and LDLR was observed by laser confocal microscope. Results 25 μmol/L curcumin did not affect the growth of HepG2 cells. Compared with the negative control group, the levels of total cholesterol and free cholesterol in HepG2 cells were significantly increased, the uptake of DiI-LDL by cells was significantly increased, the expressions of KIF16B and LDLR proteins in the cells were significantly increased, and the fluorescence co-localization of KIF16B and LDLR proteins in the cells was significantly increased in the curcumin group (P＜0.05). Conclusion The increase of LDL lipid uptake and LDLR expression caused by curcumin acting on HepG2 cells is related to the interaction between KIF16B and LDLR.

Abstract:The low density lipoprotein receptor-related protein 6 (LRP6) is one of the important receptor proteins that perform the canonical Wnt/β-catenin pathway. Mutations in the protein are often associated with a variety of human diseases, including metabolic syndrome, tumor, Alzheimers disease and osteoporosis. The mutation of low-density lipoprotein receptor-related protein 6 will have important guiding significance in the clinical diagnosis. In this paper, the function of low-density lipoprotein receptor-related protein 6 and the occurrence and development of related diseases will be summarized to provide useful resources for future research.

Abstract:Aim To investigate the effect of miR-224-5p on the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the lipid uptake of HepG2 cells. Methods The position, conservativeness and seed sequence of miR-224-5p gene were analyzed by bioinformatics method. The binding sites and binding free energies of miR-224-5p to PCSK9 were analyzed in databases such as Targetscan, miRanda, miRDB and RNAhybrid. Direct targeted binding of miR-224-5p to PCSK9 mRNA 3′UTR was verified by double luciferase reporter gene. Western blot was used to detect the effects of miR-224-5p mimic and miR-224-5p inhibitor on PCSK9 and low density lipoprotein receptor (LDLR) proteins. LDLR on the cell membrane was directly observed with cellular immunofluorescence. Oil red O staining and DiI-LDL were respectively used to observe the effects of miR-224-5p on lipid droplet content and lipid uptake in HepG2 cells. Results Bioinformatics analysis revealed that the human miR-224-5p gene was located in Xq28 and highly conserved among different species. MiR-224-5p and PCSK9 mRNA 3′UTR had the basis of targeted binding, and the binding free energy was low. The double luciferase reporter gene assay showed that miR-224-5p mimic could inhibit the luciferase activity of PCSK9-WT 3′UTR, but not PCSK9-Mut 3′UTR, suggesting that the PCSK9 mRNA 3′UTR was the target of miR-224-5p. Further experiments showed that the miR-224-5p mimic could significantly inhibit the expression of PCSK9 protein and increase the content of LDLR protein. The expression of PCSK9 increased and the level of LDLR decreased after the down-regulation of miR-224-5p. In addition, oil red O staining showed that lipid droplets in HepG2 cells decreased significantly in the miR-224-5p mimic group, while lipid droplets in HepG2 cells increased significantly in the miR-224-5p inhibitor group. High expression of miR-224-5p significantly promoted LDLC uptake by HepG2 cells. Conclusion miR-224-5p targeting PCSK9 mRNA 3′UTR inhibits the expression of PCSK9, thereby reducing the content of lipid droplets in HepG2 cells and increasing the uptake of lipid by HepG2 cells.