LncRP11-675F6.3 responds to rapamycin treatment and reduces triglyceride accumulation via interacting with HK1 in hepatocytes by regulating autophagy and VLDL-related proteins

Abstract

Long noncoding RNAs (lncRNAs) have been widely proven to be involved in liver lipid homeostasis. Herein, we identify an upregulated lncRNA named lncRP11-675F6.3 in response to rapamycin treatment using a microarray in HepG2 cells. Knockdown of lncRP11-675F6. 3 leads to a significant reduction in apolipoprotein 100 (ApoB100), microsomal triglyceride transfer protein (MTTP), ApoE and ApoC3 with increased cellular triglyceride level and autophagy. Furthermore, we find that ApoB100 is obviously colocalized with GFP-LC3 in autophagosomes when lncRP11-675F6. 3 is knocked down, indicating that elevated triglyceride accumulation likely related to autophagy induces the degradation of ApoB100 and impairs very low-density lipoprotein (VLDL) assembly. We then identify and validate that hexokinase 1 (HK1) acts as the binding protein of lncRP11-675F6.3 and mediates triglyceride regulation and cell autophagy. More importantly, we find that lncRP11-675F6.3 and HK1 attenuate high fat diet induced nonalcoholic fatty liver disease (NAFLD) by regulating VLDL-related proteins and autophagy. In conclusion, this study reveals that lncRP11-675F6.3 is potentially involved in the downstream of mTOR signaling pathway and the regulatory network of hepatic triglyceride metabolism in cooperation with its interacting protein HK1, which may provide a new target for fatty liver disorder treatment.

Keywords: autophagy; hexokinase 1; lipoprotein; lncRP11-675F6.3; nonalcoholic fatty liver disease; triglycerides.

Figure 1

Characterization of lncRP11-675F6.3 in the human genome and cellular distribution (A) Localization in the human genome and multiz alignment from the UCSC Genome Browser. (B) Alternative lncRP11-675F6.3 transcripts were identified by RLM-RACE. (C) Predicted secondary structure for lncRP11-675F6.3 using the RNA fold web server. (D) Subcellular localization of lncRP11-675F6.3 using RNA FISH and (E) ratio of cytoplasmic/nuclear using qPCR. GAPDH , β-actin and 18S rRNA were used as the internal controls for cytoplasmic RNA, and U6 for nuclear RNA.

Figure 2

LncRP11-675F6.3 regulates triglyceride level in HepG2 cells (A) LncRP11-675F6.3 expression in response to oleic acid at different concentrations for 24 h. (B) Oil Red O staining, (C) TG enzymatic assay, and (D) transmission electron microscopy (red arrows indicate the lipid droplets) showed significantly increased triglyceride contents in HepG2 cells compared with the control following siRNA treatment (10 μM) for 24 h. (E) Oil Red O staining and (F) TG enzymatic assay showed that lentivirus-mediated overexpression of lncRP11-675F6.3 suppressed triglyceride accumulation in HepG2 cells treated with oleic acid (0.4 mM) for 24 h. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. *P<0.05, **P<0.01, *** P<0.001. Lenti-lncRP11-675F6.3 represents the stable lncRNA-overexpressing HepG2 cell lines; Lenti-GFP represents GFP-expressing HepG2 cell lines.

Figure 3

LncRP11-675F6.3 regulates autophagy in HepG2 cells (A) GFP-LC3 fluorescence indicated enhanced autophagy in HepG2 cells after GFP-LC3 plasmid transient transfection for 24 h, followed by lncRP11-675F6.3 siRNA treatment. (B) Knockdown of lncRP11-675F6.3 induced autophagy in HepG2 cells with siRNA treatment (10 μM) for 24 h, represented by the amount of p62 and LC3II/I determined by western blot analysis. (C) HepG2 cells were treated with chloroquine (CQ, 4 μM) for 12 h and (D) 3-methyladenine (3-MA, 1 mM) for 12 h following knockdown of lncRP11-675F6.3. LC3II/I was evaluated by western blot analysis. (E) Lentivirus-mediated overexpression of lncRP11-675F6.3 repressed autophagy, as determined by western blot analysis. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. * P<0.05, **P<0.01.

Figure 4

LncRP11-675F6.3 regulates VLDL-related proteins and links ApoB100 to autophagy (A) Western blot analysis showed that the proteins related to VLDL, ApoB100, ApoE, MTTP, and Apoc3 were significantly reduced in HepG2 cells treated with siRNA (10 μM) for 48 h. (B) Intracellular VLDL was determined in HepG2 cells treated with siRNA (10 μM) for 48 h by ELISA. (C) Lentivirus-mediated overexpression of lncRP11-675F6.3 in HepG2 cells showed elevated levels of ApoB100, ApoE and Apoc3. (D) Intracellular VLDL was quantified by ELISA in lncRP11-675F6.3-overexpressing HepG2 cells. (E) Immunofluorescence microscopy showed the colocalization of LC3 (green) and ApoB100 (red) in HepG2 cells treated with siRNA (10 μM) for 48 h. Blue indicates DAPI. The zoom-in box showed the merged zone. White and pink arrows point to the LC3 dots (green) and merged (yellow), respectively. The bottom histogram indicates the fluorescence intensity of GFP-LC3 (green) and ApoB100 (red). Data were analyzed by one-way ANOVA with Tukey’s post hoc test. * P<0.05, **P<0.01, ***P<0.001.

Figure 5

LncRP11-675F6.3 binds to HK1 (A) RNA pull-down assay showed that one band (~100 kDa) existed only in lncRP11-675F6.3 precipitates. HK1 was identified by LC-MS/MS. The red arrow indicates the additional band. (B) Western blot analysis confirmed the binding of HK1 with lncRP11-675F6.3 following RNA pulldown. (C) RIP assay showed that HK1 interacts with lncRP11-675F6.3 with high affinity. (D) Knockdown or (E) overexpression of lncRP11-675F6.3 resulted in the downregulation or upregulation of HK1 as determined by western blot analysis. (F) Knockdown of HK1 in HepG2 cells led to downregulation of lncRP11-675F6.3 as revealed by RT-qPCR. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. **P<0.01, ****P<0.0001.

Figure 6

HK1 regulates triglyceride level and autophagy and is required for lncRP11-675F6.3 (A) Oil Red O staining showed that triglyceride accumulation was significantly increased in HepG2 cells treated with HK1 siRNA for 48 h. (B) TG enzymatic assay indicated that triglyceride level was elevated with HK1 knockdown. (C) ELISA indicated that the levels of intracellular VLDL were decreased in HepG2 cells treated with HK1 siRNA. (D) Western blot analysis showed that ApoB100, ApoE, ApoC3 and MTTP were reduced with HK1 knockdown. (E) Western blot analysis showed reduced p62 and increased LC3II/I with HK1 knockdown. (F) GFP-LC3 fluorescence was enhanced with HK1 knockdown. (G) Western blot analysis indicated that ApoB100, ApoE, ApoC3 and MTTP were reduced with HK1 knockdown in lenti-lncRP11-675F6.3 HepG2 cells. (H) ELISA indicated that intracellular VLDL levels were reduced with HK1 knockdown in lncRP11-675F6.3-overexpressing HepG2 cells. (I) Western blot analysis showed p62 reduction and LC3II/I elevation with HK1 knockdown in lncRP11-675F6.3-overexpressing HepG2 cells. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. * P<0.05, **P<0.01, ***P <0.001.

Figure 7

lncRP11-675F6.3 and HK1 alleviate hepatic steatosis in vivo (A) LncRP11-675F6.3 and (B) HK1 RNA levels were significantly increased in livers with AAV-mediated lncRP11-675F6.3 and HK1 delivery. (C) HK1 expression was confirmed by western blot analysis. (D) TG and (E) TC levels in livers were detected using enzymatic assay kits and showed a reduction in both TG and TC in mice with AAV-mediated lncRP11-675F6.3 and HK1 delivery. (F) HE staining (upper) and oil red O staining (below) showed alleviated hepatic steatosis in mice with AAV-mediated lncRP11-675F6.3 and HK1 delivery. (G) Western blot analysis showed elevated expressions of ApoB48, ApoE, MTTP and Apoc3, increased p62 and decreased LC3II/I in mice with AAV-mediated lncRP11-675F6.3 and HK1 delivery. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. **P<0.01, ****P<0.0001.