Long Noncoding RNA lncRHPL Regulates Hepatic VLDL Secretion by Modulating hnRNPU/BMAL1/MTTP Axis

As a central metabolic organ, the liver plays an important role in maintaining lipid metabolism. Under normal physiological conditions, hepatic lipid homeostasis is regulated by the interplay between the delivery of lipids to liver and hepatic uptake, synthesis, oxidation, and secretion of lipids (1). Dysregulated hepatic lipid homeostasis causes excessive accumulation of lipids in liver, which results in hepatic steatosis and contributes to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). NAFLD has been associated with hyperlipidemia, obesity, insulin resistance, and hyperglycemia (2). In NAFLD, hyperlipidemia is manifested as increased serum triglyceride and LDL levels and decreased HDL levels, all of which are key risk factors for cardiovascular disease (CVD) (1). Previous studies have demonstrated that CVD is the leading causes of mortality in patient with NAFLD (3,4). The characteristic mixed hyperlipidemia phenotype observed in NAFLD patients is a consequence of increased secretion of triacylglycerol (TAG)-rich VLDL particles (5). VLDL particles are formed in the endoplasmic reticulum, where apolipoprotein (apo)B100 is lipidated in a process catalyzed by the enzyme microsomal triglyceride transfer protein (MTTP) (6–8). Consequently, APOB100 and MTTP are key components in hepatic VLDL secretion and in maintaining hepatic lipid homeostasis. As such, hepatic steatosis, secondary to compromised triglyceride export, is common in patients with genetic defects in the ApoB or Mttp gene (9,10).

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides, which do not have protein-coding potential. lncRNAs are emerging regulators that are involved in gene expression and diverse physiological and pathological processes. Accumulating studies have revealed that lncRNAs participate in lipid metabolism by influencing the expression of key genes, networks, and pathways that are involved in cholesterol and triglyceride biosynthesis, cholesterol transport, lipid uptake, and efflux (11–16). For example, lncLSTR, a liver-specific triglyceride regulator, maintains systemic lipid homeostasis via a TDP-43/FXR/apoC2-dependent pathway in liver (13). Another lncRNA, lncRNA suppressor of hepatic gluconeogenesis and lipogenesis (lncSHGL), which is reduced in human liver with steatosis, has been shown to suppress hepatic gluconeogenesis and lipogenesis by recruiting heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) to enhance calmodulin mRNAs translation in an insulin- and calcium-independent manner (17). However, the roles of lncRNAs involved in hepatic VLDL secretion during NAFLD progression remain elusive. Therefore, to further characterize new lncRNAs that regulate lipid metabolism is of great importance. Particularly, identifying new lncRNAs that regulate hepatic VLDL secretion will shed light on the pathogenesis of NAFLD.

In this study, we identified a novel lncRNA, lncRNA regulator of hyperlipidemia (lncRHPL), as an important regulator participating in hepatic VLDL secretion both in vivo and in vitro, and revealed that lncRHPL interacts with hnRNPU to form a ribonucleoprotein complex. Deficiency of lncRHPL leads to an accelerate protein degradation of hnRNPU without affecting its mRNA level, which results in a suppressed expression of Bmal1 and its downstream genes associated with hepatic VLDL secretion. Our findings illustrate a novel protein-lncRNA regulatory network in hepatic lipid metabolism, revealing a new molecular mechanism underlying the intrahepatic and plasma lipid homeostasis, thereby providing an opportunity for further understanding of the pathogenesis of NAFLD and dyslipidemia.

C57BL/6J mice used in this study were purchased from Beijing Vital River Laboratory Animal Technology (Beijing, China). The mice were housed in a pathogen-free barrier facility of the Nanjing Medical University in 12-h light/dark cycles at 25°C with free access to food and water. All animal protocols were approved by the institutional animal care and use committee of Nanjing Medical University.

Eight-week-old wild-type male C57BL/6J mice (n = 6) were maintained on a normal diet (ND) or high-fat diet (HFD) (60% kcal from fat) (D12492; Research Diets, New Brunswick, NJ). After 23 weeks of HFD administration, mice were sacrificed under 4-h fasting condition and livers were collected for high-throughput RNA sequencing. We constructed sequencing libraries from the fragmented cDNA using NEBNext Ultra DNA Library Prep Kit according to the manufacturer’s instructions (E7370; New England Biolabs, Ipswich, MA). Paired-end 150-bp sequencing was performed on HiSeq X Ten by Annoroad Gene Technology Company (China). We used a cutoff of normalized array values (fold change >2 or <0.5) to enrich for transcripts present in ND and HFD. RNA-sequencing (RNA-seq) data of this study have been deposited at the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) (GSE157482).

For identification of the full-length sequence of lncRHPL, 5′ and 3′ rapid amplification of cDNA ends (RACE) experiments were performed with the GeneRacer Kit (Invitrogen) according to the manufacturer’s protocol. The gene-specific primers used for RACE analysis are listed in Supplementary Table 4.

Hepa1-6 cells were purchased from ATCC and maintained in high-glucose DMEM (Life Technologies) containing 10% FBS (Gibco) and 1% penicillin-streptomycin. Mouse primary hepatocytes were isolated from 6- to 8-week-old C57BL/6J mice as previously described (18). Primary hepatocytes (2 × 10⁵ cells) were cultured in low-glucose DMEM supplemented with 10% FBS (Gibco) at 37°C and 5% CO₂. For knockdown experiments, primary hepatocytes at ∼70% confluence were transfected with negative control Smart Silencer (ssNC) or lncRHPL Smart Silencer (sslncRHPL) (100 nmol/L) (Ri-boBio, Guangzhou, China) by Lipofectamine 2000 (Smart Silencer: Lipofectamine 2000 = 1: 0.5) after 4 h isolation. sslncRHPL was a mixture of three siRNAs and three antisense oligo nucleotides. The target sequences of siRNAs are listed in Supplementary Table 4. Cells and supernatant were collected at 48 h posttransfection.

Adenoviruses, of either scrambled (Ad-scramle) or shRNA for mouse lncRHPL (Ad-shlncRHPL), were constructed with use of the Ad-Track Adenoviral Vector System (StrataGene) and purified as previously described (19). Adenoviruses of shRNA sequence specifically targeted for lncRHPL and MTTP (Ad-shMTTP) are listed in Supplementary Table 4. C57BL/6J mice 6–8 weeks old were injected with 2.5 × 10¹¹ recombinant adenoviral particles of Ad-shlncRHPL, Ad-shMTTP, or Ad-Scramble through tail veins. After injection, mice were fed an ND or HFD for 7 days and the mice were sacrificed after a 16-h fast. Primary mouse hepatocytes were isolated from C57BL/6J mice and treated with adenovirus (6 × 10⁹ particles/well) for 6 h. Cells and supernatant were then collected at 48 h postinfection.

RNA isolation, cDNA synthesis, and quantitative PCR were performed as previously described (19). The sequences of the specific primers used in this study are listed in Supplementary Table 4. Western blot analysis was performed as previously described (19). The antibodies used in this study are listed in Supplementary Table 5. Protein signals were detected by incubation with horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch), followed by enhanced chemiluminescent detection reagent (Pierce).

RNA pull-down and mass spectrometry assays were performed via a method previously described (20). Briefly, we prepared lncRHPL sense or antisense RNA using in vitro transcription of appropriately linearized plasmid templates (cat. no. AM1344; Invitrogen). Then biotin-labeled RNA were synthesized and used for RNA pull-down assay according to the manufacturer’s instructions (cat. no. 20164, Magnetic RNA-Protein Pull-Down Kit; Thermo Fisher Scientific). RNA-associated proteins were eluted, followed by SDS-PAGE and silver staining, and then subjected to liquid chromatography–mass spectrometry analysis.

For RNA immunoprecipitation (RIP) experiments, nuclear lysates were isolated from primary hepatocytes and treated with 300 units/mL RNase inhibitor, followed by incubation with mouse anti-hnRNPU antibody or mouse IgG for 3 h at 4°C with continuous rotation. Antibody/protein/RNA complexes were immunoprecipitated with protein A/G beads (B23202; Bimake.com). RNA associated with hnRNPU were extracted with Trizol and analyzed with quantitative RT-PCR (RT-qPCR) as previously described (21).

Fresh liver tissue (∼100 mg) was homogenized with use of a glass Dounce homogenizer on ice with 1 mL extraction buffer containing 20 mmol/L HEPES, 1 mmol/L EDTA, 250 mmol/L sucrose, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 2 mmol/L dithiothreitol, 100 μmol/L NaVO₄, 100 μmol/L phenylmethylsulfonyl fluoride (PMSF), and complete protease inhibitor cocktail (11697498001; Roche). Separation of nuclear and cytosolic RNA fractions was performed as published previously (22).

Liver lipids were extracted from 100–200 mg frozen liver samples with the method of Folch et al. (23). TAG and total cholesterol (TC) levels were measured with a TAG assay kit and TC assay kit (Wako, Richmond, VA) and normalized to sample weight as previously described (24). Plasma TAG, free fatty acid, TC, LDL, and HDL levels were measured with an enzymatic kit (Wako). Intracellular TAG content in cultured primary hepatocytes was measured with a TAG enzymatic assay kit (Applygen Technologies, Beijing, China) and normalized to total protein amount.

For in vivo VLDL secretion in mice, 6- to 8-week-old C57BL/6J male mice were infected with 2.5 × 10¹¹ recombinant adenovirus particles of Ad-Scramble or Ad-shlncRHPL through tail vein injection for 7 days. After 8 h fasting, mouse were injected with the lipase inhibitor tyloxapol (Triton WR-1339, 500 mg/kg i.v.; Sigma-Aldrich) and 20 mL blood was collected from the retro-orbital plexus in heparinized tubes at the indicate time point. The collected blood samples were used for TAG measurements, and the VLDL-TAG production rate was calculated from the slope of the plasma TAG versus time curve as previously described (24). For in vitro VLDL secretion experiments, after 4-h attachment, primary mouse hepatocytes isolated from 6- to 8-week-old C57BL/6J mice were transfected with ssNC or sslncRHPL for 6 h. After that, those primary hepatocytes were incubated for 6 h with DMEM containing 0.4 mmol/L oleic acid/0.5% fatty acid–free BSA. Cells were then washed with PBS and incubated for a further 16 h with serum-free and phenol red–free DMEM; then the cells and medium were collected and assayed for TAG with an enzymatic TAG assay kit (Wako) and mouse VLDL ELISA kit (Jiancheng Bioengineering Research Institute, Jiangsu, China), respectively, as previously described (19).

Protein stability of hnRNPU in primary hepatocytes or Hepa1-6 cells was performed with a cycloheximide (CHX)-based protein chase experiment as previously described (25). For in vivo ubiquitination assay of hnRNPU, primary hepatocytes were transfected with sslncRHPL or infected with recombinant adenovirus particles expressing full-length lncRHPL (Ad-lncRHPL) for 12 h and then transfected with HA-ubiquitin (2 mg). After 18 h, cells were treated with 10 μmol/L MG132 for 4 h, harvested, and lysed in immunoprecipitation (IP) buffer. Total cell lysate (1 mg) was used for IP with 50 µL protein A/G beads and anti-hnRNPU antibody. The immunoprecipitates or total cell lysates were analyzed by Western blot with indicated antibodies.

RNA-fluorescence in situ hybridization (RNA-FISH) for lncRHPL was performed on primary mouse hepatocytes. Cell slides fixed with 4% paraformaldehyde were treated with several buffers in RNA-FISH Kit (Ri-boBio, Guangzhou, China). After denaturing at 37°C for 5 min, probe mixture was hybridized in a cell slide overnight in darkness at 37 °C. CY3-labeled nucleic acid probes were targeting lncRHPL. After washing, the signal was observed under confocal microscope. Detailed procedures were followed according to the manufacturer’s protocol.

After transfection with sslncRHPL or ssNC for 48 h, primary hepatocytes were collected for the detection of cell apoptosis. The cell apoptosis of primary hepatocytes was measured with use of Annexin V-FITC/PI Apoptosis Detection Kit (MedChemExpress, Shanghai, China), and the apoptotic rate was evaluated with FACS. TUNEL analysis was performed with TUNEL Apoptosis Detection Kit (MedChemExpress) according to the manufacturer protocol (26).

Statistical analysis was determined with two-tailed unpaired Student t test for single comparisons and ANOVA followed by Bonferroni-Dunn for multiple comparisons. All statistical analyses were performed with GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA). Data are expressed as mean ± SEM. Differences were considered significant when P was <0.05 (*P < 0.05; **P < 0.01; ***P < 0.001).

RNA-seq data were deposited in NCBI’s GEO with accession no. GSE157482. All other data are available from the corresponding author on reasonable request.

For comprehensive investigation of the lncRNAs expression profile in the liver of HFD and ND mice, specifically those involved in hepatic lipid metabolism, the Illumina HiSeq X Ten platform was used to obtain a comprehensive view of lncRNAs in liver. HFD for 23 weeks resulted in significantly higher liver fat accumulation compared with that of mice fed normal chow. HFD-fed mice showed a significant increase of hepatic TAG and TC levels both in serum (Supplementary Fig. 1A) and liver (Supplementary Fig. 1B) compared with ND. An average of 84.1 million high-quality paired-end sequencing reads was obtained for each sample (range 74.5–93.7 million). Thus, an average of 93.6% of the total reads (range 91.29–95.91%) could be mapped to the mouse genome. As a result, a total of 886 genes (614 annotated lncRNAs and 272 unknown lncRNAs) with sufficiently high expression levels were identified (summarized reads information shown in Supplementary Table 1). Specifically, we found that there were 77 lncRNAs with induction folds of >2 or <0.5 in response to ND (Supplementary Table 2). We next performed a cluster analysis by setting another three criteria to further screen these lncRNAs: annotation as authentic lncRNAs in Ensembl, relatively high abundance in the mouse liver, and relatively large fold change. Fifteen uncharacterized lncRNAs were then identified as dysregulated candidates in the livers from HFD mice (Fig. 1A). For confirmation of these results, an independent RT-qPCR assay was performed. Consistent with our RNA-seq results, the hepatic expression of Gm26584 was significantly reduced (Fig. 1B) while Gm26714 and Gm9968 were induced by HFD (Supplementary Fig. 1C).

We named Gm26584 as lncRHPL (lncRNA regulator of hyperlipidemia) based on its function characterized in this report. RACE assay revealed a full length of 3,923 nt for lncRHPL (Supplementary Fig. 2A), slightly longer than that annotated in Ensembl (Supplementary Fig. 2B). Coding Potential Calculator (CPC) (http://cpc.gao-lab.org/) (27) analysis indicated a lack of coding potential for this transcript (Supplementary Fig. 2C). Furthermore, the lncRHPL expression vector failed to produce any protein when transfected into 293T cells (Fig. 1C). RT-qPCR analysis indicated that lncRHPL was specifically enriched in the mouse liver (Fig. 1D) and mainly distributed in the nucleus fraction of liver (Fig. 1E). The result of RNA-FISH further confirmed that lncRHPL was localized in the nucleus of primary hepatocytes and Hepa1-6 cells (Fig. 1F and Supplementary Fig. 2D). We further validated that lncRHPL was downregulated in primary hepatocytes with oleic acid treatment (Supplementary Fig. 2E).

These data indicate that lncRHPL is a liver-enriched and metabolic stimuli–regulated lncRNA, suggesting that it may serve as a potential regulator of hepatic lipid metabolism.

To directly address the physiological role of lncRHPL in hepatic lipid metabolism, we specifically knocked down lncRHPL in C57BL/6J mice liver using adenoviral shRNA. The RT-qPCR results indicated that tail vein injection of Ad-lncRHPL shRNA effectively silenced the expression level of lncRHPL by >50% in mouse liver compared with Ad-Scramble in both ND and HFD conditions (Fig. 2A). lncRHPL knockdown in vivo did not markedly affect body weight but increased liver weight correlated with increased gonadal fat (Table 1). Interestingly, the knockdown of lncRHPL significantly increased serum level of TAG, TC (Fig. 2B), nonesterified fatty acid (NEFA), and LDL cholesterol but decreases HDL cholesterol as compared with that of control mice under both ND and HFD conditions (Supplementary Fig. 3). In contrast, Oil Red O and hematoxylin-eosin staining revealed a dramatic reduction in lipid droplets accumulation in the liver of lncRHPL knockdown mice (Fig. 2C). These results were further confirmed by reduced hepatic TAG, TC, and NEFA levels in lncRHPL knockdown mice (Fig. 2D). These data suggest that liver-specific knockdown of lncRHPL induces hyperlipidemia accompanied by lower hepatic lipid contents in vivo.

It is therefore interesting to investigate the cause of hyperlipidemia in lncRHPL knockdown mice. The RT-qPCR results indicated that liver-specific knockdown of lncRHPL did not affect expression level of genes involved in fatty acid synthesis, β-oxidation, or TAG synthesis or hydrolysis in liver of these mice (Supplementary Fig. 4A). The protein level of proprotein convertase subtilisin/kexin type 9 (PCSK9) and lipoprotein lipase (LPL), key regulators of plasma lipid metabolism, remained unchanged in plasma, suggesting that lncRHPL knockdown did not influence lipid clearance (Supplementary Fig. 4B). Of note, in lncRHPL knockdown mice, the tyloxapol-induced TAG increment was ∼30% and 25% higher than those of control mice under ND and HFD condition, respectively (Fig. 3A). Consistently, plasma APOB100 and APOB48 levels were significantly increased in lncRHPL knockdown mice, but APOE, APOA-I, and ALBUMIN were not influenced (Fig. 3B). Unlike plasma APOB level, hepatic expression of APOB did not show any change in the liver of lncRHPL knockdown mice, whereas the mRNA and protein level of Mttp were induced approximately two- to threefold in the liver of lncRHPL knockdown mice, respectively (Fig. 3C and D). These results revealed that the manifestation of hyperlipidemia observed in mice with liver-specific knockdown of lncRHPL was due to enhanced hepatic TAG-VLDL secretion.

For confirmation that the increased VLDL-TAG in the lncRHPL knockdown mice was cell autonomous and not dependent on factors extrinsic to the liver such as free fatty acids, we knocked down lncRHPL with sslncRHPL in primary hepatocytes to measure intracellular and secreted TAG (Fig. 4A). As shown in Fig. 4B, the intracellular levels of TAG were decreased by ∼37% in hepatocytes following lncRHPL knockdown, whereas the secreted VLDL-TAG in medium was ∼1.48-fold higher than that of control cells with treatment of 0.4 mmol/L oleic acid (60.42 ± 9.73 vs. 24.29 ± 8.28 nmol/mg protein, respectively) (Fig. 4C). Accordingly, knockdown lncRHPL increases the content of APOB100 in the culture medium of primary hepatocytes (Fig. 4D). In addition, the knockdown of lncRHPL increased mRNA and protein levels of MTTP in primary hepatocytes, although apoB expression was unchanged (Fig. 4E and F). Consistent with the observations in mice liver, the mRNA levels of genes involved in lipogenesis, lipolysis, and fatty acid β-oxidation such as Srebp-1c, Acc1, Fasn, and Atgl remained unchanged in lncRHPL-knockdown hepatocytes. These data suggest that the inhibition of lncRHPL resulted directly in increased VLDL-TAG secretion from hepatocytes rather than TAG synthesis or hydrolysis (Supplementary Fig. 5A). Furthermore, the results of Annexin V-FITC/PI Apoptosis Detection Kit analysis and TUNEL staining showed that the percentage of apoptotic cells in primary hepatocytes was ∼11% after 48-h culture in vitro. There was no difference in cell mortality between lncRHPL knockdown primary hepatocytes and control cells (Supplementary Fig. 5B and C).

To determine the role of MTTP in lncRHPL-regulated VLDL secretion, we further knockdown lncRHPL in primary hepatocytes isolated from mice injected with Ad-shMTTP. We found that knockdown lncRHPL–induced increases in the mRNA level of Mttp were significantly abolished by depletion of MTTP in primary hepatocytes, and no changes in the mRNA level of Apob (Supplementary Fig. 6A). Importantly, the decreased intracellular TAG levels (Supplementary Fig. 6B) and increased VLDL secretion (Supplementary Fig. 6C) as well as apoB content in culture medium (Supplementary Fig. 6D) and protein level of MTTP (Supplementary Fig. 6E) were also restored by depletion of MTTP in lncRHPL knockdown primary hepatocytes. These results indicate that the depletion of Mttp significantly attenuated the effects of lncRHPL knockdown on VLDL secretion and hepatic lipid accumulation in primary hepatocytes.

lncRNAs generally coordinate with protein partners to exert their specific function. To uncover the molecular mechanism of lncRHPL function, we sought to identify protein partners that directly bind to lncRHPL using an RNA pull-down assay by incubating full-length lncRHPL or its antisense (negative control) RNA in lysates of primary hepatocytes. The proteins retrieved by the RNA pull-down in lncRHPL sense line were subjected to SDS-PAGE and visualized with sliver staining followed by mass spectrometry analysis (Supplementary Fig. 7A). A total of 151 candidates were identified, including 16 nucleus proteins and 47 cytosolic proteins (Supplementary Table 3). The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the interacting proteins of lncRHPL were mainly involved in metabolic pathways (Supplementary Fig. 7B).

Among them, we noticed that the heterogenous nuclear ribonucleoprotein (hnRNP) family members were enriched (Supplementary Fig. 7C). Interestingly, we found multiple putative hnRNPU binding motifs within lncRHPL with RBPmap (28) using default parameters with high stringency levels (Supplementary Fig. 7D), suggesting a possible interaction between hnRNPU and lncRHPL. RIP assay was subsequently performed with nuclear extracts from primary hepatocytes using hnRNPU antibodies, and the results demonstrated that lncRHPL was strongly associated with the hnRNPU (Fig. 5A). In parallel with RIP experiments, RNA pull-down assay further confirmed lncRHPL specifically interacted with hnRNPU but not hnRNPA2B1 or hnRNPL (Fig. 5B). These findings suggested that lncRHPL and hnRNPU form a ribonucleoprotein complex.

Interestingly, liver-specific knockdown of lncRHPL reduced hnRNPU protein levels, especially in nucleus fractions (Fig. 5C), without affecting its mRNA level (Supplementary Fig. 8A). More importantly, the half-life of endogenous hnRNPU protein was markedly shortened in lncRHPL knockdown hepatocytes treated with CHX (Fig. 5D). Concomitantly, the reduced protein level of hnRNPU was restored by MG132, a proteasome inhibitor (Fig. 5E). These results indicated that lncRHPL may regulate hnRNPU protein level at the posttranslational level.

Furthermore, the ubiquitination and co-IP assays revealed that MG132 treatment and lncRHPL knockdown led to a robust increase in endogenous hnRNPU ubiquitination in primary hepatocytes (Fig. 5F). Conversely, hnRNPU ubiquitination level was markedly reduced in lncRHPL-overexpressed hepatocytes (Fig. 5G).

Taken together, these results support that lncRHPL physically interacts with hnRNPU and regulates its protein stability by influencing the proteasome-mediated ubiquitination of hnRNPU.

We next sought to explore the downstream targets of lncRHPL/hnRNPU ribonucleoprotein complex in lipid metabolism. Recent hnRNPU chromatin IP with massively parallel DNA sequencing (ChIP-seq) identified 5,285 peaks in AML12 hepatocytes. Of these peaks, at least four were located within Bmal1 gene body (29). In addition, using the electrophoretic mobility shift assay also confirmed the binding of 3′-flanking region of Bmal1 gene promoter by SAF-A/hnRNPU protein complex (30). Together, the results of these experiments supported the transcriptional regulation of hnRNPU on Bmal1. Global and hepatocytes-specific ablation of BMAL1 induces hyperlipidemia and enhances atherosclerosis through upregulation of MTTP and downregulation of SHP (31). It is therefore interesting to confirm the role of hnRNPU in the regulation of Bmal1 in hepatocytes. As expected, knockdown of hnRNPU significantly decreased mRNA and protein levels of BMAL1 in primary hepatocytes (Supplementary Fig. 8B and 8C). In agreement, we observed significantly reduced BMAL1 expression accompanied by decreased protein levels of hnRNPU in the liver of lncRHPL knockdown mice (Fig. 6A and B). Furthermore, lncRHPL deficiency reduced liver mRNA and protein levels of ABCG5, SHP, and GATA4, correlating with the changes in BMAL1 levels (Fig. 6A and B). These results indicated that lncRHPL knockdown in liver exerts its functional influence at least partially by repressing the transcriptional activity of hnRNPU/BMAL1 axis.

In vitro studies further confirmed that manipulation of lncRHPL did not affect hnRNPU mRNA levels (Supplementary Fig. 9A), whereas the protein levels of hnRNPU were significantly decreased on lncRHPL knockdown and were restored in response to lncRHPL overexpression in primary hepatocytes (Supplementary Fig. 9B). Accordingly, the mRNA levels of Bmal1, Shp, and Gata4 were significantly reduced, and Mttp was increased in lncRHPL knockdown hepatocytes. Moreover, lncRHPL overexpression reversed their levels (Supplementary Fig. 9A and 9B). In agreement with this, silencing of lncRHPL in primary hepatocytes resulted in a significant induction of VLDL secretion (Fig. 6C), associated with reduced intracellular TAG content (Fig. 6D). Importantly, the secreted VLDL and intracellular TAG levels in these lncRHPL knockdown primary hepatocytes were recovered with exogenous expression of lncRHPL following Ad-lncRHPL infection (Fig. 6C and D).

We overexpressed Bma1 to further determine the effect of Bmal1 on the enhanced VLDL secretion in lncRHPL knockdown primary hepatocytes. Again, mRNA and protein levels of Bmal1, Shp, and Gata4 were decreased and Mttp were increased in lncRHPL knockdown hepatocytes, whereas restoring Bmal1 reverted Shp, Gata4, and Mttp mRNA and protein expression (Fig. 7A and B). Importantly, the increased VLDL secretion and decreased intracellular TAG levels were also restored by overexpression of BMAL1 in lncRHPL knockdown primary hepatocytes (Fig. 7C and D). Collectively, these results demonstrate that the deficiency of lncRHPL promotes VLDL secretion by attenuating the transcriptional activity of hnRNPU/BMAL1/MTTP axis.

The liver is an essential metabolic organ, and metabolic function of liver is controlled by nutritional and hormonal signaling (32). NAFLD is the most common liver disease due to overnutrition and obesity. The presence of hyperlipidemia was reported in 20%–80% of cases of NAFLD (33). The increased serum TAG, cholesterol, and LDL are known risk factors for CVD. Therefore, identifying new regulatory factors participating in hepatic VLDL secretion is crucial for understanding the pathogenesis of NAFLD and dyslipidemia.

Recent studies show an emerging role of lncRNAs in hepatic lipid metabolism (34). lncRNAs are expressed in a more tissue-specific manner than protein-coding genes (35). In this study, lncRHPL was identified as a novel lncRNA specifically enriched in liver and downregulated in HFD-fed mice as well as in oleic acid–treated primary hepatocytes. Gain- and loss-of-function studies revealed that lncRHPL suppressed hepatic VLDL secretion without affecting lipogenesis and fatty acid oxidation. These findings strongly suggest that expression of lncRHPL is regulated by nutritional signaling to meet the requirements for metabolic homeostasis and participates in the pathogenesis of NAFLD and dyslipidemia.

Compared with mRNAs, a great proportion of lncRNAs are localized in the nucleus. Depending on nucleus localization, lncRNAs often function by partnering with RNA binding proteins (RBPs) to form ribonucleoprotein complexes, which is critical for lncRNAs to exert their function as gene regulators (36,37). In this study we found that lncRHPL was mainly distributed in nucleus and directly interacted with hnRNPU, an RNA binding protein participating in mRNA stabilization and transcriptional or translational regulation of downstream targets (38) to form ribonucleoprotein complex. Interestingly, depletion of lncRHPL decreased the hnRNPU protein levels without affecting its transcription. Through in vivo and in vitro studies, lncRHPL-regulated hnRNPU protein levels most likely resulted from posttranslational regulation because knockdown of lncRHPL promoted proteasome-mediated ubiquitination and degradation of hnRNPU, whereas overexpression of lncRHPL stabilized the protein levels of hnRNPU. Therefore, the interaction between lncRHPL and hnRNPU is essential and responsible for protein stability and nuclear retention of hnRNPU, just as lncRNA DINO regulates p53 stability (39).

Recently, increasing evidence has indicated a tight connection between circadian rhythms and energy metabolism. Synchronizing of a physiological circadian rhythm is crucial for metabolic health and an important strategy for treatment of obesity and NAFLD (40–42). BMAL1 was initially identified as an essential component of the molecular circadian clock in mammals (43). Beside the role in circadian regulation, an influence of BAML1 on lipid metabolism has been reported (31,44,45). Depletion of Bmal1 induces hyperlipidemia by enhancing VLDL assembly and secretion with upregulation of Mttp and downregulation of Shp. Meanwhile, ablation of Bmal1 inhibits cholesterol secretion to bile acid with downregulation of Abcg5/8 and Gata4 (31). In agreement, we found that knockdown of lncRHPL significantly decreased mRNA and protein levels of BMAL1, accompanied by induced VLDL secretion in vivo and in vitro. Consistent with previous observation, downregulation of Baml1 resulted in reduced Shp expression and increased Mttp expression. The expression patterns of Gata4, Abcg5, and Abcg8, downstream targets of BMAL1, were also regulated accordingly. Importantly, restoring Bmal1 reversed the induced VLDL secretion and expression of downstream targets in lncRHPL knockdown primary hepatocytes. Thus, our findings suggest that BMAL1 is the downstream target of lncRHPL in the regulation of hepatic VLDL secretion.

As a member of the hnRNPU subfamily, hnRNPU mainly localized in the nucleus, where it is able to bind pre-mRNA through RGG box in the glycine-rich COOH terminus (46). Previous studies showed transcriptional regulation of hnRNPU on Bmal1 (29,30). Indeed, knockdown of hnRNPU significantly impaired Bmal1 expression in primary hepatocytes. The critical role of lncRHPL in mediating the transcriptional function of hnRNPU/BMAL1/MTTP axis was illustrated by shRNA knockdown studies. We found that shRNA-mediated depletion of lncRHPL markedly reduced hnRNPU protein level and BMAL1 expression, which in turn impaired the transcriptional regulation effects of BMAL1 on gene expression of Mttp, Shp, Gata4, Abcg5, and Abcg8 in hepatocytes. In contrast, restoring lncRHPL recovers the protein level of hnRNPU and reverted the expression level of BMAL1 as well as downstream targets of BMAL1. These studies raise the possibility that lncRHPL may engage the BMAL1 expression through its association with hnRNPU. Recently, hnRNPU has been shown to facilitate the assembly of brown fat lncRNA 1 (Blnc1) with transcription factors EBF2 and ZBTB7B to form ribonucleoprotein transcriptional complexes to stimulate the thermogenic gene expression in adipose tissue (47–49). This indicates that lncRHPL recruiting hnRNPU to regulate the transcription efficiency of other mRNAs besides Bmal1 is also possible. Further identification of the targets of the lncRHPL/hnRNPU axis is of great interest. Further work will be needed to clarify whether lncRHPL may also regulate the hepatic lipid metabolism via other mechanisms beyond hnRNPU/BMAL1/MTTP pathway.

Taken together, the results of our study provide the first evidence that lncRHPL is involved in the regulation of hepatic VLDL-TAG secretion by modulating hnRNPU/BMAL1/MTTP axis. Specifically, the expression levels of lncRHPL were strongly downregulated on HFD treatment, indicating that lncRHPL plays a major role in the development of HFD-induced hyperlipidemia. The reduction of lncRHPL leads to enhanced protein degradation of hnRNPU, which further reduces BMAL1 expression and affects the transcription of its downstream target genes, thus increasing the VLDL secretion and development of hyperlipidemia (Fig. 7E). VLDL secretion is a hepatic-specific defense against the excessive liver TAG accumulation that occurs in nutritional overload or metabolism. Decreased levels of lncRHPL in liver may therefore represent a potential risk factor for CVD. These results highlight a novel molecular mechanism of hepatic lipid secretion regulated by lncRNA, thereby providing an opportunity for further understanding of the pathogenesis of the development of NAFLD and dyslipidemia.

Acknowledgments. The authors are grateful to Dr. Chang Liu (China Pharmaceutical University) for kindly providing adenoviruses Ad-Bmal1and Ad-GFP, Dr. Zheng Chen (Harbin Institute of Technology) for kindly providing adenoviruses Ad-shlncRHPL and Ad-Scramble, Dr. Tong-Jin Zhao (Fudan University) for critical reading for the manuscript, and members of the J.Z.L. laboratory for technical assistance and helpful discussion.

Funding. This work was supported by grants from the National Key R&D Program of China (grant 2018YFA0506904 to J.Z.L.), the National Natural Science Foundation of China (grants 32130050, 31771307, 91957115, and 81471079 to J.Z.L. and 81770859 to Q.W.), and the Jiangsu Provincial Innovation Team Program Foundation.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. X.S. and Y.Z. designed the experiments. X.S., Y.Z., X.J., B.L., Y.W., Y.H., X.Z., J.Y., R.Z., and D.Q. performed the experiments. X.S., Y.Z., Q.W., and J.Z.L. analyzed data. H.Z., Q.W., and J.Z.L. contributed to discussion and wrote the manuscript. All authors approved the final manuscript. Q.W. and J.Z.L. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.