Lnc027912 Reduces Lipid Accumulation Through Akt/MTOR/SREBP1C Axis in Hepatic Cells


 Background: Various metabolism diseases are closely related to lipid metabolism disorder, but long noncoding-RNAs (lncRNA) involve in regulating function of lipid was limited elucidated. Previous our work have found that lnc027912 involve in cholesterol metabolism. Here, we further explore the role of lipid metabolism-associated lncRNA-lnc027912 in oleic acid- (OA) and palmitic acid (PA)-induced hepatic cells. Methods: The overexpression of lnc027912 cell model was constructed by using virus particles transfection, and the level of lnc027912 in AML12 cells were detected by RT-qPCR. High fat cell model was established by treating AML12 cells with OA and PA, and the level of lipid drops was detected by Oil red O staining and triglyceride analyze Kit. The lipid metabolism related-genes, such as SREBP1C, FAS, PPARγ, MTTP, ApoE and ApoC3 level, was detected using RT-qPCR and Western blot. The role of SREBP1C in lipid metabolism was further analyzed using double luciferase reporter gene assay and Immunofluorescence. The Akt/mTOR signal pathway related genes was detected by Western blot. Results: We found that TG level was inhibited in overexpression of lnc027912 cell. Upregulated lnc027912 of AML12 cells treated with OA and PA showed a significant decrease in lipid accumulation and TG levels. Furthermore, overexpression of lnc027912, the lipid biosynthesis genes of SREBP1C, FAS and PPARγ was significantly decreased and a significant increase in expression of MTTP and ApoE. Interestingly, lnc027912 inhibited Akt/mTOR signaling axis and decreased SREBP1C transit into nucleus and the promoter activity of SREBP1C and regulated expression of its targets. Conclusions: Our study revealed a new insights into the molecular function of lnc027912 in lipid metabolism by Akt/mTOR/SREBP1C signaling axis and highlights the potential of lnc027912 as a new therapeutic target for lipid disorder diseases (such as, NAFLD).

Long nocoding-RNAs (lncRNAs) are non-coding transcripts more than 200 nucleotides in length with no protein-coding capacity [8]. As the function of modulated by lncRNAs continues to grow, it has become more important that they are key regulators of a wide range of biological processes, in particular lipids homeostasis. A large number of studies have indicated that lncRNAs involved in lipid metabolism various processes, such as synthesis, transport and secretion [9]. LncHULC (highly upregulated in liver cancer), is the rst identi ed liver-speci c lncRNA, which was acted on PPARA to regulate the transcriptional factor of long-chain acy-CoA synthetase 1(ACSL1) involved in cellular fatty acid metabolism [10]. Moreover, lncRNAs, including MALAT-1 [10], HOTAIR [11], H19 [12], HR1 [13], LeXis [14] and Gm16551 [15] were found to be related with lipid metabolic disorder. Recent studies have demonstrated that lncH19 suppressed lipid metabolism which may act as therapeutic target of atherosclerosis [16]. These results suggested that lncRNAs played a vital role in remaining lipid homeostasis. Previous our work have found that lncRNA, NONMUG027912 (after abbreviate it to lnc027912), is implicated in the process of cholesterol biosynthesis [17]. Depending on cholesterol levels, metabolic pathways are activated resulting in fatty acid or cholesterol synthesis. However, the function of lnc027912 in regulating lipid metabolism have not be investigated.
In this study, we explore the role of lnc027912 in regulating lipid metabolism. Up-regulated lnc027912 signi cantly reduced lipid accumulation in AML12 cells. Mechanistically, lnc027912 negatively modulated the expression of SPEBP-1C, PPARγ and FAS, meanwhile, positively regulated the expression of MTTP and ApoE, thereby reducing lipid accumulation induced by oleic acid (OA) and palmitic acid (PA). Furthermore, our results further con rmed that lnc027912 inhibited the SREBP1C promoter activity and reduced SREBP1C transit into nucleus through restraining Akt/mTOR signaling axis. Together, our study provides new insights into the molecular function of lnc027912 in lipid metabolism and highlights the potential of lnc027912 as a new therapeutic target for lipid disorder diseases (such as, NAFLD).

Construction of overexpression cell line
After mixing the plasmids (pCDH-GFP-lnc027912 and pCDH-GFP) and pPACKH1-plasmid Mix, they were transfected into HEK-293T cells, the culture medium was collected, and the virus particles were precipitated with PEG-it. Puromycin was added to AML12 cells to screen out the cell line stably expressing lnc027912, and the concentration of puromycin in AML12-GFP or AML12-lnc027912 cells was 0.8 μg/ml. Quantitative RT-PCR Total RNA was isolated from cells using the TRIzol reagent (Invitrogen). Remove genomic DNA with 4×gDNA wiper Mix, and then add 5×HiScript qRT SuperMix (Vazyme;) to synthesize cDNA. Quantitative RT-PCR was performed on the CFX96TM real-time PCR system (Applied Biosystems). The PCR reagent mix was obtained from Vazyme. The data was analyzed using the ΔΔCT method. GAPDH expression was an control to normalize the data. All primers sequences used are listed in the Table S1.

Western blot analysis
Cellular protein was extracted with cell lysis buffer and measured using an BCA Protein Assay Kit (Beyotime). Then, the proteins were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblotting was performed using select primary antibodies, followed by probing with the recommended secondary antibodies, GAPDH was used as a control. Finally, the signals were detected using a ChemiDoc TM Imaging System (Bio-Rad).The antibodies used are listed in the Table   S2.

Luciferase reporter assays
The SREBP1C promoter containing the far-upstream element sequence (about-2000-0 bp) was subcloned and inserted into the pGL3-basic vector (Promega, Madison, WI, USA). The recombinant plasmid (pGL3-SREBP1C-promoter vector) was obtained by MiaoLingBio. HEK-293T cells were seeded onto 48well plate and transfected with pCDH-lnc027912, pGL3-SREBP1C, and phRL-TK vectors. After incubation for 36 h, the cells were treated with OA (200 μM) and PA (200 μM) for 24 h. Then, the cells were lysed in 1×passive lysis. A Dual-luciferase reporter assay system (Promega) was performed according the manufacturer's protocols, with Renilla luciferase serving as a transfection control.

Statistical analysis
All experiments were repeated at least three times, and the results were presented as mean ± SD.
Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA). The P values were calculated using a one-way ANOVA. A P value of <0.05 was considered statistically signi cant.

Results
Up-regulated lnc027912 reduced the total triglyceride level in hepatic cells Our previous studies have demonstrated that lnc027912 played a key role in regulating of cholesterol homeostasis [17]. Cholesterol level is closely related to lipid metabolism, inducing lipid biosynthesis, transport or secretion, etc [19]. To further explore the function of lnc027912 in regulating lipid metabolism, we used virus particles (obtaining pCDH-lnc027912-GFP and pCDH-GFP vectors) to infect AML12 cells, as shown in Fig. 1a, above 95% cells showing green uorescence. Compared with the control group, the CT value of lnc027912 was decreased by 10 (Fig. 1b). These results suggest that overexpression of lnc027912 cell line was successfully constructed. At the same time, the total triglyceride (TG) level were analyzed. As showing in Fig.1c, overexpression of lnc027912, the TG level was signi cantly inhibited in AML12 cells. To further analyze the mechanism of lnc027912 in regulating lipid metabolism, these lipid metabolism related genes (SREBP1C, PPARγ, FAS, MTTP, ApoE, ApoC3) mRNA expression were measured. As showing in Fig.S1, the key lipogenesis genes of SPEBP1C and FAS were obviously decreased, simultaneously, the gene of MTTP was increased in AML12-lnc027912 cells. These results further suggested lnc027912 involved in lipid metabolism.
Lnc027912 could alleviate OA and PA induced lipid accumulation To further demonstrate the effect of lnc027912 on lipid level, cells were treatment with OA and PA, then the total triglyceride (TG) level was assessed by oil red O staining and TG kit. Firstly, oil red O staining was performed to analyses the cellular lipid level. As shown in Fig. 2a, compared with control group, upregulation of lnc027912 in cells resulted in a decrease in oil red O positive lipids. Importantly, overexpression of lnc027912 could signi cantly reduce lipid accumulation induced by OA and PA. Then, the number of lipid drops was measured using image Pro plus 6.0, showing that lipid drops signi cantly reduced in upregulated of lnc027912 groups (Fig. 2b). Furthermore, we used TG kit to measure the TG level, results showing that the TG decreased by 32.7% (OA-treated, Fig. 2c) and 32.3% (PA-treated, Fig.  2d), respectively, than the control group. To further explore the role of lnc027912, silencing lnc027912 was performed in Hepa1-6 cells, results showing that lnc027912 level decreased by 54% (Fig. S1a). While, we noticed that the TG level was no change (Fig. S1b). Together, these results strongly suggested that lnc027912 inhibited lipid accumulation in cells.

Lnc027912 regulate lipid metabolism by inhibiting lipid biosynthesis and improving lipid transport
To determine the mechanism by which lnc027912 in uences lipid level, we analyzed the expression of lipid metabolism related-genes by qPCR and Western Blot. Overexpression of lnc027912, the lipogenesis genes of SREBPC1 and FAS was signi cantly inhibited, concurrently, VLDL metabolism genes of MTTP was induced by RT-qPCR (Fig. S2). Importantly, we found that lnc027912 could inhibit lipid biosynthesis and increase lipid transport, when cells was treated with OA ( Fig.3a, b and Fig.S2). These results suggested that lnc027912 could alleviate OA induced lipid accumulation. Additionally, the lipid level was analyzed under PA treatment. The decreased of SREBPC1 and FAS, and increased of MTTP was found by RT-qPCR (Fig. S2) and Western blot (Fig. 3c, d). These results su ciently con rmed that lnc027912 could inhibit OA-or PA-induced lipid accumulation in hepatic cell. To further con rm the lnc027912 role of lipid metabolism, the FAS level was detected by immuno uorescence (Fig. 3e). We found that the FAS, which the lipid biosynthesis key gene, was signi cantly inhibited in overexpressed of lnc027912 cells. Concurrently, we found that lnc027912 can effectively reduce the elevation of FAS caused by OA or PA (Fig. 3e, f). Together, these ndings suggest that lnc027912 could inhibit lipid accumulation by reducing lipid biosynthesis and inducing lipid transport.
Overexpression of lnc027912 could inhibit SREBP1C activity SREBP1C is a key transcription regulator of fatty acid synthesis, a process known as de novo lipogenesis [20]. Therefore, we reasoned that lnc027912 may involve in regulating of SREBP1C activity to modulate the lipid metabolism. Firstly, to investigate the effect of lnc027912 on promoter activity of SREBP1C, the dual-luciferase reporter assay was performed. As shown in Fig. 4a, the SREBP1C promoter activity was signi cantly inhibited when lnc027912 was upregulated. Interestingly, SREBP1C promoter activity was activated under the treatment with OA and PA, while, overexpression of lnc027912 can signi cantly reduce the activation of SREBP1C promoter induced by OA and PA. These results suggested that lnc027912 may affect lipid deposition by inhibiting SREBP1C activity. Additionally, we also found that the uorescence density of SREBP1C was signi cantly inhibited in unregulated of lnc027912 cells (Fig. 4b and Fig. S3). Furthermore, we found that overexpression of lnc027912 reduced the expression of SREBP1C in nuclei (Fig. 4c), which may reduce the expression of lipid-related genes. To further explore the mechanism of lnc027912 regulation of SREBP1C, the Akt and mTOR levels were detected. As shown in Fig. 4d, the p-mTOR (Ser2448) and p-Akt (Ser473 and Thr308) level were inhibited after overexpression of lnc027912, while, the levels of mTOR and Akt remained unchanged. These results suggested that lnc027912 inhibited the expression lipid synthesis genes and lipid accumulation through Akt/mTOR/SREBP1C signaling axis.
In summary, our ndings investigated that lnc027912 inhibited SREBP1C transferred into the nuclei through reducing phosphorylation of Akt and mTOR, then decreased of the activity of SREBP1C promoter, resulted in decreased of lipid synthesis and increased of the transport of lipid, and alleviated to lipid accumulation (Fig. 5).

Discussion
Excessive lipids in the liver could disrupt lipid synthesis and transport balance, and lipid metabolism can be perturbed [21,22]. Lipid homeostasis disorders not only causes NAFLD, but is also a component of metabolic syndrome with obesity, type 2 diabetes, dyslipidemia, and arteriosclerotic cardiovascular disease [23,24]. Recent studies have suggested that lncRNAs played a key role in regulating lipid metabolism, such as lncHC, lncLSTR, lncHULC and lncMALAT1 [25]. LncRNAs often target several transcription factors that play essential roles in the regulation of lipid metabolism, such as sterol regulatory element binding proteins (SREBPs). Previous our work have found that lnc027912 involved in regulation of cholesterol metabolism via modulating the expression of farnesyl-diphosphate farnesyltransferase 1(Fdft1), which is key enzyme for cholesterol biosynthesis [17]. While, lnc027912 involving in lipid metabolic diseases has not been elucidated yet.
SREBP1C is major transcription factors control fatty acid synthesis and play key role in the process of lipid metabolism. Some works have demonstrated that lncRNAs could affect lipid synthesis via regulating the level of SREBP1C. LncMALAT1 could inhibit PA-induced upregulate of SREBP1C and hepatic lipid accumulation through inhibition of FAS, which is the principle target of SREBP1C [26]. Overexpression of lncHR1 could decrease triglyceride (TG) through inhibiting activation of SREBP1C and FAS in oleic acid (OA)-induced hepatic cells [13]. These results indicated that lncRNAs played an important role in inhibiting lipid accumulation in OA-and PA-treated cells. In our study, we found that lnc027912 could abrogates PA-or OA-induced lipid accumulation through a mechanism of action that increase SREBP1C protein. Further the mechanism exploration, we found that lncRNA027912 could inhibit promoter activity of SREBP1C. Additionally, studied have revealed that the SREBP1C transited into cell nuclei and involved in regulating lipid biosynthesis [27]. However, lncRNAs induced SREBP1C transferred into the nucleus have been rarely reported. Interestingly, in this study, our ndings showing that, lnc027912 reduced SREBP1C transit into the nucleus. Furthermore, Akt activation appears to be both necessary for the induction of hepatic SREBP1C [28], and recently studies have shown Akt activated mTOR was through the phosphorylation [29].Our ndings were consistent with previous studies showing lnc027912 inhibited Akt and mTOR of phosphorylation, then reduced lipid accumulation.
Furthermore, PPARγ plays an important role in regulation of lipid metabolism. PPARγ as well as SREBP1 and FAS are important transcription factors that modulate genes involved in fatty acid metabolism and TG synthesis [30]. In this study, lnc027912 inhibited PPARγ protein level and reduced hepatic lipid accumulation. In addition, lipid transport is also vital process in lipid metabolism. Some research groups have con rmed that lncRNAs modulate lipid homeostasis via regulating the VLDL metabolism key genes expression of APOA4, APOC3, MTTP, and APOE [31]. Down regulation of lncAPOA4-AS inhibited APOA4 expression, resulting in decreased triglyceride (TG) concentrations, indicating that lncAPOA4-AS may be a potential target for fatty liver disease [32]. LncMTTP-AS affected the MTTP stability and translation, which may be involve in lipid transport [33]. These results demonstrated that lncRNAs play a vital for lipid metabolism e ux. Interestingly, in this study, we found that lnc027912 promoted the expression of MTTP and ApoE, resulting in decreased lipid accumulation in hepatic cells.

Conclusions
In this study, we found that lnc027912 inhibited SREBP1C activity through Akt/mTOR signal pathway and involved in lipid metabolism balance. Lnc027912 can not only reduce lipid synthesis, but also promote lipid transport, thereby reducing intracellular lipid accumulation. Therefore, lnc027912 played a dual regulatory role in the regulation of lipid metabolism homeostasis and may be an effective target for the regulation of lipid metabolism diseases, such as lipid disorder diseases (NAFLD).

Availability of data and materials
All study data are contained within the manuscript and additional materials. The data that support the ndings of this study are available from the corresponding author upon reasonable request.

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