The lncRNA Gm15622 stimulates SREBP-1c expression and hepatic lipid accumulation by sponging the miR-742-3p in mice

Excessive lipid deposition is a hallmark of nonalcoholic fatty liver disease (NAFLD). Although much has been learned about the enzymes and metabolites involved in NAFLD, few studies have focused on the role of long non-coding RNAs (lncRNAs) in hepatic lipid accumulation. Here, using in vitro and in vivo models of NAFLD, we found that the lncRNA Gm15622 is highly expressed in the liver of obese mice fed a high-fat diet (HFD) and in murine liver (AML-12) cells treated with free fatty acids. Investigating the molecular mechanism in the liver-enriched expression of Gm15622 and its effects on lipid accumulation in hepatocytes and on NAFLD pathogenesis, we found that Gm15622 acts as a sponge for the microRNA miR-742-3p. This sponging activity increased the expression of the transcriptional regulator sterol regulatory element–binding transcription factor 1c (SREBP-1c) and promoted lipid accumulation in the liver of the HFD mice and AML-12 cells. Moreover, further results indicated that metformin suppresses Gm15622 and alleviates NAFLD-associated lipid deposition in mice. In conclusion, we have identified an lncRNA Gm15622–miR-742-3p–SREBP-1c regulatory circuit associated with NAFLD in mice, a finding that significantly advances our insight into how lipid metabolism and accumulation are altered in this metabolic disorder. Our results also suggest that Gm15622 may be a potential therapeutic target for managing NAFLD. knockdown can reduce SREBP-1c expression and inhibit lipid accumulation in the liver. Gain-of-function and loss-of-function experiments demonstrated that Gm15622 can regulate the miR-742-3p/SREBP-1c axis to improve lipid accumulation in vitro and in vivo . These findings support the rationale that Gm15622 acts as a novel biomarker and may serve as a therapeutic target for NAFLD.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is a metabolic syndrome with a widespread histological distribution, from simple hepatic steatosis to nonalcoholic steatohepatitis (NASH) with or without fibrosis. NASH may eventually develop into cirrhosis and hepatocellular carcinoma (1)

. A World
Health Organization survey found that the number of obese people in the world has nearly tripled since 1975 (https://www.who.int/zh). With the rising rate of obesity and type 2 diabetes, NAFLD has become one of the most prominent liver diseases in the world, with an incidence rate of 25% (2). The highest incidence rate is in the Middle East and South America at between 30% and 35%, followed by Asia, where the incidence is between 25% and 30% (3). The major causes of NAFLD include the metabolic diseases associated with type 2 diabetes, genetic predisposition, environmental factors and gender (3).
Therefore, timely intervention of NAFLD is of critical importance for alleviating the public health burden.
Lipids obtained from the diet or synthesized by the liver and stored in adipose tissue are in an equilibrium state in terms of synthesis and energy utilization (4).
Chylomicrons transport food lipids to the liver either via the low-density lipoprotein receptor or in the form of free fatty acids (FFAs) that enter liver cells via CD36 (5). The de novo synthesis of lipids is mainly regulated by transcription factors, such as carbohydrate reactive element binding protein and cholesterol regulating element binding protein (SREBP), which control the transcription levels of the rate-limiting enzyme, acetyl-CoA carboxylase, and fatty acid synthase (FASN) (6). Excessive lipids in the liver are transported out of the liver by ATP-binding cassette transporters. If this delicate balance is disrupted, lipid metabolism in the liver can be perturbed, leading to disease (7,8). Therefore, the regulation of lipid homeostasis is crucial.
Metformin is a first-line drug in the treatment of type 2 diabetes (14). In recent years, various studies have demonstrated that in addition to treating type 2 diabetes, it also has anti-cancer properties (15), can reverse pulmonary fibrosis and can ameliorate NAFLD (16,17). Although several studies have explored the molecular mechanism of metformin, its role in the pathological process of NAFLD needs to be further elucidated.
In this study, we found that Gm15622 was significantly up-regulated in the liver of high-fat diet (HFD)-induced obese mice. Up-regulated Gm15622 significantly increased lipid accumulation, whereas silencing Gm15622 significantly reduced lipid accumulation in AML-12 cells. Mechanistically, Gm15622 positively modulated the expression of SREBP-1c by acting as an miRNA sponge for miRNA-742-3p. We also found that metformin could target Gm15622 to regulate lipid accumulation in HFD mice. Therefore, our study provides new insights into the molecular function of the Gm15622/miR-742-3p/SREBP-1c signaling pathway in the pathogenesis of NAFLD and highlights the potential of Gm15622 as a new therapeutic target for NAFLD.

Animals
All animal procedures conformed to the Guide for the Care and Use of

Cell viability assay
Cell viability was measured in triplicate using a commercially available kit (Cell

Statistical analysis
Data are presented as the mean ± standard deviation (SD) from multiple samples. All experiments were repeated at least three times. Group differences were considered statistically significant at p<0.05, as assessed by Student's t-test, one-way or two-way ANOVA followed by Fisher's least significant difference post-hoc test using Origin 2017 (Origin Lab Corporation, USA).

lncRNA Gm15622 is highly expressed in mouse liver and is up-regulated in the ob/ob, db/db and Fasted models
We examined the basal expression of Gm15622 in seven tissues of C57BL/6 male mice by RT-qPCR. Gm15622 was expressed in all tissues, but the highest expression was in the liver in our study ( Figure 1A). Different studies archived in the Expression Atlas database also showed that Gm15622 was specifically expressed in the liver of C57BL/6 mice ( Figure 1B). We then examined the expression of Gm15622 in the ob/ob, db/db and fasted models by RT-qPCR, and Gm15622 was up-regulated in these models in our study ( Figure 1C-1E). These results indicated that Gm15622 was a liver-enriched lncRNA and was up-regulated in the liver of ob/ob, db/db and fasted models.

Gm15622 is elevated in the liver of HFD-fed obese mice
Mice fed the HFD for 18 weeks had a significantly increased body weight compared with the ND groups ( Figure 2A). Serum levels of TG, TC, ALT, and AST were also significantly increased in the HFD mice ( Figure 2B, 2C). The liver of HFD-fed mice was larger than normal and yellow, indicating the occurrence of lipid accumulation and liver steatosis. H&E and Oil Red O staining of liver sections also confirmed lipid accumulation ( Figure 2D). Indeed, the HFD significantly increased the hepatic levels of TG and TC ( Figure 2E).
The HFD also increased expression of lncRNA Gm15622 and Gomafu (positive control). Correspondingly, the GEO database (GSE35961) also showed that Gm15622 was up-regulated in the liver of HFD-fed C57BL/6 mice ( Figure 2F). Therefore, Gm15622 was elevated in the liver of HFD-fed mice.

Gm15622 regulates the expression of SREBP-1c and FASN in AML-12 cells
Gm15622 was liver-enriched and the HFD changed Gm15622 expression. We
Surprisingly, the StarBase database also predicted that SREBP-1c is a target gene for miRNA-742-3p ( Figure 4B). These bioinformatic results indicated that Red O staining showed that metformin can attenuate the increase in lipid accumulation caused by FFAs ( Figure 5F). Taken together, metformin may reduce lipid accumulation and expression of lipid synthetic genes in AML-12 cells by modulating Gm15622 or miR-742-3p.

Metformin inhibits SREBP-1c and FASN expression in AML-12 cells by targeting Gm15622
To explore the specific molecular mechanism by which metformin reduces lipid accumulation through lncRNAs, we constructed the pcDNA3.0-Gm15622 overexpression plasmid and synthesized a Gm15622 interference sequence

Metformin reduces body weight gain, alleviates hyperlipidemia and decreases lipid accumulation in the liver of HFD-fed obese mice
To explore the role of metformin in vivo, we next used mice that had been fed a HFD for 18 weeks and then gavaged with different concentrations of metformin.
In the 9 weeks of metformin treatment, the obesity of control mice receiving saline gavage became more severe. In contrast, the average body weight gain of HFD-fed obese mice treated with metformin at 150 mg/kg/d was decreased, and a high dose of metformin (300 mg/kg/d) completely inhibited HFD-induced obesity ( Figure 7A). To determine the possible hypolipidemic effect of metformin on HFD-fed obese mice, the serum lipid profile was examined. As  Figure   7F).

Metformin inhibits the expression of lipid synthesis genes in HFD-fed obese mice by inhibiting the expression of Gm15622
HFD significantly increased the ratio of liver weight to body weight, and the hepatic levels of TG and TC ( Figure 8A, 8B). These liver abnormalities were alleviated by metformin. HFD-fed mice had a robust increase in hepatic expression levels of Gm15622, but a substantial decrease in the level of miR-742-3p. Conversely, metformin reversed these changes in expression in a dose-dependent manner ( Figure 8C). To confirm the regulatory role of metformin in lipogenic gene expression, we examined protein levels in liver samples and found a trend similar to that of mRNA levels. Metformin inhibited HFD-feeding-induced upregulation of the hepatic protein levels of SREBP-1c and FASN, in a dose-dependent manner ( Figure 8D, 8E). In conclusion, we found that metformin effectively antagonized HFD-induced hyperlipidemia and hepatic lipid accumulation in mice. These effects were achieved by regulating the Gm15622/miR-742-3p/SREBP-1c axis ( Figure 8F).

DISCUSSION
Excessive accumulation of lipids caused by unbalanced lipid metabolism in the liver is the main cause of NAFLD (8). NAFLD not only causes hepatocellular carcinoma, but is also a component of metabolic syndrome together with obesity, type 2 diabetes, arteriosclerotic cardiovascular disease, and dyslipidemia (1, 2). Therefore, it imposes a huge clinical and economic burden on patients and society. Although the molecular mechanism of NAFLD has not been clearly elucidated, lncRNAs can be used as potential targets for the diagnosis and treatment of NAFLD (9,19). In the present study, increased expression of the lncRNA Gm15622 in HFD-induced NAFLD was investigated.
We found Gm15622 to be capable of sponging miR-742-3p, thereby increasing the amount of SREBP-1 protein, a transcription factor that regulates the expression of genes that control fatty acid lipid and cholesterol synthesis. Thus, we speculate that SREBP-1c is a downstream target of Gm15622 in NAFLD.
lncRNAs located in the cytoplasm and nucleus have different mechanisms of action, which can be taken into account when investigating their functions (28).  (29,30). We then used bioinformatics to predict that Gm15622 contains a target site for and can interact with miR-742-3p. In addition, SREBP-1c is a target gene of miR-742-3p. Finally, we found that overexpression of Gm15622 increased the expression of SREBP-1c, which was offset by overexpression of miR-742-3p. Similarly, knockdown of Gm15622 reduced the expression of SREBP-1c, which was reversed by down-regulation of miR-742-3p expression. Therefore, we propose that Gm15622 acts as a ceRNA in AML-12 cells to regulate SREBP-1c expression by sponging miR-742-3p, which may be a mechanism by which Gm15622 acts as a critical regulator in NAFLD.
Different pathological processes have been described for NAFLD (31).
Therapeutic drugs for NAFLD are still in the development stage, and the most developed drugs, such as pioglitatone and GFT 505, are only in phase III clinical trials (32,33). Metformin is the preferred drug for treating diabetes mellitus, but it can also affect the pathological process of some cancers (15,16).

Conflict of interest
The authors declare no conflict of interests and no permission is required for publication.     Data are presented as the mean ± SD, n = 6 mice/group. * p < 0.05, ** p < 0.01, *** p < 0.001.  Experiments were repeated in triplicate and data are presented as the mean ± SD. * p < 0.05, ** p <0.01 and *** p <0.001 vs. BSA, ### p < 0.001 vs. FFAs without metformin.  intuitively that metformin can improve lipid accumulation in the liver of NAFLD mice, the results in ND group was reused from Figure 2D.
by guest, on May 8, 2020