Long non-coding RNA GAS5 contributes to the progression of nonalcoholic fatty liver disease by targeting the microRNA-29a-3p/NOTCH2 axis

ABSTRACT Long non-coding RNAs (lncRNAs) have been widely recognized as critical players in the development of nonalcoholic fatty liver disease (NAFLD), one of the most prevalent liver diseases globally. In this study, we established a HFD-induced NAFLD mouse model and explored the role of lncRNA GAS5 in NAFLD progression and its possible underlying mechanisms. We showed that NAFLD activity score was elevated in the HFD mice. GAS5 knockdown attenuated HFD-induced hepatic steatosis and lipid accumulation and reduced NAFLD activity score in HFD mice. In addition, GAS5 knockdown reduced serum triglyceride cholesterol levels and inhibited alanine aminotransferase and aspartate aminotransferase activities in HFD mice. Moreover, GAS5 overexpression enhanced NOTCH2 levels in liver cells and promoted NAFLD progression by sponging miR-29a-3p in vivo. Furthermore, miR-29a-3p inhibited NAFLD progression by targeting NOTCH2 in vivo. Overall, our results indicated that GAS5 acts as a sponge of miR-29a-3p to increase NOTCH2 expression and facilitate NAFLD progression by targeting the miR-29a-3p/NOTCH2 axis and demonstrated a new GAS5-mediated mechanism underlying NAFLD development, suggesting that GAS5 could be a potential therapeutic target of NAFLD. Abbreviations: Alanine aminotransferase: ALT; Aspartate aminotransferase: AST; Enzyme linked immunosorbent assay: ELISA; Hepatocellular carcinoma: HCC; High-fat diet: HFD; Long non-coding RNA: Lnc RNA; Long non-coding RNA GAS5: GAS5; MicroRNAs: MiRNAs; Nonalcoholic fatty liver disease: NAFLD; Quantitative reverse transcription PCRs: RT-qPCRs; siRNA negative control: si-NC; Total cholesterol: TC; Triglyceride: TG


Background
Nonalcoholic fatty liver disease (NAFLD) is one of the most prevalent hepatic diseases globally and is often correlated with type 2 diabetes, obesity, and harmful diet [1,2]. NAFLD is initiated by irregular triglyceride (TG) increase in the liver and can progress to severe hepatic diseases, including cirrhosis and hepatocellular carcinoma (HCC) [3]. NAFLD accounts for over 13% of hepatocellular carcinoma patients and increases year by year [4]. Therefore, exploring the underlying mechanisms of NAFLD progression and identifying essential treatment targets are crucial to the drug development and therapy of NAFLD [5,6].
MicroRNAs (miRNAs) are another form of extensively studied non-coding RNAs with 20-25 nucleotides. They are well-recognized as important regulators of various biological processes [17]. MiRNAs commonly function through binding to the 3′ untranslated region (3′ UTR) of targeted mRNAs to disrupt their stability or impede their translation [18]. Noteworthy, studies have demonstrated that miRNAs participate in the development of NAFLD. MiR-873-5p modulates the mitochondrial GNMT-Complex II interface, leading to NAFLD [19]. Plasma miR-122 and miR-29a serve as potential markers of NAFLD [20]. Moreover, miR-29a-3p is involved in the modulation of NAFLD. For example, miR-29a-3p controls cholesterol metabolism and triglyceride level by targeting HMGCR in NAFLD [21]. NOTCH2 is a suppressor of transforming growth factor (TGF)-β1 signaling, which is correlated to the maintenance of chronic inflammation and involved in modulating various pathological processes, including NAFLD [22]. Moreover, NOTCH2 has been reported as one of the targets of miR-29a-3p [23,24]. Nevertheless, the connection between GAS5 and miR-29a-3p/ NOTCH2 regulatory axis remains unclear. This study aimed to decipher the function of GAS5 during NAFLD pathogenesis and demonstrated the relationship between GAS and the miR-29a-3p/ NOTCH2 axis in NAFLD progression.

nonalcoholic fatty liver disease mouse model
The NAFLD mouse model was generated by administrating a high-fat diet (HFD) to C57BL/6 mice. Briefly, male C57BL/6 mice (12-14 weeks old) were assigned into the high-fat diet (HFD) and control groups with 5 mice in each group and maintained at 23 ± 3°C in a humidified atmosphere with a 12-h circadian rhythm and free access to water and high-fat diet (HFD, Dyets Bethlehem, PA, USA) or standard diet (#CE-2, CLEA Japan Inc., Shizuoka, Japan), respectively. The standard diet provided 3.4 kcal per gram and contained 46 g/kg of crude fat, and the HFD provided 5.2 kcal per gram and contained 320 g/kg of lard-based fat. After 56 days, mice in the HF and control groups were weighted 45 ± 4 g and 21 ± 3 g, respectively, and their serum and liver tissues were collected for further analysis. Lentivirus containing shRNAs targeting GAS5 or NOTCH2, miR-29a-3p inhibitors and mimics, and controls were GenePharma (China). For exploring the effects of GAS5 knockdown, NOTCH2 inhibition, and miR-133a-inhibition, lentivirus vector with si-GAS5, si-NOTCH2, and si-NC, miR-133a-inhibitor or its NC, were administrated into mice via tail vein injection. The NAFLD activity score in the mice was quantified as previously reported [25]. The levels of total cholesterol (TC), triglyceride (TG), alanine aminotransferase (ALT), and aspartate Aminotransferase (AST) in serum or liver tissues were analyzed using enzyme-linked immunosorbent assay (ELISA) kits (Applygen, China). The levels of GAS5, NOTCH2, and miR-29a-3p in mouse tissues were detected using qPCR. NOTCH2 protein level was evaluated using Western blotting assay with NOTCH2 antibody (1:1000) from Abcam, USA. Lipid accumulation was analyzed using Oil Red O staining. All animal experiments were approved by the Ethic Committee of the First Affiliated Hospital of Anhui Medical University (No. 34234#HYRE) and operated in compliance with the guidelines of the American Animal Protection Legislation and the Animal Research Reporting In Vivo Experiments.

Western blotting assay
Total proteins were obtained from LO2 cells and mouse liver tissues by homogenization in icecold RIPA lysis solution (Beyotime, China) and quantified using a BCA kit (Thermo). An equal amount of samples were separated by SDS-PAGE and transferred onto nitrile cellulose membranes. The membranes were blocked in 5% nonfat milk and incubated with first specific primary antibodies against NOTCH2 (1:1000, Santa Cruz, USA) and GAPDH (1:1000, Santa Cruz) at 4°C overnight and then with corresponding secondary anti-mouse or anti-rabbit antibodies (1:2000, Santa Cruz). The protein signals were visualized by an ECL solution in a Gel Imaging system (BD Biosciences, USA) and quantified using ImageJ software.

Luciferase reporter gene assay
Wild-type and mutated sequences of GAS5 and NOTCH2 were cloned into pGL3-basic vectors purchased from Promega (USA). The obtained vectors were co-transfected with miR-29a-3p mimic or negative control into cells using Lipofectamine 2000. At 48 h of posttransfection, luciferase activity was detected using a dual-luciferase reporter assay kit (Promega) [26].

RNA pull-down assay
Cells were transfected with biotin-labeled RNAs from GenePharma. At 24 h of post-transfection, cells were lysed and incubated with magnetic beads (Thermo) following the manufacturer's protocol [27]. The obtained samples were analyzed by qRT-PCR.

Statistical analysis
Data were shown as mean ± standard deviation (SD) and analyzed by SPSS 22.0 software. The differences were determined by one-way ANOVA or unpaired Student's t-test and were considered significant with P < 0.05.

LncRNA GAS5 and NOTCH2 are elevated, while miRNA-29a-3p is decreased in the NAFLD mouse model
To evaluate the correlation of GAS5, NOTCH2, and miR-29a-3p with NAFLD, a NAFLD model was established using C57BL/6 mice by feeding HFD. The success of NAFLD mice was evaluated by ELISA and Oil Red staining. The results revealed that the HFD mice had hepatic steatosis (Figure 1(a)), increased lipid accumulation (Figure 1(b)), enhanced NAFLD activity score (Figure 1 RT-qPCR analyses showed that GAS5 RNA level was notably enhanced, whereas miR-29a-3p the level of was declined in liver tissues from the HFD mice (Figure 1(f,g)). Besides, NOTCH2 mRNA and protein levels were enhanced in the liver tissues from the HFD mice (Figure 1(h,i)). Similar changes were also observed in the adipose tissues (Supplement data1). Together, these data imply that GAS5, NOTCH2, and miR-29a-3p may participate in NAFLD modulation.

LncRNA GAS5 promotes the progression of NAFLD in vivo
The role of GAS5 in NAFLD development was evaluated in the NAFLD mouse model with GAS5 depletion by injecting lentivirus containing shGAS5 or NC. The effective transfection of sh-GAS5 was determined to evaluate GAS5 RNA level in liver tissues (Figure 2(a)). As shown in (Figure 2 (b)), the HFD-induced hepatic steatosis was attenuated by t GAS5 knockdown in the HFD mice. GAS5 depletion reduced lipid accumulation in the mice (Figure 2(c)) and reversed the elevated NAFLD activity score in the HFD mice (P < 0.05) (Figure 2(d)). Moreover, GAS5 knockdown attenuated HFD-enhanced serum TG and TC levels (Figure 2(e)) and relieved HFD-caused elevation of serum AST and ALT levels (Figure 2 (f)). These results demonstrate that GAS5 promotes NAFLD progression in vivo.

Discussion
NAFLD is one of the most frequently occurring chronic hepatic disorders, affecting nearly 20% of the population worldwide [28,29]. It is generally believed that NAFLD consists of four histological steps, including simple steatosis, nonalcoholic steatohepatitis, fibrosis, and cirrhosis. Despite the complicated pathogenesis of NAFLD, lncRNAs have been extensively involved in NAFLD regulation. For instance, lncRNA NEAT1 enhanced liver lipid accumulation by controlling miR-146a-5p/ROCK1 in NAFLD [30]. LncRNA-AK012226 participates in fat accumulation in fatty liver of DB/DB mice and  NAFLD cell model [31]. Repression of lncRNA HULC contributes to hepatocyte apoptosis and hepatic fibrosis by repressing MAPK signaling in NAFLD rats [32]. The silencing lncRNA SNHG20 restrains NAFLD progression to hepatocellular carcinoma by modulating liver Kupffer cell polarization [33]. LncRNA Mirt2 upregulates USP10 to repress hepatic steatosis by targeting miR-34a-5p [34]. LncRNA NONMMUT010685 performs a vital function in NAFLD based on microarray analysis [35]. LncRNA NEAT1 controls inflammatory and fibrosis response in NAFLD by mediating miR-506/GLI3 [36]. Moreover, it has been reported that GAS5 inhibits NAFLD development to hepatocellular carcinoma by controlling the Kupffer cell M1/M2 polarization [16]. Our results indicated that GAS5 level was elevated in the HFD mouse model, and this elevation was capable of promoting NAFLD development and revealed a novel role of GAS5 during NAFLD development, thereby providing vital evidence to establish the function of lncRNAs in the pathogenesis of NAFLD.
NOTCH2 has been indicated in the development of hepatic diseases. For example, it is reported that FBXO31 inflects liver fibrogenesis and hepatic stellate cell activation through increasing NOTCH2 ubiquitination [49]. Hepatic Notch2 deletion in mice contributes to aggravating alcoholic liver injury and mechanical liver damage [50]. NOTCH2 modulates compensatory hepatocyte proliferation in the damaged liver of mice and is positively related to more favorable clinical outcomes of hepatocellular carcinoma [51]. MiR-29a-3p2 increases liver fibrosis and stimulates liver stellate cells by modulating NOTCH2 [52]. The elevated NOTCH2 expression is associated with cholestasis-induced liver fibrogenesis [53]. Moreover, NOTCH2 plays a critical role in TGFβ signaling and is recognized as a miR-29a-3p target [23,24]. Significantly, many investigations have proved that TGF-β signaling is essential for modulating fibrogenesis in NAFLD [54,55]. This study revealed that GAS5 enhances NOTCH2 expression by sponging miR-29a-3p and further inhibits NAFLD progression via targeting NOTCH2, consistent with the previously reported role of NOTCH2 in regulating NAFLD.
To summarize, our work deciphered that GAS5 facilitates NAFLD development via regulating the miR-29a-3p/NOTCH2 regulatory axis. These findings may provide novel evidence for underlying the mechanisms that activate NAFLD progression and present GAS5/miR-29a-3p/NOTCH2 regulatory axis as promising therapeutic targets for NAFLD.

Conclusions
LncRNA GAS5 and NOTCH2 expression levels are increased, and miR-29a-3p is decreased in the NAFLD mouse model. In addition, lncRNA GAS5 may sponge miR-29a-3p to attenuate the inhibitory role of NOTCH2, thereby promoting NAFLD.

Ethics approval and consent
All animal experiments were authorized by the Ethnic Committee of the First Affiliated Hospital of Anhui Medical University (No. 34234#HYRE) and conducted following the guidance of the American Animal Protection Legislation and in compliance with the ARRIVE guidelines.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.