Inhibition of miR‐499‐5p expression improves nonalcoholic fatty liver disease

1 INTRODUCTION

Nonalcoholic fatty liver disease (NAFLD) is a disease associated with abnormal lipid accumulation in hepatocytes, which might develop into fibrosis, cirrhosis, and even canceration under certain circumstances (Alghamdi, 2017; Atarashi et al., 2018). Steatosis (accumulation of fat droplets in hepatocytes), inflammation, balloon damage, fibrosis, and cirrhosis are the pathological features of NAFLD (Bedossa & Kleiner, 2016). Causes of NAFLD mainly include changes in fat metabolism and proinflammatory and anti-inflammatory molecular imbalances (Chen, Montagner, Tan, & Wahli, 2018). Generally, 30% of the American population are affected by NAFLD (Rinella & Sanyal, 2016), and the prevalence of NAFLD in Asia is about 25% (Fan, Kim, & Wong, 2017b). In fact, NAFLD is a liver manifestation of metabolic and inflammatory liver disease; a high-fat diet (HFD) triggers steatosis and induces lipotoxicity, inflammation, and insulin resistance (Patton et al., 2018). Correspondingly, the traditional treatments for NAFLD focuses on lifestyle interventions (control diet and exercise) and drug therapy (Charytoniuk, Drygalski, Konstantynowicz-Nowicka, Berk, & Chabowski, 2017; Zhu, Baker, Zhu, & Baker, 2018). With the widespread outbreak of NAFLD, molecular targeting technology has also become a hot spot for the treatment of this disease.

MicroRNAs (miRNAs) are a class of noncoding RNAs found in eukaryotes with regulatory functions, which circulate freely in human plasma and are closely related to different pathologies, including NAFLD. For instance, up-regulation of miR-181b alters lipid metabolism in patients with NAFLD, while inhibition of miR-181b expression reduces hepatic steatosis (Wang et al., 2017). Moreover, the levels of miR-34a and miR-122 in the serum of patients with NAFLD are higher than those in the normal population, which could be used as a potential biomarker for distinguishing between NAFLD patients and healthy people (Salvoza, Klinzing, Gopez-Cervantes, & Baclig, 2016). As a biomarker of diabetes mellitus, miR-499-5p is associated with the risk and prognosis of diabetes mellitus (Ma et al., 2014). NAFLD could cause impaired glucose tolerance, leading to type 2 diabetes (Kim et al., 2017). Additionally, elevated levels of miR-499-5p could participate in innate immune responses and viral replication, whereas decreased miR-499-5p in early fibrosis may mediate chronic hepatitis (Salvoza et al., 2016). Although the expression of miR-499-5p was closely related to the pathology of NAFLD-related diseases, the regulation mechanism of miR-499-5p in NAFLD was still unclear.

In this study, models of NAFLD were established based on C57BL/6 mice and HL-7702 cells, and then the quantitative real-time polymerase chain reaction (qRT-PCR) analysis, hematoxylin–eosin (HE) staining, oil red O staining, as well as GPO Trinder enzymatic assay were investigated. Our findings suggest that miR-499-5p may represent a novel indicator of fat metabolism in NAFLD and may be a potential target for diagnosis and therapy.

2 MATERIALS AND METHODS 2.1 Construction of NAFLD cell model

Human normal liver cells HL-7702 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were seeded in 6-well plates at 5 × 105 cells/well. They were divided into a control group and NAFLD model group (free fatty acid [FFA] group). After the adherent cell growth, cells in the control group were added with RPMI1640 medium containing 1% bovine serum albumin, and cells in the FFA group were added with HF medium containing 1 mmol/L FFA. Subsequently, cells were cultured in a 37°C incubator containing 5% CO2. After induction of high-fat culture solution for 24 hr, the cells were subjected to oil red O staining and triglyceride (TG) determination, respectively.

2.2 Cell transfection

HL-7702 cells were divided into the control group (normal liver cells), FFA group (NAFLD cells), FFA + NC group (NAFLD cells transfected with miR-499-5p negative-control inhibitor), and FFA + miR-499-5p inhibitor group (NAFLD cells transfected with miR-499-5p inhibitor). The control group was cultured in the usual medium, while the other three groups were cultured in the FFA high-fat medium. The normal liver cells and FFA-treated HL-7702 cells were first collected, resuspended and counted, then inoculated into a 24-well plate in equal amounts, and cultured in a 37°C and 5% CO2 incubator. When the cells were confluent to 60%–80%, siRNA was transfected by Lipofectamine 2000. Total 3 µl Lipofectamine 2000 and 1 µl siRNA was diluted by 50 µl Opti-MEM Reduced-Serum Medium, and the liquid was kept at room temperature for 5 min after dilution. The diluted siRNA and Lipofectamine 2000 were then gently mixed and incubated for 20 min at room temperature. Finally, miR-499-5p inhibitor and Lipofectamine 2000 complex were added to each well, and the plates were gently shaken and incubated at 37°C in a 5% CO2 incubator for 24 hr.

2.3 Construction and grouping of NAFLD mouse models

Twenty-four 4-week-old specific pathogen-free (SPF) male C57BL/6 mice, weighing 18–20 g, were purchased from the Experimental Animal Center of Zhejiang Academy of Medical Sciences (Zhejiang, China). Ordinary feed contained 8% rice bran, 51% corn, 30% soy flour, 3% bone meal, 1.3% multivitamin, and 6.7% mineral, and an HFD contained 80.5% basic feed, 2% cholesterol, 7% lard, 10% egg yolk powder, and 0.5% bile salt.

After 7 days of adaptive feeding, 24 mice were randomly divided into a standard chow diet (SCD) group (n = 8) and HFD group (n = 16), with a temperature range of 20–22°C, a humidity range of 50%–55%, and a brightness of 12 hr each. Mice were free to eat and drink, and four mice were kept in one cage. After feeding for 4 weeks, mice in the control group and the model group were sacrificed to detect whether the NAFLD model was successfully constructed. Then, the NAFLD model group (n = 12) was subdivided into an HFD group, HFD + negative control (NC) group, and HFD + miR-499-5p inhibitor group (n = 4 in each group). Mice in the HFD group (n = 4) were injected with 100 µl physiological saline in the tail vein. Lentiviral expression vector containing 100 µl unrelated sequence (viral quantity was 2 × 107 transducing units [TUs]) was injected into tail vein of mice in the HFD + NC group. Mice in the HFD + miR-499-5p inhibitor group were injected with a lentiviral expression vector containing 100 µl inhibitory sequence in the tail vein (viral quantity was 2 × 107 TU). Then, the HFD was continued for 4 weeks, and all mice were sacrificed after 8 weeks. After anesthesia, the eyeballs of mice were extirpated for blood collecting. Next, the middle left lobe of the liver was treated with HE staining for observation of pathological changes in the liver. Finally, the remaining liver tissue was washed with phosphate-buffered saline (PBS) and preserved in liquid nitrogen. All animal experiments followed the guidelines for the management and use of laboratory animals.

2.4 Measurement of total cholesterol (TC) and aspartate aminotransferase (AST)

After feeding for 8 weeks, the mice were fasted overnight and anesthetized by intraperitoneal injection of 10% chloral hydrate. The mice blood was collected with a 1.5 ml Eppendorf tube, and centrifuged at 3,000 g for 15 min at 4°C to separate the serum. Finally, the levels of TC and AST in serum were measured using a Cobas 8000 automatic biochemical analyzer (Roche, South San Francisco, CA).

2.5 qRT-PCR

Total RNA of normal liver cells HL-7702 and NAFLD cells was extracted by TRIzol reagent (Thermo Fisher Scientific, New York, NY). Simultaneously, total RNA of normal mouse liver tissues and liver tissues of NAFLD mice were extracted by RNAprep Pure Tissue Kit (TIANGEN Biotech, Beijing, China). First, 1 µg total RNA was used as the initial template, and the total reaction system was 20 µl. The reverse transcription synthesis cDNA was performed on the Gene Amp PCR System 9700 using the FastQuant cDNA First Chain Synthesis Kit (FastQuant RT Kit [with gDNase], KRl06, TIANGEN). Subsequently, qRT-PCR was performed on a Rotor-Gene 3000 real-time PCR instrument (Corbett Research, South San Francisco, CA) using a SuperReal Fluorescence Quantitative Premix Kit (SuperReal PreMix Plus, FP205, TIANGEN). The reaction conditions were pre-denaturation at 95°C for 15 min, denaturation at 95°C for 15 s, annealing at 60°C for 60 s for 40 cycles, and U6 (F:5′-ATTGGAACGATACAGAGAAGATT-3′; R:5′-GGAACGCTTCACGAATTTG-3′) as an internal reference for miR-499-5p (F:5′-ACTGCTTAAGACTTGGAGTGA-3′; R:5′-TACATTGGTGTCGTGGAGTCGGCAA-3′). Small nuclear U6 RNA has been used as an internal control as U6 fulfilled the following criteria: detectable in all samples, low dispersion of expression levels and null association with NAFLD, and it has been used in many recently published researches in NAFLD (Ding et al., 2015; Okamoto et al., 2016; Xu, Zheng, Jiang, & Qiu, 2018). The relative expression of miR-499-5p was calculated using the 2−∆∆Ct formula. The primers used in the experiment were synthesized by Invitrogen (Carlsbad, CA).

2.6 HE staining

For HE staining, mice thoracic liver tissue was first taken and the blood was washed away by PBS. Then, liver tissue was paraffin-embedded, the sections were dewaxed by xylene and dehydrated by alcohol gradient, then stained with hematoxylin stain for 1 min, soaked in PBS for 1 min, and rinsed with pure water until the sections were fully blue. Next, the sections were stained with eosin solution for 1 min, and then placed in gradient alcohol and xylene for dehydration and transparency. Finally, the neutral resin was mounted and the histopathological changes were observed under an ordinary light microscope.

2.7 Oil red O staining

The dry powder of 0.25 g oil red O was dissolved in isopropanol to 50 ml and stored at 4°C in the dark. Before the cells and liver tissue were stained, 4 ml oil red O stock solution was diluted with 6 ml pure water to form oil red O staining solution. Immediately, the cell culture was removed and the cells were fixed with 10% neutral formaldehyde for 15 min. Then, the cells were stained with oil red O staining solution for 10 min and counterstained with hematoxylin for 5 min. After rinsing with ddH2O, the cells were observed and photographed under the microscope.

The liver tissue stored in liquid nitrogen was taken out and cut into 5-µm thick sections at −34°C. The steps of the liver tissue staining were the same as the cells.

2.8 Determination of triglycerides

TGs were mainly determined by the GPO Trinder enzymatic method. The cell culture medium was aspirated, and 200 µl lysate was added to each well of a 6-well plate to lyse the cells. Next, the standard glycerin and cell lysate were mixed with the working solution, respectively, and the mixture was allowed to stand at 37°C for 10 min. The TG concentration of the cells was measured at 570 nm using a microplate reader (Model 680, Bio-Rad, Hercules, CA). Finally, to detect the content of TGs in the liver tissue of mice, 50 mg liver tissue was weighed, and then 1 ml of the lysate was added and ground into a uniform mixture. The remaining detection steps for the content of TG in mouse liver tissue were the same as the procedure for cells.

2.9 Statistical analysis

Statistical analysis was performed using SPSS 19.0 (SPSS, Chicago, IL). The results were expressed as mean ± standard deviation (M ± SD). Statistical analysis was carried out using Student's t-test between two groups. One-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was used for comparison among multiple groups. All experiments were repeated at least three times. P < 0.05 indicated that the difference was statistically significant.

3 RESULTS 3.1 Construction of NAFLD cell model

To determine whether the NAFLD cell model was constructed, we performed oil red O staining. The results revealed that red-stained lipid droplets were observed in the cytoplasm in the FFA group and barely red-stained lipid droplets were seen in the control group (Figure 1a). In addition, the content of intracellular TG was obviously increased in the FFA group compared with the control group (P < 0.01, Figure 1b). The results indicated that the NAFLD cell model was successfully established by culturing with FFA high-fat culture medium for 24 hr.

image Nonalcoholic fatty liver disease (NAFLD) cells model was constructed and the level of intracellular triglycerides (TGs) was detected. (a) Lipid droplet deposition in control group and free fatty acid (FFA) group (oil red O staining, 400×), (b) The level of triglycerides (TGs) in the control group and FFA group (n = 3). Data are presented as mean ± standard deviation. **P µm [Color figure can be viewed at wileyonlinelibrary.com] 3.2 Construction of NAFLD mouse model

Oil red O staining showed that a large number of red-stained lipid droplets were observed in the cytoplasm of hepatocytes in the HFD group (Figure 2a). HE staining showed that the hepatic lobules in the SCD group were clear, the liver cells cable was neat and orderly, the liver cells had no obvious lesions, and the nuclear structure was clear. In the HFD group, diffuse hepatic steatosis occurred in the liver of mice, which showed microvesicular steatosis, and no obvious inflammatory cell infiltration, hepatocyte necrosis, and fibrosis (Figure 2b). Simultaneously, compared with the control group, the content of TGs in the liver of the HFD group was observably increased (P < 0.01, Figure 2c). The results suggested that the NAFLD model was successfully established after 4 weeks of HFD feeding.

image Nonalcoholic fatty liver disease (NAFLD) mouse model was constructed and observed using oil red O staining and hematoxylin–eosin (HE) staining, followed by the detection of level of triglycerides (TGs). (a) Lipid deposition in mouse hepatocytes (oil red O staining, 400×). (b) Performance of liver pathology in mice (HE staining, 400×). Arrows point to microvesicular steatosis. (c) The level of TG in mouse liver tissue (n = 4), Data are presented as mean ± standard deviation. **P µm [Color figure can be viewed at wileyonlinelibrary.com] 3.3 Expression of miR-499-5p in NAFLD cell model and mouse model

The expression level of miR-499-5p in the FFA group was significantly higher than that in the control group (P < 0.05, Figure 3a). Meanwhile, the level of miR-499-5p in the HFD group was distinctly increased from that in the SCD group (P < 0.01, Figure 3b).

image

Expression of miR-499-5p in nonalcoholic fatty liver disease (NAFLD) cell and mouse models. (a) Expression of miR-499-5p in normal hepatocytes and the NAFLD cell model was detected by quantitative real-time polymerase chain reaction (qRT-PCR) (n = 3). (b) Expression of miR-499-5p in normal mice and NAFLD model mice was measured by qRT-PCR (n = 4). Data are presented as mean ± standard deviation. FFA, free fatty acid; HFD, high-fat diet. *P < 0.05 versus control group, **P < 0.01 versus the standard chow diet (SCD) group

3.4 Transfection of miR-499-5p inhibitor

The expression of miR-499-5p in HL-7702 cells is shown in Figure 4a. Compared with the control group, the expression levels of miR-499-5p in FFA, FFA + NC, and FFA + miR-499-5p inhibitor groups were higher (P < 0.05 or P < 0.01), while the expression levels of miR-499-5p in the FFA + miR-499-5p inhibitor group were lower than those in FFA or FFA + NC groups (P < 0.05). There was no significant difference between FFA and FFA + NC groups (P > 0.05).

image

miR-499-5p inhibitor was transfected in vivo and in vitro. (a) miR-499-5p inhibitor was transfected into HL-7702 cells (n = 3). ##P < 0.01 versus control group, *P < 0.05 versus free fatty acid (FFA) or FFA + negative control (NC) group (n = 4). (b) MiR-499-5p inhibitor was successfully transfected in mice. ##P < 0.01 versus control group, *P < 0.05 versus high-fat diet (HFD) or HFD + NC group. Data are presented as mean ± standard deviation

Compared with the control group, the expression levels of miR-499-5p in HFD, HFD + NC, and HFD + miR-499-5p inhibitor groups were higher (P < 0.05 or P < 0.01) (Figure 4b), while the expression levels of miR-499-5p in HFD + miR-499-5p inhibitor group were lower than those in HFD or HFD + NC groups (P < 0.05). There was no significant difference between HFD and HFD + NC groups (P > 0.05).

3.5 Inhibition of miR-499-5p expression attenuated hydroxy steatosis in HL-7702 cells

Hepatic steatosis is commonly observed in histopathological evaluation of patients with NAFLD. Also, it is a hallmark of NAFLD, which is defined as a high intrahepatic glycerol (TG) content (Lee, 2014). In this study, we investigated the effect of miR-499-5p on hydroxy steatosis. After oil red O staining, we found that a large number of red-stained lipid droplets appeared in the cytoplasm of FFA and FFA + NC groups and little was observed in the FFA + miR-499-5p inhibitor group (Figure 5a). GPO Trinder assay showed that the content of TG in the FFA + miR-499-5p inhibitor group was observably lower than that in the FFA or FFA + NC groups (P < 0.05, Figure 5b).

image Inhibition of miR-499-5p expression attenuated steatosis in HL-7702 cells. (a) Changes in the degree of lipidation of HL-7702 cells after transfection with miR-499-5p inhibitor. (b) Changes in the content of triglycerides (TGs) after transfection with miR-499-5p inhibitor. Data are presented as mean ± standard deviation (n = 3). *P µm [Color figure can be viewed at wileyonlinelibrary.com] 3.6 Inhibition of miR-499-5p expression mitigated liver cell steatosis in mice

After feeding with HFD for 8 weeks, diffuse hepatocyte steatosis was found in the liver of HFD and HFD + NC mice, and the degree of steatosis was visibly worse than 4 weeks. Moreover, there was mixed steatosis predominantly vesicular without obvious inflammatory cell infiltration, hepatocyte necrosis, and fibrosis. The degree of steatosis of the liver tissue of the miR-499-5p inhibitor group was markedly improved, and there were no other histological manifestations such as inflammatory cell infiltration, hepatocyte necrosis, and fibrosis in comparison with the HFD + NC group (Figure 6a). Additionally, as shown in oil red O staining, the degree of lipid droplet deposition in the cytoplasm of liver tissue cells of HFD and HFD + NC groups was almost the same. The mice treated with the miR-499-5p inhibitor showed clearly reduced lipid droplet deposition in the cytoplasm of liver tissue cells when compared with corresponding control groups (Figure 6b). The GPO Trinder assay showed that no significant difference was found between HFD and HFD + NC groups (P > 0.05). Compared with the HFD or HFD + NC groups, the content of TG in the liver tissue of the HFD + miR-499-5p inhibitor group was significantly decreased (P < 0.05, Figure 6c).

image Down-regulating the expression of miR-499-5p mitigated liver steatosis in mice with nonalcoholic fatty liver disease (NAFLD). (a) Liver pathology of mice in different groups (hematoxylin–eosin [HE] staining, 400×). Arrows point to steatosis. (b) Lipid deposition in mouse liver tissue (oil red O staining, 400×). (c) The contents of triglycerides (TGs) in mouse liver tissue (n = 4). Data are presented as mean ± standard deviation. *P µm [Color figure can be viewed at wileyonlinelibrary.com] 3.7 Inhibition of miR-499-5p expression improved liver damage

A high AST level is an indicator of moderate-to-severe fibrosis in NAFLD patients (Noreen et al., 2009). Moreover, high TC levels are associated with a greater risk of NAFLD (Wu et al., 2016). Compared with the SCD group, the levels of TC and AST in serum of mice in HFD, HFD + NC, and HFD + miR-499-5p inhibitor groups were evidently higher (P < 0.01), while in the miR-499-5p inhibitor group, they were markedly more decreased than those in HFD or HFD + NC groups (P < 0.05, Figure 7).

image

Down-regulating the expression of miR-499-5p increased total cholesterol (TC) and aspartate aminotransferase (AST) levels in mouse with nonalcoholic fatty liver disease (NAFLD). (a) A Cobas 8000 automatic biochemical analyzer was used to detect the content of TC. (b) The content of AST in mouse serum was detected by a Cobas 8000 automatic biochemical analyzer. Data are presented as mean ± standard deviation (n = 4). **P < 0.05 versus standard chow diet (SCD) group, #P < 0.05 versus high-fat diet (HFD) or HFD + negative control (NC) group

4 DISCUSSION

Recently, miRNAs have been found to play important roles in several animal models of NAFLD (Sayed & Abdellatif, 2011). Dysregulated miRNAs are involved in the transition from hepatic steatosis to steatohepatitis in rat models of NAFLD (Jin et al., 2012). In this study, qRT-PCR showed that the expression of miR-499-5p increased in the NAFLD model group compared with the control group and miR-499-5p inhibitor could alleviate the steatosis of liver cells with NAFLD and reduce the content of TC and AST in the serum, suggesting that reduced miR-499-5p levels contribute to alleviate the steatosis and improve the liver damage of mice. MiR-499-5p might become a new target for the treatment of NAFLD.

miRNAs have been found as useful serum biomarkers in the diagnosis and treatment of various diseases (Chen et al., 2008). Previous study has reported that serum levels of miR-122 were correlated with severity of liver steatosis, and may act as a useful screening biomarker for NAFLD (Yamada et al., 2013). MiR-21 could regulate TG and cholesterol metabolism by inhibiting the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the serum of patients with NAFLD (Chuanzheng et al., 2015). Ding et al. (2015) have reported that miR-34a inhibitors counteract NAFLD by inhibiting lipid accumulation. In this study, the expression of miR-499-5p increased in the NAFLD model group and inhibition of miR-499-5p expression attenuated liver cell steatosis, suggesting that miR-499-5p may be suited to assess early NAFLD and could serve as a tool for understanding the progression of NAFLD.

Based on our results, reducing the accumulation of TG content can alleviate the steatosis of NAFLD. Similar to our findings, a previous research has found that miR-30c-5p targeting regulates fatty acid synthase, reduces TG accumulation and lipid deposition, and improves NAFLD (Fan et al., 2017a). In fact, abnormal accumulation of TG in the liver is one of the characteristics of NAFLD (Goh & Silver, 2013; Hur et al., 2016). Studies reveal that a large amount of lipid deposition is observed in the cell model of NAFLD, and the levels of TG are elevated (Liu et al., 2017; Zhu, Cheng, Zhou, Zhao, & Infection, 2017). Besides, when lipid accumulation in the liver cells of NAFLD mice is increased, the TG content in liver tissue is also increased (Gao, Zhang, Yu, Tan, & Wang, 2016). Consistent with previous studies, the miR-499-5p inhibitor was also found to reduce the TG content of the NAFLD models, suggesting that miR-499-5p might improve the fatty degeneration of NAFLD hepatocytes. In addition to the mechanisms mentioned above, the expression of genes involved in lipid metabolism is regulated by affecting the methylation modification of adipose DNA to reduce the content of TG and TC, and thus liver damage caused by NAFLD is effectively alleviated (Chen et al., 2016). Recent research has indicated that compared with the non-NAFLD population, the levels of TC in NAFLD patients evidently increase (Peng, Mo, & Tian, 2017; Wei et al., 2018). Moreover, as NAFLD is aggravated, the content of TC is evidently increased (Zhang, Wang, & Wang, 2018). A previous study has found that the levels of TC and AST in patients with NAFLD are obviously higher than those in healthy subjects (Qiang, Wang, & Yanhua, 2016). Naturally, the levels of TC and AST in the HFD group are significantly elevated (Fenglin, Bao, & Shixia, 2016; Xiang, 2018). In this study, we also found that inhibition of miR-499-5p expression reduced serum TC and AST levels in NAFLD mice, suggesting that decrease of miR-499-5p levels might ameliorate liver damage caused by NAFLD. In agreement with our results, through improving liver function in rats with NAFLD by reducing serum TC and AST levels, researchers have achieved the effect of alleviating NAFLD induced by HFD (Zhang et al., 2013). In conclusion, we have shown that decreased expression of miR-499-5p in mice models of NAFLD likely plays a critical homeostatic role to prevent excessive lipid accumulation in livers, which could ultimately give rise to liver damage. Future studies determining how miR-499-5p interacts with lipid, fibrosis, and inflammatory pathways in NAFLD are needed and could offer new insights into the pathogenesis of NAFLD.

CONFLICTS OF INTEREST

All authors have no conflict of interest to declare.

AUTHORS CONTRIBUTIONS

H.L. and T.W., conception and design and analysis of data, drafting the article; X.C., S.X., and J.J., drafting the article; N.S., R.L, and Y.X., revising the article critically for important intellectual content.

All data are available through corresponding author Shiying Xuan.

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