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Inhibition of miR-324-5p increases PM20D1-mediated white and brown adipose loss and reduces body weight in juvenile mice
European Journal of Pharmacology 863 (2019) 172708
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Inhibition of miR-324-5p increases PM20D1-mediated white and brown adipose loss and reduces body weight in juvenile mice
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Dandan Li, Yang Liu, Wei Gao, Jiakai Han, Rongrong Yuan, Mengdi Zhang, Wuyan Pang∗ Department of Endocrinology, Huaihe Hospital of Henan University, Kaifeng, 475000, Henan Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-324-5p PM20D1 Obesity Fat consumption Body weight
Obesity is a serious public health problem characterized by abnormal or excessive fat accumulation, which is caused by an energy imbalance between calories consumed and calories expended. MiRNAs have been involved in the regulation of occurrence and progression of obesity. This study aims to investigate the role of miR-324-5p in regulating the adipose tissue mass and preliminarily probe into its effect on progression of obesity. MiR-3245p was upregulated in the epididymal white adipose tissues (eWAT), inguinal white adipose tissues (iWAT) and brown adipose tissues (BAT) of the mice fed with high fat diet (HFD). Under room temperature (RT) or thermoneutrality (TN) condition, when tail intravenously injected with miR-324-5p antagomir (anta-miR-324-5p), the fat mass and total weight of mice were both significantly suppressed. The suppressive effect was more distinct under TN than RT. The weight of iWAT and BAT were both inhibited by anta-miR-324-5p under TN. Moreover, PM20D1 was a direct target gene of miR-324-5p. In primary iWAT cells, the expression of PM20D1 was significantly increased by anta-miR-324-5p, whereas decreased by the miR-324-5p mimic. Furthermore, anta-miR-324-5p noticeably increased the cellular oxygen consumption in primary BAT and iWAT cells. Our findings indicated that inhibition of miR-324-5p increased PM20D1-mediated fat consumption and reduced body weight in mice, suggesting that miR-324-5p may be a novel therapeutic target against obesity.
1. Introduction Nowadays, childhood obesity and overweight has become a serious public health problem all over the word. The prevalence of childhood obesity was reported to increase by 47.1% between 1980 and 2013 (Ng et al., 2014). According to the data of Chinese National Surveys on Students Constitution Health (CNSSCH), the prevalence rates of overweight of children nearly doubled from 4.1% in 2000 to 8.1% in 2010 (Sun et al., 2015a). Childhood obesity is associated with various comorbidities, such as cardiovascular diseases (Nielsen et al., 2015), type II diabetes (Hayden et al., 2016), metabolic syndrome (Liang et al., 2015) and sleep apnea (Bazzano et al., 2016). So, it is urgent to find an effective approach for the prevention of childhood obesity. During the past thirty or forty years, it has been acknowledged that, in addition to coding protein genes, most of the mammalian genomes are transcribed into a great variety of types of non-coding RNAs (ncRNAs). NcRNAs are emerging as important regulatory factors in the pathogenesis of diseases. MiRNAs are a class of short non-coding RNA molecules that regulate gene expression at the post transcriptional level (Sandhir et al., 2014). They have been proven to control various biological progresses, such as cell growth, cell differentiation and
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apoptosis (Sun et al., 2015b; Wang et al., 2015; Yu et al., 2014). Many studies have demonstrated that miRNA plays an important role in the progression of obesity, such as miR-21 (Seeger et al., 2014), miR-103 (Vinnikov et al., 2014), miR-34a (Fu et al., 2014). Obesity is a complex disease that is regulated by a variety of factors including genetics, endocrine, drugs, lifestyle, and adipocytokines. When the body consumes more calories than calories, the excess calories are stored in the body in the form of fat, which exceeds the normal physiological requirements, and when it accumulates a certain value, it evolves into obesity (Jordania et al., 2017). In the processes of lipogenesis and consumption, some small RNAs play important roles. For example, Martinelli et al. reported that miR519 was up-regulated and associated with metabolic imbalance and subsequent adipocyte hypertrophy through suppressing the expression of PPARA in subcutaneous adipose tissues (Martinelli et al., 2010); Shi et al. reported that miR-148 was associated with obesity and modulated adipocyte differentiation of mesenchymal stem cells through Wnt signaling (Shi et al., 2015). MiR-324-5p is a relatively well characterized onco-miRNA in many types of carcinomas. It was recently shown to be associated with metabolism in liver and adipose tissues (Guenther et al., 2007; Szkolnicka
Corresponding author. Department of Endocrinology, Huaihe Hospital of Henan University, No. 1 Baobei Road, Kaifeng, 475000, Henan Province, China. E-mail address: [email protected] (W. Pang).
https://doi.org/10.1016/j.ejphar.2019.172708 Received 22 August 2019; Received in revised form 25 September 2019; Accepted 26 September 2019 Available online 27 September 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
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et al., 2016). Moreover, miR-324-5p was differentially expressed in visceral adipose tissues of patients with abnormal metabolism (Estep et al., 2010). A couple of recent studies showed that miR-324 was dysregulated during tissue injury and revealed that miR-324 was involved in energy metabolism in cell stress (Diaz et al., 2017; Liu et al., 2019). Our previous microarray study showed that miR-324-5p was significantly up-regulated in the heart, liver, fat and various secretory organs of juvenile animals under cold conditions. Our team has been studying endocrinal and metabolic disorders in children, such as childhood obesity. Herein, we investigated the role of miR-324-5p in regulating the adipose tissue mass and preliminarily probe into its effect on progression of obesity. 2. Materials and methods 2.1. Animals and diets Animal experiments were approved in accordance with the guidelines by Huaihe Hospital of Henan University (Kaifeng, China). Three week old weaned BALB/C mice were purchased from BetterBiotechnology Co., Ltd (Nanjing, China) and were maintained in 12 h light: dark cycles at 22 °C. The mice were divided into two groups (n = 10 for each group): normal group (mice that were fed with a standard irradiated rodent chow diet), high fat diet (mice that were fed with high fat diet) group. After mice were fed with chow diet or high fat diet for 8 weeks (each one was fed 3 g at a time), the epididymal white adipose tissues (eWAT), inguinal white adipose tissues (iWAT) and brown adipose tissues (BAT) were collected from each mouse. The contents of major macronutrients in basic feed and high-fat feed are shown in Table 1 as follows. 2.2. Real-time PCR The total RNA of eWAT, iWAT and BAT were isolated using Trizol reagent (Invitrogen, Carlsbad, CA) according to the instruction of manufacture. Then the first strand of cDNA was synthesized using MMLV Reverse Transcriptase kit (TaKaRa, Da Lian, China). Real-time PCR was conducted by using SYBR Premix Ex TaqTM Kit (TaKaRa). The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). Briefly, samples were incubated at 95 °C for 5 min for initial denaturation, and followed by 40 cycles at 95 °C for 10 s and 60 °C for 34 s. The primers used in this study were synthesized from Sangon Biotech (Shanghai, China). The relative expression of genes was measured by 2-△△CT method. 2.3. Tail intravenous injection MiR-324-5p antagomir and relative control were all obtained from Biomics Biotechnologies (Nantong, China). The mice in high fat diet group were maintained at room temperature (21–23 °C) or thermoneutrality (30 °C) condition. Then mice were tail intravenous injected with miR-324-5p antagomir or control antagomir (5 mg/kg). The body weight was measured every week after injection.
Fig. 1. The expression of miR-324-5p was up-regulated in eWAT, iWAT and BAT. After mice were fed with chow diet or high fat diet for 8 weeks, the eWAT, iWAT and BAT were collected from each mouse. Then, the total RNA was isolated from eWAT, iWAT and BAT. A. The expression of miR-324-5p in eWAT was measured by Real-time PCR. B. The expression of miR-324-5p in iWAT was measured by Real-time PCR. C. The expression of miR-324-5p in BAT was measured by Real-time PCR. *P < 0.05 vs chow diet. **P < 0.01 vs chow diet.
2.4. Measurement of body composition Body composition was measured by DXA (Delphi A; Hologic, Inc., Table 1 Macronutrient content of the two groups of feeds (in 100 g). Feedstuffs
Constituents (%)
Normal Chow High-fat diet
Protein 17.02 19.43
Fat 7.91 25.77
Bedford, MA), using the small animal software. This software allows for whole body and subregion analysis of body composition. The coefficients of variation (%CV) are 0.54% for BMC, 0.58% for BMD, 0.40% for lean mass, 1.66% for fat mass. Lean and fat mass was measured for the total body (Carter et al., 2004).
Total calories Carbohydrates 48.02 35.06
(KJ) 1423.52 1937.48
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Fig. 2. Inhibition of miR-324-5p promotes fat metabolism and thermogenesis in vivo. After mice in high fat diet group were tail intravenous injected with miR324-5p antagomir or control antagomir, the mice were maintained in room temperature or thermoneutrality condition for 8 weeks, separately. A. The body weight of mice that in room temperature group was measured. B. The body weight of mice that in thermoneutrality group was measured. C. Body composition of mice that in room temperature group was measured by DXA (Delphi A; Hologic, Inc., Bedford, MA), using the small animal software. D. Body composition of mice that in thermoneutrality group was measured. *P < 0.05 vs ctrl-anta, **P < 0.01 vs ctrl-anta. Mice in high fat diet group were tail intravenous injected with miR-324-5p antagomir or control antagomir, and the mice were maintained in thermoneutrality condition. E. The effect of anta-miR-324-5p on the weight of iWAT and BAT was measured. F. The effect of anta-miR-324-5p on the expression of thermogenic genes in BAT was measured by Real-time PCR. G. The effect of anta-miR-324-5p on the expression of thermogenic genes in iWAT was measured by Real-time PCR. H. The effect of anta-miR-324-5p on thermogenic genes in BAT was evaluated by Western blotting. I. The effect of anta-miR-324-5p on thermogenic genes in iWAT was evaluated by Western blotting. *P < 0.05 vs ctrl-anta, **P < 0.01 vs ctrl-anta.
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Fig. 3. PM20D1 was a direct target of miR324-5p. A. The potential target of miR-324-5p was measured using TargetScan 7.1 software. B. The sequences of wild type or mutant type of 3′UTR of PM20D1 gene. C. A dual luciferase assay was conducted in HEK293 cells, **P < 0.01 vs PM20D1 WT 3′UTR + ctrl-mimic group or PM20D1 MUT 3′UTR +324-5p-mimic group. D. The effect of 324-5p mimic or 324-5panta on the expression of PM20D1 was measured by Western blotting, **P < 0.01 vs ctrlmimic or ctrl-anta group.
2.5. Cellular respiration measurements
2.8. Statistical analysis
The primary BAT cells and iWAT cells were isolated and transfected with miR-324-5p antagomir. After the transfected cells were incubation with C18 : 1, cellular oxygen consumption rates (OCR) were measured using an XF24 Extracellular Flux Analyzer according to previous study described (Long et al., 2016).
All data were analyzed using SPSS16.0 and were expressed as mean ± SD. P < 0.05 was considered statistically significant. 3. Results 3.1. The expression of miR-324-5p was up-regulated in eWAT, iWAT and BAT
2.6. Dual luciferase activity assay
To analyze whether miR-324-5p may play a role in regulating the mass of adipose tissue, the mice were fed with high fat diet and the expression of miR-324-5p in different fat tissues was measured. The results of real-time PCR showed that high fat diet significantly induced miR-324-5p expression in eWAT, iWAT and BAT compared with chow diet group (P < 0.05, Fig. 1A), especially in iWAT and BAT (P < 0.01, Fig. 1B and C), These results indicated that miR-324-5p may involve in the process of fat metabolism and thermogenesis.
For dual luciferase activity assay, the HEK293 cells were co-transfected with miR-324-5p mimic and p-MIR-report plasmid (Ambion, Austin, TX, USA) containing the wide type of 3′-UTR of PM20D1, or cotransfected with miR-324-5p mimic and p-MIR-report plasmid (Ambion, Austin, TX, USA) containing the mutant type of 3′-UTR of PM20D1NC, or transfected with mimic control and p-MIR-report plasmid (Ambion, Austin, TX, USA) containing the 3′-UTR of PM20D1. The control group was transfected with only p-MIR-report plasmid. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega, WI, USA) after 48 h of transfection.
3.2. Inhibition of miR-324-5p promotes fat metabolism and thermogenesis in vivo
2.7. Western blot To investigate if miR-324-5p was involved in the process of fat metabolism in vivo, anta-miR-324-5p was injected into the high fat diet induced mice. The results showed that when the mice were injected with anta-miR-324-5p for 7 weeks, the body weight gain was significantly suppressed in the condition of room temperature (P < 0.05, Fig. 2A), and was dramatically suppressed in the condition of thermoneutrality (P < 0.01, Fig. 2B). The results of body composition showed a decrease in fat mass and total weight gain was noticeably decreased in the condition of room temperature, and the effect was more pronounced in the thermoneutrality group (P < 0.01, Fig. 2C and D). It is noteworthy that anta-miR-324-5p has no effect on lean body mass, suggesting that the treatment does not compromise the development or growth of the animal. To further analyze if miR-324-5p was involved in the process of thermogenesis in vivo, the mice in the group of high fat diet were
The primary iWAT cells were isolated and transfected with miR324-5p mimic, control mimic, anta-miR-324-5p or control-anta for 48 h, the total proteins were extracted by radioimmunoprecipitation assay (RIPA) protein extraction. Then, 30 μg of protein was separated by SDS-PAGE gel electrophoresis and transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). The membrane was incubated overnight in PM20D1 antibody (Abcam, Cambridge, UK). The membrane was washed with tris buffered saline and Tween-20 (TBST) three times. Then the secondary antibody was added for 1 h incubation at room temperature. The imaging was performed with electron chemiluminescence (ECL) emitting solution. The proteins were visualized with an Anmobilon Western Chemiluminescent HRP Substrate system (Millipore Corp., Billerica, MA, USA). 4
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MUT group (Fig. 3C). The results of Western blot showed that overexpression of miR-324-5p significantly suppressed the expression of PM20D1, whereas inhibition of miR-324-5p dramatically increased the expression of PM20D1 (P < 0.01, Fig. 3D). These results indicated that PM20D1 was the direct target of miR-324-5p.
3.4. Inhibition of miR-324-5p increased cellular oxygen consumption in primary BAT and iWAT cells Finally, the effect of miR-324-5p on the oxygen consumption rates (OCR) was measured. The results showed that anta-miR-324-5p significantly increased OCR of primary BAT and iWAT cells compared with control group (P < 0.05, Fig. 4A and B).
4. Discussion In recent years, many studies have estimated that miRNAs were involved in the progression of obesity. The present study showed that miR-324-5p was significantly overexpressed in iWAT and BAT after mice were fed a high fat diet. Traditional, WAT has been regarded as an organ for energy storage and BAT was responsible for thermogenesis (Jia et al., 2016; Minji et al., 2015; Sánchez-Infantes et al., 2016). There is also a study reported that brown adipocyte-like adipocytes, termed beige adipocytes, are found in the WAT, including the iWAT of humans (Aldhahi, 2009; Minji et al., 2015; Wd et al., 2009). In addition, beige adipocytes also expressed uncoupling protein 1(UCP1) and were specialized to dissipate stored chemical energy in the form of heat. Therefore, it is well explained why miR-324-5p was also up-regulated in iWAT. The further experiments proved that inhibition of miR-324-5p dramatically suppressed the body weight gain, the fat and total weight gain both in room temperature and thermoneutrality condition. Interestingly, we also found that the inhibition effect of miR-324-5p was higher in thermoneutrality condition than room temperature. Inhibition of miR-324-5p also suppressed the weight gain of iWAT and BAT in the condition of thermoneutrality. Thus, we speculated that miR-324-5p might involve in the process of fat metabolism and thermogenesis. It was reported that activation of brown adipose is stimulated by hormones (mainly adrenaline) controlled by the sympathetic signals originating in the hypothalamus (Cai et al., 2019; Derghal et al., 2018). However, regulation on brown adipose by genes (including proteincoding genes and non-coding transcripts) can be direct. For example, Roger et al. recently found that Argonaute-2, a core component in the machinery of miRNA regulating gene expression, was directly associated with brown adipose tissue activation (Roger et al., 2019). Another more direct evidence is that miR-494-3p regulates thermogenesis signaling in mouse beige adipocytes under mild cold exposure (Lemecha et al., 2018). Here, our findings also indicated that manipulation of miR-324-5p could directly regulate the activation of in brown adipocytes. Peptidase M20 domain containing 1 (PM20D1) is a newly identified secreted enzyme that is enriched in UCP1+ versus UCP1- adipocytes. Previous study showed that mice with increased PM20D1 have augmented respiration and increased N-acyl amino acids in blood, and the N-acyl amino acids improved glucose homeostasis and increased energy expenditure (Long et al., 2016). Our results showed that PM20D1 was the direct target of miR-324-5p. Inhibition of miR-324-5p significantly increased the expression of PM20D1. Furthermore, the cellular oxygen consumption of primary BAT and iWAT cells were noticeably increased by anta-miR-324-5p. In conclusion, the present study first provides the evidence that inhibition of miR-324-5p increases PM20D1-mediated fat consumption and reduces body weight in mice. MiR-324-5p may be a novel therapeutic target for the prevention and treatment of childhood obesity.
Fig. 4. Inhibition of miR-324-5p increased cellular oxygen consumption in primary BAT and iWAT cells. The primary BAT and iWAT cells were isolated and transfected with miR-324-5p antagomir. A. The cellular oxygen consumption rate of BAT was measured. A. The cellular oxygen consumption rate of iWAT was measured. *P < 0.05 vs ctrl-anta.
injected with anta-miR-324-5p and was housed in the condition of thermoneutrality. The results showed that anta-miR-324-5p significantly inhibited the weight of iWAT and BAT (P < 0.05, Fig. 2E). Furthermore, we also found that anta-miR-324-5p dramatically upregulated the expression of UCP1, Ckmt2 and PM20D1 in BAT (P < 0.05, Fig. 2F). The expression of UCP1, Cox4, Ckmt2 and PM20D1 in iWAT was also up-regulated by anta-miR-324-5p (P < 0.05, Fig. 2G). Interestingly, UCP1, Ckmt2 and Cox4 are thermogenic genes, and their expression levels were significantly increased under the action of anta-mir-324-5p, which indicated that anta-miR324-5p had an effect on thermogenesis in vivo. 3.3. PM20D1 was a direct target gene of miR-324-5p To further investigate how miR-324-5p regulated the progress of fat metabolism and thermogenesis, the target gene of miR-324-5p was measured. First, the TargetScan 7.1 was used to search the target gene of miR-324-5p. The results showed that among the genes of UCP1, Ckmt2, PM20D1, Cox4, miR-324-5p could only target the 3′UTR region of PM20D1 (Fig. 3A). Then, dual luciferase reporter vectors including the wild type (WT) luciferase reporter vector and a mutated (MUT) luciferase reporter vector with a mutation at the binding sites were established (details shown in Fig. 3B). The dual luciferase activity assay proved that miR-324-5p mimic could significantly suppress the luciferase activity in the WT group but had no effect on and the that of the 5
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Declaration of competing interest
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Full length article
Inhibition of miR-324-5p increases PM20D1-mediated white and brown adipose loss and reduces body weight in juvenile mice
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Dandan Li, Yang Liu, Wei Gao, Jiakai Han, Rongrong Yuan, Mengdi Zhang, Wuyan Pang∗ Department of Endocrinology, Huaihe Hospital of Henan University, Kaifeng, 475000, Henan Province, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-324-5p PM20D1 Obesity Fat consumption Body weight
Obesity is a serious public health problem characterized by abnormal or excessive fat accumulation, which is caused by an energy imbalance between calories consumed and calories expended. MiRNAs have been involved in the regulation of occurrence and progression of obesity. This study aims to investigate the role of miR-324-5p in regulating the adipose tissue mass and preliminarily probe into its effect on progression of obesity. MiR-3245p was upregulated in the epididymal white adipose tissues (eWAT), inguinal white adipose tissues (iWAT) and brown adipose tissues (BAT) of the mice fed with high fat diet (HFD). Under room temperature (RT) or thermoneutrality (TN) condition, when tail intravenously injected with miR-324-5p antagomir (anta-miR-324-5p), the fat mass and total weight of mice were both significantly suppressed. The suppressive effect was more distinct under TN than RT. The weight of iWAT and BAT were both inhibited by anta-miR-324-5p under TN. Moreover, PM20D1 was a direct target gene of miR-324-5p. In primary iWAT cells, the expression of PM20D1 was significantly increased by anta-miR-324-5p, whereas decreased by the miR-324-5p mimic. Furthermore, anta-miR-324-5p noticeably increased the cellular oxygen consumption in primary BAT and iWAT cells. Our findings indicated that inhibition of miR-324-5p increased PM20D1-mediated fat consumption and reduced body weight in mice, suggesting that miR-324-5p may be a novel therapeutic target against obesity.
1. Introduction Nowadays, childhood obesity and overweight has become a serious public health problem all over the word. The prevalence of childhood obesity was reported to increase by 47.1% between 1980 and 2013 (Ng et al., 2014). According to the data of Chinese National Surveys on Students Constitution Health (CNSSCH), the prevalence rates of overweight of children nearly doubled from 4.1% in 2000 to 8.1% in 2010 (Sun et al., 2015a). Childhood obesity is associated with various comorbidities, such as cardiovascular diseases (Nielsen et al., 2015), type II diabetes (Hayden et al., 2016), metabolic syndrome (Liang et al., 2015) and sleep apnea (Bazzano et al., 2016). So, it is urgent to find an effective approach for the prevention of childhood obesity. During the past thirty or forty years, it has been acknowledged that, in addition to coding protein genes, most of the mammalian genomes are transcribed into a great variety of types of non-coding RNAs (ncRNAs). NcRNAs are emerging as important regulatory factors in the pathogenesis of diseases. MiRNAs are a class of short non-coding RNA molecules that regulate gene expression at the post transcriptional level (Sandhir et al., 2014). They have been proven to control various biological progresses, such as cell growth, cell differentiation and
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apoptosis (Sun et al., 2015b; Wang et al., 2015; Yu et al., 2014). Many studies have demonstrated that miRNA plays an important role in the progression of obesity, such as miR-21 (Seeger et al., 2014), miR-103 (Vinnikov et al., 2014), miR-34a (Fu et al., 2014). Obesity is a complex disease that is regulated by a variety of factors including genetics, endocrine, drugs, lifestyle, and adipocytokines. When the body consumes more calories than calories, the excess calories are stored in the body in the form of fat, which exceeds the normal physiological requirements, and when it accumulates a certain value, it evolves into obesity (Jordania et al., 2017). In the processes of lipogenesis and consumption, some small RNAs play important roles. For example, Martinelli et al. reported that miR519 was up-regulated and associated with metabolic imbalance and subsequent adipocyte hypertrophy through suppressing the expression of PPARA in subcutaneous adipose tissues (Martinelli et al., 2010); Shi et al. reported that miR-148 was associated with obesity and modulated adipocyte differentiation of mesenchymal stem cells through Wnt signaling (Shi et al., 2015). MiR-324-5p is a relatively well characterized onco-miRNA in many types of carcinomas. It was recently shown to be associated with metabolism in liver and adipose tissues (Guenther et al., 2007; Szkolnicka
Corresponding author. Department of Endocrinology, Huaihe Hospital of Henan University, No. 1 Baobei Road, Kaifeng, 475000, Henan Province, China. E-mail address: [email protected] (W. Pang).
https://doi.org/10.1016/j.ejphar.2019.172708 Received 22 August 2019; Received in revised form 25 September 2019; Accepted 26 September 2019 Available online 27 September 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
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et al., 2016). Moreover, miR-324-5p was differentially expressed in visceral adipose tissues of patients with abnormal metabolism (Estep et al., 2010). A couple of recent studies showed that miR-324 was dysregulated during tissue injury and revealed that miR-324 was involved in energy metabolism in cell stress (Diaz et al., 2017; Liu et al., 2019). Our previous microarray study showed that miR-324-5p was significantly up-regulated in the heart, liver, fat and various secretory organs of juvenile animals under cold conditions. Our team has been studying endocrinal and metabolic disorders in children, such as childhood obesity. Herein, we investigated the role of miR-324-5p in regulating the adipose tissue mass and preliminarily probe into its effect on progression of obesity. 2. Materials and methods 2.1. Animals and diets Animal experiments were approved in accordance with the guidelines by Huaihe Hospital of Henan University (Kaifeng, China). Three week old weaned BALB/C mice were purchased from BetterBiotechnology Co., Ltd (Nanjing, China) and were maintained in 12 h light: dark cycles at 22 °C. The mice were divided into two groups (n = 10 for each group): normal group (mice that were fed with a standard irradiated rodent chow diet), high fat diet (mice that were fed with high fat diet) group. After mice were fed with chow diet or high fat diet for 8 weeks (each one was fed 3 g at a time), the epididymal white adipose tissues (eWAT), inguinal white adipose tissues (iWAT) and brown adipose tissues (BAT) were collected from each mouse. The contents of major macronutrients in basic feed and high-fat feed are shown in Table 1 as follows. 2.2. Real-time PCR The total RNA of eWAT, iWAT and BAT were isolated using Trizol reagent (Invitrogen, Carlsbad, CA) according to the instruction of manufacture. Then the first strand of cDNA was synthesized using MMLV Reverse Transcriptase kit (TaKaRa, Da Lian, China). Real-time PCR was conducted by using SYBR Premix Ex TaqTM Kit (TaKaRa). The reaction was run in ABI7500 Real-time PCR system (Applied Biosystems, Carlsbad, CA). Briefly, samples were incubated at 95 °C for 5 min for initial denaturation, and followed by 40 cycles at 95 °C for 10 s and 60 °C for 34 s. The primers used in this study were synthesized from Sangon Biotech (Shanghai, China). The relative expression of genes was measured by 2-△△CT method. 2.3. Tail intravenous injection MiR-324-5p antagomir and relative control were all obtained from Biomics Biotechnologies (Nantong, China). The mice in high fat diet group were maintained at room temperature (21–23 °C) or thermoneutrality (30 °C) condition. Then mice were tail intravenous injected with miR-324-5p antagomir or control antagomir (5 mg/kg). The body weight was measured every week after injection.
Fig. 1. The expression of miR-324-5p was up-regulated in eWAT, iWAT and BAT. After mice were fed with chow diet or high fat diet for 8 weeks, the eWAT, iWAT and BAT were collected from each mouse. Then, the total RNA was isolated from eWAT, iWAT and BAT. A. The expression of miR-324-5p in eWAT was measured by Real-time PCR. B. The expression of miR-324-5p in iWAT was measured by Real-time PCR. C. The expression of miR-324-5p in BAT was measured by Real-time PCR. *P < 0.05 vs chow diet. **P < 0.01 vs chow diet.
2.4. Measurement of body composition Body composition was measured by DXA (Delphi A; Hologic, Inc., Table 1 Macronutrient content of the two groups of feeds (in 100 g). Feedstuffs
Constituents (%)
Normal Chow High-fat diet
Protein 17.02 19.43
Fat 7.91 25.77
Bedford, MA), using the small animal software. This software allows for whole body and subregion analysis of body composition. The coefficients of variation (%CV) are 0.54% for BMC, 0.58% for BMD, 0.40% for lean mass, 1.66% for fat mass. Lean and fat mass was measured for the total body (Carter et al., 2004).
Total calories Carbohydrates 48.02 35.06
(KJ) 1423.52 1937.48
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Fig. 2. Inhibition of miR-324-5p promotes fat metabolism and thermogenesis in vivo. After mice in high fat diet group were tail intravenous injected with miR324-5p antagomir or control antagomir, the mice were maintained in room temperature or thermoneutrality condition for 8 weeks, separately. A. The body weight of mice that in room temperature group was measured. B. The body weight of mice that in thermoneutrality group was measured. C. Body composition of mice that in room temperature group was measured by DXA (Delphi A; Hologic, Inc., Bedford, MA), using the small animal software. D. Body composition of mice that in thermoneutrality group was measured. *P < 0.05 vs ctrl-anta, **P < 0.01 vs ctrl-anta. Mice in high fat diet group were tail intravenous injected with miR-324-5p antagomir or control antagomir, and the mice were maintained in thermoneutrality condition. E. The effect of anta-miR-324-5p on the weight of iWAT and BAT was measured. F. The effect of anta-miR-324-5p on the expression of thermogenic genes in BAT was measured by Real-time PCR. G. The effect of anta-miR-324-5p on the expression of thermogenic genes in iWAT was measured by Real-time PCR. H. The effect of anta-miR-324-5p on thermogenic genes in BAT was evaluated by Western blotting. I. The effect of anta-miR-324-5p on thermogenic genes in iWAT was evaluated by Western blotting. *P < 0.05 vs ctrl-anta, **P < 0.01 vs ctrl-anta.
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Fig. 3. PM20D1 was a direct target of miR324-5p. A. The potential target of miR-324-5p was measured using TargetScan 7.1 software. B. The sequences of wild type or mutant type of 3′UTR of PM20D1 gene. C. A dual luciferase assay was conducted in HEK293 cells, **P < 0.01 vs PM20D1 WT 3′UTR + ctrl-mimic group or PM20D1 MUT 3′UTR +324-5p-mimic group. D. The effect of 324-5p mimic or 324-5panta on the expression of PM20D1 was measured by Western blotting, **P < 0.01 vs ctrlmimic or ctrl-anta group.
2.5. Cellular respiration measurements
2.8. Statistical analysis
The primary BAT cells and iWAT cells were isolated and transfected with miR-324-5p antagomir. After the transfected cells were incubation with C18 : 1, cellular oxygen consumption rates (OCR) were measured using an XF24 Extracellular Flux Analyzer according to previous study described (Long et al., 2016).
All data were analyzed using SPSS16.0 and were expressed as mean ± SD. P < 0.05 was considered statistically significant. 3. Results 3.1. The expression of miR-324-5p was up-regulated in eWAT, iWAT and BAT
2.6. Dual luciferase activity assay
To analyze whether miR-324-5p may play a role in regulating the mass of adipose tissue, the mice were fed with high fat diet and the expression of miR-324-5p in different fat tissues was measured. The results of real-time PCR showed that high fat diet significantly induced miR-324-5p expression in eWAT, iWAT and BAT compared with chow diet group (P < 0.05, Fig. 1A), especially in iWAT and BAT (P < 0.01, Fig. 1B and C), These results indicated that miR-324-5p may involve in the process of fat metabolism and thermogenesis.
For dual luciferase activity assay, the HEK293 cells were co-transfected with miR-324-5p mimic and p-MIR-report plasmid (Ambion, Austin, TX, USA) containing the wide type of 3′-UTR of PM20D1, or cotransfected with miR-324-5p mimic and p-MIR-report plasmid (Ambion, Austin, TX, USA) containing the mutant type of 3′-UTR of PM20D1NC, or transfected with mimic control and p-MIR-report plasmid (Ambion, Austin, TX, USA) containing the 3′-UTR of PM20D1. The control group was transfected with only p-MIR-report plasmid. Luciferase activity was measured using a dual-luciferase reporter assay system (Promega, WI, USA) after 48 h of transfection.
3.2. Inhibition of miR-324-5p promotes fat metabolism and thermogenesis in vivo
2.7. Western blot To investigate if miR-324-5p was involved in the process of fat metabolism in vivo, anta-miR-324-5p was injected into the high fat diet induced mice. The results showed that when the mice were injected with anta-miR-324-5p for 7 weeks, the body weight gain was significantly suppressed in the condition of room temperature (P < 0.05, Fig. 2A), and was dramatically suppressed in the condition of thermoneutrality (P < 0.01, Fig. 2B). The results of body composition showed a decrease in fat mass and total weight gain was noticeably decreased in the condition of room temperature, and the effect was more pronounced in the thermoneutrality group (P < 0.01, Fig. 2C and D). It is noteworthy that anta-miR-324-5p has no effect on lean body mass, suggesting that the treatment does not compromise the development or growth of the animal. To further analyze if miR-324-5p was involved in the process of thermogenesis in vivo, the mice in the group of high fat diet were
The primary iWAT cells were isolated and transfected with miR324-5p mimic, control mimic, anta-miR-324-5p or control-anta for 48 h, the total proteins were extracted by radioimmunoprecipitation assay (RIPA) protein extraction. Then, 30 μg of protein was separated by SDS-PAGE gel electrophoresis and transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). The membrane was incubated overnight in PM20D1 antibody (Abcam, Cambridge, UK). The membrane was washed with tris buffered saline and Tween-20 (TBST) three times. Then the secondary antibody was added for 1 h incubation at room temperature. The imaging was performed with electron chemiluminescence (ECL) emitting solution. The proteins were visualized with an Anmobilon Western Chemiluminescent HRP Substrate system (Millipore Corp., Billerica, MA, USA). 4
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MUT group (Fig. 3C). The results of Western blot showed that overexpression of miR-324-5p significantly suppressed the expression of PM20D1, whereas inhibition of miR-324-5p dramatically increased the expression of PM20D1 (P < 0.01, Fig. 3D). These results indicated that PM20D1 was the direct target of miR-324-5p.
3.4. Inhibition of miR-324-5p increased cellular oxygen consumption in primary BAT and iWAT cells Finally, the effect of miR-324-5p on the oxygen consumption rates (OCR) was measured. The results showed that anta-miR-324-5p significantly increased OCR of primary BAT and iWAT cells compared with control group (P < 0.05, Fig. 4A and B).
4. Discussion In recent years, many studies have estimated that miRNAs were involved in the progression of obesity. The present study showed that miR-324-5p was significantly overexpressed in iWAT and BAT after mice were fed a high fat diet. Traditional, WAT has been regarded as an organ for energy storage and BAT was responsible for thermogenesis (Jia et al., 2016; Minji et al., 2015; Sánchez-Infantes et al., 2016). There is also a study reported that brown adipocyte-like adipocytes, termed beige adipocytes, are found in the WAT, including the iWAT of humans (Aldhahi, 2009; Minji et al., 2015; Wd et al., 2009). In addition, beige adipocytes also expressed uncoupling protein 1(UCP1) and were specialized to dissipate stored chemical energy in the form of heat. Therefore, it is well explained why miR-324-5p was also up-regulated in iWAT. The further experiments proved that inhibition of miR-324-5p dramatically suppressed the body weight gain, the fat and total weight gain both in room temperature and thermoneutrality condition. Interestingly, we also found that the inhibition effect of miR-324-5p was higher in thermoneutrality condition than room temperature. Inhibition of miR-324-5p also suppressed the weight gain of iWAT and BAT in the condition of thermoneutrality. Thus, we speculated that miR-324-5p might involve in the process of fat metabolism and thermogenesis. It was reported that activation of brown adipose is stimulated by hormones (mainly adrenaline) controlled by the sympathetic signals originating in the hypothalamus (Cai et al., 2019; Derghal et al., 2018). However, regulation on brown adipose by genes (including proteincoding genes and non-coding transcripts) can be direct. For example, Roger et al. recently found that Argonaute-2, a core component in the machinery of miRNA regulating gene expression, was directly associated with brown adipose tissue activation (Roger et al., 2019). Another more direct evidence is that miR-494-3p regulates thermogenesis signaling in mouse beige adipocytes under mild cold exposure (Lemecha et al., 2018). Here, our findings also indicated that manipulation of miR-324-5p could directly regulate the activation of in brown adipocytes. Peptidase M20 domain containing 1 (PM20D1) is a newly identified secreted enzyme that is enriched in UCP1+ versus UCP1- adipocytes. Previous study showed that mice with increased PM20D1 have augmented respiration and increased N-acyl amino acids in blood, and the N-acyl amino acids improved glucose homeostasis and increased energy expenditure (Long et al., 2016). Our results showed that PM20D1 was the direct target of miR-324-5p. Inhibition of miR-324-5p significantly increased the expression of PM20D1. Furthermore, the cellular oxygen consumption of primary BAT and iWAT cells were noticeably increased by anta-miR-324-5p. In conclusion, the present study first provides the evidence that inhibition of miR-324-5p increases PM20D1-mediated fat consumption and reduces body weight in mice. MiR-324-5p may be a novel therapeutic target for the prevention and treatment of childhood obesity.
Fig. 4. Inhibition of miR-324-5p increased cellular oxygen consumption in primary BAT and iWAT cells. The primary BAT and iWAT cells were isolated and transfected with miR-324-5p antagomir. A. The cellular oxygen consumption rate of BAT was measured. A. The cellular oxygen consumption rate of iWAT was measured. *P < 0.05 vs ctrl-anta.
injected with anta-miR-324-5p and was housed in the condition of thermoneutrality. The results showed that anta-miR-324-5p significantly inhibited the weight of iWAT and BAT (P < 0.05, Fig. 2E). Furthermore, we also found that anta-miR-324-5p dramatically upregulated the expression of UCP1, Ckmt2 and PM20D1 in BAT (P < 0.05, Fig. 2F). The expression of UCP1, Cox4, Ckmt2 and PM20D1 in iWAT was also up-regulated by anta-miR-324-5p (P < 0.05, Fig. 2G). Interestingly, UCP1, Ckmt2 and Cox4 are thermogenic genes, and their expression levels were significantly increased under the action of anta-mir-324-5p, which indicated that anta-miR324-5p had an effect on thermogenesis in vivo. 3.3. PM20D1 was a direct target gene of miR-324-5p To further investigate how miR-324-5p regulated the progress of fat metabolism and thermogenesis, the target gene of miR-324-5p was measured. First, the TargetScan 7.1 was used to search the target gene of miR-324-5p. The results showed that among the genes of UCP1, Ckmt2, PM20D1, Cox4, miR-324-5p could only target the 3′UTR region of PM20D1 (Fig. 3A). Then, dual luciferase reporter vectors including the wild type (WT) luciferase reporter vector and a mutated (MUT) luciferase reporter vector with a mutation at the binding sites were established (details shown in Fig. 3B). The dual luciferase activity assay proved that miR-324-5p mimic could significantly suppress the luciferase activity in the WT group but had no effect on and the that of the 5
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Declaration of competing interest
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