- Email: [email protected]
Overexpression of microRNA-9 inhibits 3T3-L1 cell adipogenesis by targeting PNPLA3 via activation of AMPK
Journal Pre-proofs Research paper Overexpression of microRNA-9 inhibits 3T3-L1 cell adipogenesis by targeting PNPLA3 via activation of AMPK Beibei Xu, Jianhua Shen, Dan Li, Baoshuo Ning, Liping Guo, Hao Bing, Jiayu Chen, Yiling Li PII: DOI: Reference:
S0378-1119(19)30919-9 https://doi.org/10.1016/j.gene.2019.144260 GENE 144260
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
31 May 2019 6 November 2019 7 November 2019
Please cite this article as: B. Xu, J. Shen, D. Li, B. Ning, L. Guo, H. Bing, J. Chen, Y. Li, Overexpression of microRNA-9 inhibits 3T3-L1 cell adipogenesis by targeting PNPLA3 via activation of AMPK, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene.2019.144260
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Overexpression of microRNA-9 inhibits 3T3-L1 cell adipogenesis by targeting PNPLA3 via activation of AMPK
Beibei Xua,1, Jianhua Shenb,1, Dan Lia, Baoshuo Ninga, Liping Guoa, Hao Binga, Jiayu Chena, Yiling Lia,*,[email protected]
aDepartment
of Gastroenterology, the First Affiliated Hospital of China Medical
University, Shenyang 110001, China bDepartment
of Gastroenterology, the Affiliated Hospital of Qingdao University,
Qingdao 266000, China *Corresponding
authors. Department of Gastroenterology, the First Affiliated Hospital
of China Medical University, NO. 155, North Nanjing Street, Heping District, Shenyang City, Liaoning 110001, China.
1The
authors contributed equally to the work.
Abstract Adipocyte differentiation, which plays an important role in the development of obesity, involves complex molecular networks in which microRNAs (miRNAs) are essential. Here, we show that miR-9 expression was upregulated during adipogenesis in 3T3-L1 cells. miR-9 overexpression reduced the accumulation of lipid droplets and the content of triglycerides by downregulating patatin-like phospholipase domain-containing protein 3 (PNPLA3). PNPLA3 knockdown or miR-9 overexpression downregulated the protein expression of peroxisome proliferator-activated receptor γ, oxidative tissueenriched PAT, and LC3, and promoted the phosphorylation of AMP-activated protein kinase (AMPK), which was inhibited by treatment with the AMPK inhibitor compound C. These results indicate that miR-9 inhibits adipocyte differentiation by targeting PNPLA3 through the AMPK pathway.
Keywords: miR-9; adipogenesis; PNPLA3; AMPK; autophagy
Abbreviations 3′-UTR, 3′ untranslated region AMPK, AMP-activated protein kinase DM, differentiation medium FBS, fetal bovine serum miRNAs, MicroRNAs PNPLA3, Patatin-like phospholipase domain-containing protein 3 PPARγ, peroxisome proliferator-activated receptor γ OXPAT, oxidative tissue-enriched PAT protein
1 Introduction Obesity is a serious public health problem worldwide, and it is associated with diseases such as diabetes, hyperglycemia, hyperlipidemia, and atherosclerosis (Bray et al., 2017; Soares et al., 2017). Obesity is a metabolic abnormality characterized by excessive accumulation of fat. This accumulation is caused by an imbalance of body energy associated with adipocyte hyperplasia and hypertrophy. Mature adipocytes are differentiated from preadipocytes and progenitor cells in adipose tissue, a process known as adipogenesis (Ali et al., 2013; Jeffery et al., 2016). Therefore, reducing adipose tissue accumulation can improve obesity and obesity-related diseases. MicroRNAs (miRNAs) are endogenous non-coding RNAs composed of 18–25 nucleotides that bind to the 3′ untranslated region (3′-UTR) of target mRNAs to regulate gene expression at the post-transcriptional level, thereby affecting a variety of biological processes (Hwang and Mendell, 2006; Shenoy and Blelloch, 2014). miRNAs are post-transcriptional regulators of fat development and adipogenesis (Jin et al., 2010; Ning et al., 2017; Shen et al., 2018b; Lorente-Cebrian et al., 2019). miR-185 negatively regulates the differentiation of 3T3-L1 cells by targeting sterol regulatory element binding protein 1 (Ning et al., 2017), whereas miR-144-3p promotes adipogenesis by
targeting Klf3 and CtBP2 (Shen et al., 2018b). miR-9 upregulation is associated with the progression of liver disease and the severity of nonalcoholic fatty liver disease, and upregulation of miR-9 by mimic transfection decreases intracellular lipid content in L02 cells (Ao et al., 2016). However, the molecular mechanism underlying the function of miR-9 in regulating adipogenesis remains to be elucidated. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) plays an important role in liver lipid metabolism, and changes in its expression level are closely related to lipogenesis (Huang et al., 2010). In human hepatocytes, PNPLA3 plays an important role in autophagosome formation during the process of lipophagy (Negoita et al., 2019). Autophagy is important for initiating adipocyte differentiation, and AMPactivated protein kinase (AMPK) plays a key role in autophagy and lipid metabolism (Hahm et al., 2014; Li et al., 2018). Sozio et al. (Sozio et al., 2011) showed that inhibition of AMPK in hepatoma cells can activate peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity, which can induce oxidative tissueenriched PAT protein (OXPAT) synthesis and promote the utilization of fatty acids (Wolins et al., 2006; Hadrich and Sayadi, 2018). In this study, we investigated the role of miR-9 in adipogenesis in 3T3-L1 preadipocytes and explored the underlying mechanism by identifying the factors involved. miR-9 expression was assessed in 3T3-L1 cells undergoing adipogenesis, and the regulatory relationship between miR-9 and its putative target gene PNPLA3 was examined. In addition, the potential effect of miR-9 or PNPLA3 on AMPK-related proteins during adipogenesis was examined in 3T3-L1 cells.
2 Materials and methods 2.1 Cell culture and adipogenic differentiation 3T3-L1 preadipocytes obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) were cultured in DMEM (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 5% CO2 and 37°C. To induce adipocyte differentiation, 3T3-L1 cells were incubated in differentiation medium (DM) supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-
methylxanthine, 250 nM dexamethasone, and 160 nM insulin (designed as day 0). After 2 days, the medium was replaced with DMEM containing 10% FBS and 160 nM insulin for an additional 2 days, after which the medium was replaced every other day until day 14.
2.2 Cell transfection The oligonucleotide sequences of miR-9 mimics (miR-9, sense: 5′-UGC UGC ACC UUA GUC UCU GG-3′, antisense: 5′-GGG UAC UUG CCG CGU UUA AG3′), negative control miRNA mimics (miR-NC, sense: 5′-AGA CUU GGG CAG UUC CUU UGG-3′, antisense: 5′-UUU GCC UGC UAG CUC UAA GUC-3′), miR-9 inhibitor (anti-miR-9, 5′-CUG UCU UAG UGU GUG CGG CUA-3′), negative control miRNA inhibitor (anti-miR-NC, 5′-UGG CUU UAG GGA AGC GUA GUC-3′), shRNA targeting PNPLA3 (shPNPLA3, 5′-CCC GTT CTT CAA CAT TAA CAA-3′), and negative control shRNAs (shRNA-NC, 5′-CAT TTT GCC TGC TAG CTC TAA GT-3′) were designed and synthesized by Genepharma (Shanghai, China). The 3T3-L1 preadipocytes were transfected with 50 nM miR-9, miR-NC, shPNPLA3, or shRNANC using LipofectamineTM 2000 (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Cells were collected 48 h after transfection and used for adipogenic differentiation.
2.3 Plasmid construction The wild-type 3′-UTR of PNPLA3 was amplified from genomic DNA of 3T3-L1 cells by PCR using the following primers: PNPLA3 sense, 5′-GGG TGG TGA CAG CCT TAA CA-3′; antisense, 5′- CTG TCA GCA TCG AAG GGG TT-3′. The mutant PNPLA3
3′-UTR
was
generated
using
the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. The wild-type and mutant PNPLA3 3′-UTRs were inserted downstream of the luciferase reporter gene in the psiCHECK-2 dual-luciferase reporter plasmid (Promega, Madison, USA) between XhoI and NotI restriction sites, and the constructs were designated PNPLA3-wt and PNPLA3-mut, respectively.
2.4 Luciferase reporter assay Luciferase reporter assays were performed as previously described (Shen et al., 2018a). Briefly, 3T3-L1 cells were co-transfected with 50 nM miR-9 mimics or miRNC and 100 ng recombinant PNPLA3-wt or PNPLA3-mut psiCHECK-2 vector using LipofectamineTM 2000 (Invitrogen). Luciferase activity was measured at 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions.
2.5 Oil red O staining Lipid droplets in cells were stained with Oil Red O as previously described with minor modifications (Liu et al., 2017). Briefly, 3T3-L1 cells transfected with miR-9 or shPNPLA3 were induced to differentiate into mature adipocytes in the presence or absence of 5 μM AMPK inhibitor compound C (Sigma, Louis, Mo, USA) for 14 days. Differentiated 3T3-L1 cells were washed twice with phosphate buffered saline (PBS), fixed in 4% formaldehyde for 30 min, and washed three times with PBS. The fixed cells were stained with 5% Oil red O (Sigma Aldrich, St. Louis, MO, USA) for 15 min and washed with PBS three times. The lipid droplets were visualized and photographed using a light phase contrast microscope (Nikon, Japan).
2.6 Triglyceride content assays Differentiated 3T3-L1 cells were washed with PBS and sonicated for 1 min to obtain homogenous samples. The triglyceride content in the samples was determined with a commercial kit (Biosino Bio-Technology and Science Inc., Beijing, China).
2.7 Quantitative real time PCR (qRT-PCR) Total RNA was extracted from cells using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed using Reverse Transcription Kits (Beyotime, China). qRT-PCR was performed using a SYBR Green PCR Master Mix (TaKaRa, Japan) on the Bio-RAD IQTM5 system (Bio-Rad,
Hercules, CA, USA). The PCR primer sequences were as follows: miR-9 sense, 5′TCT GCG CCT AGC ATT GGA AA-3′, antisense, 5′-AGT ATG GTG TGT GCG CTT GA-3′; U6 sense, 5′-TTG TTC CCT TCC CTT GTC GC-3′, antisense, 5′-AGA ACT TGT CAC CAT CCC GC-3′; PNPLA3 sense, 5′-CCT CCC AGA CAA TGT CCA CC3′, antisense, 5′-GGT TTT GGC ATC CAG CAC AG-3′; GAPDH sense, 5′-GCC TCG TCC CGT AGA CAA AA-3′, antisense, 5′-GCA ACA ATC TCC ACT TTG CCA-3′. The relative expression level of miR-9 was normalized to that of U6, and the relative expression level of PNPLA3 mRNA was normalized to that of the internal control GAPDH using the 2-ΔΔCt method (Cikos et al., 2007).
2.8 Western blot analysis Protein was extracted from 3T3-L1 cells using lysis buffer (Sigma), and protein concentration was determined with the BCA protein assay kit (Beyotime). For western blotting, 40 μg total cellular protein was separated by 10% SDS-PAGE and transferred to PVDF membranes (Roche, Germany). The membranes were incubated overnight at 4°C with primary antibodies against PNPLA3 (1:1500; Abcam, Cambridge, MA, USA), phospho-AMPK (Thr172) (1:2000; Cell Signaling Technology, USA), AMPK (1:2000; Cell Signaling Technology), PPARγ (1:1000; Abcam), OXPAT (1:1000; Abcam), and β-actin (1:2000; Abcam). After washing, the membranes were incubated with goat polyclonal anti-rabbit IgG–H&L-pre-adsorbed (HRP; 1:2000; Abcam) for 1 h at room temperature. The bands were developed using the enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare, USA). The densitometric values were quantified using Image J software (National Institutes of Health, USA) and normalized to β-actin.
2.9 Statistical analysis Data are expressed as the mean ± SD of three replicates in each experiment and analyzed using GraphPad Prism 6.0 software (GraphPad Inc., USA). Student's t-test was used to determine statistical differences between two groups, and comparisons between multiple groups were evaluated using one-way analysis of variance. P < 0.05
was considered statistically significant.
3 Results 3.1 miR-9 is upregulated during adipogenesis in 3T3-L1 cells Oil Red O staining showed that the number and size of lipid droplets gradually increased in a time-dependent manner in 3T3-L1 preadipocytes treated with differentiation medium (Fig. 1A), in parallel with an increase in cellular triglyceride content during differentiation (Fig. 1B). miR-9 expression during 3T3-L1 cell differentiation was examined by extracting total RNA from adipocytes on days 0, 1, 3, 7, and 14 after differentiation and measuring miR-9 levels by qRT-PCR. As shown in Fig. 1C, miR-9 expression was low on day 0 and 1 of differentiation induction, and gradually increased on day 3, peaked on day 7, and slightly decreased on day 14. These results indicated that miR-9 may play an important role during 3T3-L1 cell differentiation.
3.2 Overexpression of miR-9 inhibits 3T3-L1 adipogenesis To investigate the role of miR-9 in cell differentiation, 3T3-L1 cells were transfected with miR-9 mimics or miR-9 inhibitor during the 14 days of the differentiation period. miR-9 levels significantly increased upon miR-9 mimic transfection and decreased in response to miR-9 inhibitor transfection compared with the NC group (p < 0.01; Fig. 2A). Oil Red O staining showed that miR-9 overexpression decreased the formation of lipid droplets, whereas miR-9 knockdown increased the formation of lipid droplets (Fig. 2B). Consistent with these results, the triglyceride content in 3T3-L1 cells decreased significantly in the miR-9 mimics group and increased in the miR-9 inhibitor group compared with the NC group (p < 0.01 and P < 0.05, respectively; Fig. 2C). these results suggested that miR-9 can inhibit 3T3-L1 adipogenesis.
3.3 miR-9 directly targets the 3′-UTR of PNPLA3 To explore the mechanisms underlying the effect of miR-9 on suppressing 3T3-
L1 adipogenesis, bioinformatics analysis (TargetScan, Pic Tar and miRanda) was performed to identify the potential targets of miR-9. The result showed binding sites for miR-9 in the 3′-UTR of PNPLA3 (Fig. 3A). To determine whether PNPLA3 is a direct target of miR-9, the wild type (wt) or mutant (mut) PNPLA3 3′-UTR was subcloned into a luciferase reporter vector and co-transfected with miR-9 mimics or miR-NC into 3T3-L1 cells. The luciferase reporter assays showed that miR-9 decreased the luciferase activity of the wt 3′-UTR, but not that of the mut 3′-UTR, of PNPLA3 compared with the miR-NC group (p < 0.05; Fig. 3B). To determine whether miR-9 can regulate PNPLA3 expression, 3T3-L1 cells were transfected with miR-9 mimics, miR-NC, shRNA-NC, or shPNPLA3 as a positive control. As shown in Fig. 3C and D, PNPLA3 mRNA and protein levels were significantly decreased in the miR-9 mimics and shPNPLA3 groups compared with the NC group (p < 0.01). Taken together, these results indicated that PNPLA3 is a direct target of miR-9.
3.4 miR-9 inhibits 3T3-L1 cell adipogenesis by activating AMPK The molecular mechanism underlying the effect of miR-9 on inhibiting 3T3-L1 cell adipogenesis was further examined by inducing 3T3-L1 cells transfected with miR9 or shPNPLA3 to differentiate into mature adipocytes in the presence or absence of 5 μM of compound C (an inhibitor of AMPK) for 14 days. Oil Red O staining showed that the formation of lipid droplets and triglyceride content decreased in the miR-9 mimics and shPNPLA3 groups. Treatment with compound C abolished the effects of miR-9 mimics and shPNPLA3 (Fig. 4A and B), whereas compound C treatment had no effect on the expression of PNPLA3 (Fig. 4C). Western blot analysis of the protein levels of p-AMPK, AMPK, PPARγ, OXPAT, and LC3 in each group showed that PNPLA3 knockdown or miR-9 overexpression increased AMPK phosphorylation and downregulated PPARγ, OXPAT, and LC3 protein expression, and this effect was blocked by compound C (Fig. 4D–H). Taken together, these results suggest that miR-9 inhibits 3T3-L1 cell adipogenesis by inhibiting PNPLA3 and activating the AMPK signaling pathway.
4 Discussion In recent years, a large number of reports have shown that miRNAs are involved in preadipocyte differentiation, lipid metabolism, and lipogenesis (Jin et al., 2010; Ning et al., 2017; Shen et al., 2018b; Lorente-Cebrian et al., 2019). For example, miR-144, miR-16, and miR-214 promote adipogenesis (Shen et al., 2018b; Xi et al., 2019; Xu et al., 2019), whereas miR-152, miR-26, and miR-27 suppress preadipocyte differentiation and lipid metabolism (Acharya et al., 2019; Fan et al., 2019; Jang et al., 2019). However, the molecular mechanism underlying the role of miRNAs in lipogenesis is not fully understood. In this study, we found that miR-9 was upregulated during the adipogenesis of 3T3-L1 cells in the first 7 days, whereas its expression decreased slightly on day 14 (Fig. 1). This suggests the involvement of other genes in the regulation of miR-9 during adipogenesis, such as monocyte chemoattractant protein-1-induced protein 1 (MCPIP1)(Losko et al., 2018). We also showed that overexpression of miR-9 suppressed, whereas downregulation of miR-9 promoted 3T3L1 preadipocyte differentiation and lipid accumulation (Fig. 2). Ao et al. showed that miR-9 is upregulated during hepatic steatosis progression, and overexpression of miR-9 decreases the formation of intracellular lipid droplets in oleic acid-induced L-02 cells (Ao et al., 2016). These results indicate that miR-9 may serve as an inhibitor of adipogenesis. To screen mir-9 targets and identify binding sites involved in the interaction between miR-9 and its target, we used TargetScan, PicTar, and miRanda bioinformatics target prediction packages to predict miR-9 targets. We found that the 3′-UTR of PNPLA3 contains potential binding sites for miR-9. Luciferase report assays confirmed that miR-9 inhibited the transcriptional activity of wt PNPLA3. In addition, miR-9 overexpression and PNPLA3 knockdown inhibited adipogenesis (Fig. 3). These results suggest that PNPLA3 is the target of miR-9. PNPLA3 is a triglyceride lipase/acyltransferase that mediates triglyceride hydrolysis in adipocytes and hepatocytes (Min et al., 2014), and mutations in the PNPLA3 gene predispose obese adolescents to hepatic steatosis (Santoro et al., 2010; Sookoian and Pirola, 2011; Xu et al., 2015). The family of adipogenic-specific factors
include SREBP-1c and PPARγ(Zhang et al., 2004). AMPK can negatively regulate SREBP-1C to reduce the formation of free fatty acids (FFAs) (Seo et al., 2014). PNPLA3 is a determinant of triglyceride accumulation in the liver and is regulated by SREBP-1c (Ren et al., 2017). OXPAT is a homologous protein of Perilipin, which is mainly distributed in tissues and cells with a fast rate of lipid metabolism (Brasaemle, 2007). OXPAT activates the PPAR pathway and promotes the catabolism of fatty acids in cells (Wolins et al., 2006). Hahm et al. showed that alpha-lipoic acid attenuates adipocyte differentiation and lipid accumulation in 3T3-L1 cells via AMPKdependent autophagy(Hahm et al., 2014). To further determine whether the reduction of PNPLA3 induced by miR-9 induces the AMPK pathway, we used shRNA oligonucleotides against PNPLA3 to simulate the role of miR-9. The results showed that the content of triglycerides in 3T3-L1 cells treated with shPNPLA3 decreased compared with that in the NC group (Fig. 4A-C). In addition, the expression of marker genes for adipogenesis, such as PPARγ and OXPAT, as well as the autophagy-related gene LC3, were significantly inhibited, and these effects were blocked by the AMPK inhibitor compound C (Fig. 4D-H). These results further suggest that the antiadipogenic effect of miR-9 can be attributed to inhibition of PNPLA3 and stimulation of the AMPK signaling pathway during 3T3-L1 adipogenesis. In conclusion, our findings suggest that miR-9 activates the AMPK signaling pathway by targeting PNPLA3 and inhibits the expression of PPARγ and OXPAT as well as autophagy, resulting in blocked 3T3-L1 preadipocytes differentiation and adipogenesis. Therefore, miR-9 may serve as a new negative regulator during preadipocyte differentiation and adipogenesis and is of great significance to adipogenesis.
Acknowledgments This study was supported by The Foundation of the National Natural Science Foundation of China (General program, No. 81570519 for Jianhua Shen).
Authors' contributions
JS and YL conceived the experiments. BX and JS wrote the manuscript. DL, BN, and LG conducted the experiments. BX, HB, and JC analyzed the data. All authors read and approved the final manuscript.
Competing interests The authors declare that they have no competing interests.
References Acharya, A., Berry, D.C., Zhang, H., Jiang, Y., Jones, B.T., Hammer, R.E., Graff, J.M. and Mendell, J.T., 2019. miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19. Genes Dev 33, 1367-1380. Ali, A.T., Hochfeld, W.E., Myburgh, R. and Pepper, M.S., 2013. Adipocyte and adipogenesis. Eur J Cell Biol 92, 229-36. Ao, R., Wang, Y., Tong, J. and Wang, B.F., 2016. Altered microRNA-9 Expression Level is Directly Correlated with Pathogenesis of Nonalcoholic Fatty Liver Disease by Targeting Onecut2 and SIRT1. Med Sci Monit 22, 3804-3819. Brasaemle, D.L., 2007. Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. J Lipid Res 48, 2547-59. Bray, G.A., Kim, K.K., Wilding, J.P.H. and World Obesity, F., 2017. Obesity: a chronic relapsing progressive disease process. A position statement of the World Obesity Federation. Obes Rev 18, 715-723. Cikos, S., Bukovska, A. and Koppel, J., 2007. Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis. BMC Mol Biol 8, 113.
Fan, Y., Gan, M., Tan, Y., Chen, L., Shen, L., Niu, L., Liu, Y., Tang, G., Jiang, Y., Li, X., Zhang, S., Bai, L. and Zhu, L., 2019. Mir-152 Regulates 3T3-L1 Preadipocyte Proliferation and Differentiation. Molecules 24. Hadrich, F. and Sayadi, S., 2018. Apigetrin inhibits adipogenesis in 3T3-L1 cells by downregulating PPARgamma and CEBP-alpha. Lipids Health Dis 17, 95. Hahm, J.R., Noh, H.S., Ha, J.H., Roh, G.S. and Kim, D.R., 2014. Alpha-lipoic acid attenuates adipocyte differentiation and lipid accumulation in 3T3-L1 cells via AMPK-dependent autophagy. Life Sci 100, 125-32. Huang, Y., He, S., Li, J.Z., Seo, Y.K., Osborne, T.F., Cohen, J.C. and Hobbs, H.H., 2010. A feed-forward loop amplifies nutritional regulation of PNPLA3. Proc Natl Acad Sci U S A 107, 7892-7. Hwang, H.W. and Mendell, J.T., 2006. MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 94, 776-80. Jang, S.Y., Chae, M.K., Lee, J.H., Lee, E.J. and Yoon, J.S., 2019. MicroRNA-27 inhibits adipogenic differentiation in orbital fibroblasts from patients with Graves' orbitopathy. PLoS One 14, e0221077. Jeffery, E., Wing, A., Holtrup, B., Sebo, Z., Kaplan, J.L., Saavedra-Pena, R., Church, C.D., Colman, L., Berry, R. and Rodeheffer, M.S., 2016. The Adipose Tissue Microenvironment Regulates Depot-Specific Adipogenesis in Obesity. Cell Metab 24, 142-50. Jin, W., Dodson, M.V., Moore, S.S., Basarab, J.A. and Guan, L.L., 2010. Characterization of microRNA expression in bovine adipose tissues: a potential regulatory mechanism of
subcutaneous adipose tissue development. BMC Mol Biol 11, 29. Li, C., Chen, K., Jia, M., Ding, X., Jiang, Z., Li, L. and Zhang, D., 2018. AMPK promotes survival and adipogenesis of ischemia-challenged ADSCs in an autophagy-dependent manner. Biochim Biophys Acta Mol Cell Biol Lipids 1863, 1498-1510. Liu, Z., Qiao, Q., Sun, Y., Chen, Y., Ren, B. and Liu, X., 2017. Sesamol ameliorates dietinduced obesity in C57BL/6J mice and suppresses adipogenesis in 3T3-L1 cells via regulating mitochondria-lipid metabolism. Mol Nutr Food Res 61. Lorente-Cebrian, S., Gonzalez-Muniesa, P., Milagro, F.I. and Martinez, J.A., 2019. MicroRNAs and other non-coding RNAs in adipose tissue and obesity: emerging roles as biomarkers and therapeutic targets. Clin Sci (Lond) 133, 23-40. Losko, M., Lichawska-Cieslar, A., Kulecka, M., Paziewska, A., Rumienczyk, I., Mikula, M. and Jura, J., 2018. Ectopic overexpression of MCPIP1 impairs adipogenesis by modulating microRNAs. Biochim Biophys Acta Mol Cell Res 1865, 186-195. Min, H.K., Sookoian, S., Pirola, C.J., Cheng, J., Mirshahi, F. and Sanyal, A.J., 2014. Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells. Am J Physiol Gastrointest Liver Physiol 307, G66-76. Negoita, F., Blomdahl, J., Wasserstrom, S., Winberg, M.E., Osmark, P., Larsson, S., Stenkula, K.G., Ekstedt, M., Kechagias, S., Holm, C. and Jones, H.A., 2019. PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes. J Cell Biochem 120, 343-356. Ning, C., Li, G., You, L., Ma, Y., Jin, L., Ma, J., Li, X., Li, M. and Liu, H., 2017. MiR-185 inhibits
3T3-L1 cell differentiation by targeting SREBP-1. Biosci Biotechnol Biochem 81, 17471754. Ren, T., Zhu, J., Zhu, L. and Cheng, M., 2017. The Combination of Blueberry Juice and Probiotics Ameliorate Non-Alcoholic Steatohepatitis (NASH) by Affecting SREBP1c/PNPLA-3 Pathway via PPAR-alpha. Nutrients 9. Santoro, N., Kursawe, R., D'Adamo, E., Dykas, D.J., Zhang, C.K., Bale, A.E., Cali, A.M., Narayan, D., Shaw, M.M., Pierpont, B., Savoye, M., Lartaud, D., Eldrich, S., Cushman, S.W., Zhao, H., Shulman, G.I. and Caprio, S., 2010. A common variant in the patatinlike phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents. Hepatology 52, 1281-90. Seo, M.S., Hong, S.W., Yeon, S.H., Kim, Y.M., Um, K.A., Kim, J.H., Kim, H.J., Chang, K.C. and Park, S.W., 2014. Magnolia officinalis attenuates free fatty acid-induced lipogenesis via AMPK phosphorylation in hepatocytes. J Ethnopharmacol 157, 140-8. Shen, L., Gan, M., Li, Q., Wang, J., Li, X., Zhang, S. and Zhu, L., 2018a. MicroRNA-200b regulates preadipocyte proliferation and differentiation by targeting KLF4. Biomed Pharmacother 103, 1538-1544. Shen, L., Li, Q., Wang, J., Zhao, Y., Niu, L., Bai, L., Shuai, S., Li, X., Zhang, S. and Zhu, L., 2018b. miR-144-3p Promotes Adipogenesis Through Releasing C/EBPalpha From Klf3 and CtBP2. Front Genet 9, 677. Shenoy, A. and Blelloch, R.H., 2014. Regulation of microRNA function in somatic stem cell proliferation and differentiation. Nat Rev Mol Cell Biol 15, 565-76. Soares, A.G., de Carvalho, M.H.C. and Akamine, E., 2017. Obesity Induces Artery-Specific
Alterations: Evaluation of Vascular Function and Inflammatory and Smooth Muscle Phenotypic Markers. Biomed Res Int 2017, 5038602. Sookoian, S. and Pirola, C.J., 2011. Meta-analysis of the influence of I148M variant of patatinlike phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology 53, 1883-94. Sozio, M.S., Lu, C., Zeng, Y., Liangpunsakul, S. and Crabb, D.W., 2011. Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells. Am J Physiol Gastrointest Liver Physiol 301, G739-47. Wolins, N.E., Quaynor, B.K., Skinner, J.R., Tzekov, A., Croce, M.A., Gropler, M.C., Varma, V., Yao-Borengasser, A., Rasouli, N., Kern, P.A., Finck, B.N. and Bickel, P.E., 2006. OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization. Diabetes 55, 3418-28. Xi, F.X., Wei, C.S., Xu, Y.T., Ma, L., He, Y.L., Shi, X.E., Yang, G.S. and Yu, T.Y., 2019. MicroRNA-214-3p Targeting Ctnnb1 Promotes 3T3-L1 Preadipocyte Differentiation by Interfering with the Wnt/beta-Catenin Signaling Pathway. Int J Mol Sci 20. Xu, J., Zhang, L., Shu, G. and Wang, B., 2019. microRNA-16-5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1. Biochem Biophys Res Commun 514, 12511256. Xu, R., Tao, A., Zhang, S., Deng, Y. and Chen, G., 2015. Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis. Sci Rep 5, 9284. Zhang, F., Pan, T., Nielsen, L.D. and Mason, R.J., 2004. Lipogenesis in fetal rat lung:
importance of C/EBPalpha, SREBP-1c, and stearoyl-CoA desaturase. Am J Respir Cell Mol Biol 30, 174-83.
Fig. 1. Changes in the expression of miR-9 during the adipogenesis of 3T3-L1 cells. (A) 3T3-L1 cells were cultured in differentiation medium for 1, 3, 7, and 14 days, and lipid droplets in cells were detected by Oil red O staining. Scale bars = 50 μm. (B) Total intracellular triglyceride content at the indicated days was quantified using a kit. *p < 0.05, **p < 0.01 versus 1D group. (C) Changes in miR-9 expression in 3T3-L1 cells were analyzed by qRT-PCR on the indicated days during adipocyte differentiation, and snRNA U6 level was used as the internal control. *p < 0.05, **p < 0.01 versus 0 D group.
Fig. 2. Overexpression of miR-9 inhibits 3T3-L1 cell adipogenesis. (A) 3T3-L1 cells were transfected with 50 nM miR-9 mimics (miR-9), negative control miRNA mimics (miR-NC), miR-9 inhibitor (anti-miR-9), or negative control miRNA inhibitor (antimiR-NC) for 48 h. Untreated cells served as controls (NC). miR-9 expression was determined by qRT-PCR. Data were normalized to U6. **p < 0.01 versus NC. (B-C) Different transfected 3T3-L1 cells were induced to differentiate for 14 days. (B) Lipid droplets were examined using Oil red O staining. Scale bars = 50 μm. (C) Total intracellular triglyceride content was quantified using a kit. *p < 0.05, *p < 0.01 versus NC group.
Fig. 3. MiR-9 directly targets the 3′-UTR of PNPLA3. (A) Biological information predicted that PNPLA3 is a target gene of miR-9. (B) Dual-luciferase reporter gene assay confirmed that PNPLA3 is a target of miR-9 in 3T3-L1 cells. *p < 0.05 versus miRNA mimics negative control group (miR-NC). (C-D) 3T3-L1 cells were transfected with miR-NC, miR-9, shRNA targeting PNPLA3 (shPNPLA3), or negative control
shRNA-NC for 48 h. (C) PNPLA3 mRNA expression was determined by qRT-PCR and normalized to GAPDH. **p < 0.01 versus NC. (D) PNPLA3 protein levels were assessed by western blot analysis and normalized to β-actin. **p < 0.01 versus NC.
Fig. 4. miR-9 inhibits 3T3-L1 cell adipogenesis by activating AMPK. 3T3-L1 cells transfected with miR-9 or shPNPLA3 were cultured with or without 5 μM compound C (an inhibitor of AMPK) for 14 days. (A) Lipid droplets were examined using Oil red O staining. Scale bars = 50 μm. (B) Total intracellular triglyceride content was quantified. *p < 0.05 versus NC group. (C-H) Protein expression of PNPLA3, p-AMPK, AMPK, PPARγ, OXPAT, and LC3 was detected by western blot analysis. *p < 0.05, **p < 0.01 versus NC group.
S0378-1119(19)30919-9 https://doi.org/10.1016/j.gene.2019.144260 GENE 144260
To appear in:
Gene Gene
Received Date: Revised Date: Accepted Date:
31 May 2019 6 November 2019 7 November 2019
Please cite this article as: B. Xu, J. Shen, D. Li, B. Ning, L. Guo, H. Bing, J. Chen, Y. Li, Overexpression of microRNA-9 inhibits 3T3-L1 cell adipogenesis by targeting PNPLA3 via activation of AMPK, Gene Gene (2019), doi: https://doi.org/10.1016/j.gene.2019.144260
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier B.V.
Overexpression of microRNA-9 inhibits 3T3-L1 cell adipogenesis by targeting PNPLA3 via activation of AMPK
Beibei Xua,1, Jianhua Shenb,1, Dan Lia, Baoshuo Ninga, Liping Guoa, Hao Binga, Jiayu Chena, Yiling Lia,*,[email protected]
aDepartment
of Gastroenterology, the First Affiliated Hospital of China Medical
University, Shenyang 110001, China bDepartment
of Gastroenterology, the Affiliated Hospital of Qingdao University,
Qingdao 266000, China *Corresponding
authors. Department of Gastroenterology, the First Affiliated Hospital
of China Medical University, NO. 155, North Nanjing Street, Heping District, Shenyang City, Liaoning 110001, China.
1The
authors contributed equally to the work.
Abstract Adipocyte differentiation, which plays an important role in the development of obesity, involves complex molecular networks in which microRNAs (miRNAs) are essential. Here, we show that miR-9 expression was upregulated during adipogenesis in 3T3-L1 cells. miR-9 overexpression reduced the accumulation of lipid droplets and the content of triglycerides by downregulating patatin-like phospholipase domain-containing protein 3 (PNPLA3). PNPLA3 knockdown or miR-9 overexpression downregulated the protein expression of peroxisome proliferator-activated receptor γ, oxidative tissueenriched PAT, and LC3, and promoted the phosphorylation of AMP-activated protein kinase (AMPK), which was inhibited by treatment with the AMPK inhibitor compound C. These results indicate that miR-9 inhibits adipocyte differentiation by targeting PNPLA3 through the AMPK pathway.
Keywords: miR-9; adipogenesis; PNPLA3; AMPK; autophagy
Abbreviations 3′-UTR, 3′ untranslated region AMPK, AMP-activated protein kinase DM, differentiation medium FBS, fetal bovine serum miRNAs, MicroRNAs PNPLA3, Patatin-like phospholipase domain-containing protein 3 PPARγ, peroxisome proliferator-activated receptor γ OXPAT, oxidative tissue-enriched PAT protein
1 Introduction Obesity is a serious public health problem worldwide, and it is associated with diseases such as diabetes, hyperglycemia, hyperlipidemia, and atherosclerosis (Bray et al., 2017; Soares et al., 2017). Obesity is a metabolic abnormality characterized by excessive accumulation of fat. This accumulation is caused by an imbalance of body energy associated with adipocyte hyperplasia and hypertrophy. Mature adipocytes are differentiated from preadipocytes and progenitor cells in adipose tissue, a process known as adipogenesis (Ali et al., 2013; Jeffery et al., 2016). Therefore, reducing adipose tissue accumulation can improve obesity and obesity-related diseases. MicroRNAs (miRNAs) are endogenous non-coding RNAs composed of 18–25 nucleotides that bind to the 3′ untranslated region (3′-UTR) of target mRNAs to regulate gene expression at the post-transcriptional level, thereby affecting a variety of biological processes (Hwang and Mendell, 2006; Shenoy and Blelloch, 2014). miRNAs are post-transcriptional regulators of fat development and adipogenesis (Jin et al., 2010; Ning et al., 2017; Shen et al., 2018b; Lorente-Cebrian et al., 2019). miR-185 negatively regulates the differentiation of 3T3-L1 cells by targeting sterol regulatory element binding protein 1 (Ning et al., 2017), whereas miR-144-3p promotes adipogenesis by
targeting Klf3 and CtBP2 (Shen et al., 2018b). miR-9 upregulation is associated with the progression of liver disease and the severity of nonalcoholic fatty liver disease, and upregulation of miR-9 by mimic transfection decreases intracellular lipid content in L02 cells (Ao et al., 2016). However, the molecular mechanism underlying the function of miR-9 in regulating adipogenesis remains to be elucidated. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) plays an important role in liver lipid metabolism, and changes in its expression level are closely related to lipogenesis (Huang et al., 2010). In human hepatocytes, PNPLA3 plays an important role in autophagosome formation during the process of lipophagy (Negoita et al., 2019). Autophagy is important for initiating adipocyte differentiation, and AMPactivated protein kinase (AMPK) plays a key role in autophagy and lipid metabolism (Hahm et al., 2014; Li et al., 2018). Sozio et al. (Sozio et al., 2011) showed that inhibition of AMPK in hepatoma cells can activate peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity, which can induce oxidative tissueenriched PAT protein (OXPAT) synthesis and promote the utilization of fatty acids (Wolins et al., 2006; Hadrich and Sayadi, 2018). In this study, we investigated the role of miR-9 in adipogenesis in 3T3-L1 preadipocytes and explored the underlying mechanism by identifying the factors involved. miR-9 expression was assessed in 3T3-L1 cells undergoing adipogenesis, and the regulatory relationship between miR-9 and its putative target gene PNPLA3 was examined. In addition, the potential effect of miR-9 or PNPLA3 on AMPK-related proteins during adipogenesis was examined in 3T3-L1 cells.
2 Materials and methods 2.1 Cell culture and adipogenic differentiation 3T3-L1 preadipocytes obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China) were cultured in DMEM (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 5% CO2 and 37°C. To induce adipocyte differentiation, 3T3-L1 cells were incubated in differentiation medium (DM) supplemented with 10% FBS, 0.5 mM 3-isobutyl-1-
methylxanthine, 250 nM dexamethasone, and 160 nM insulin (designed as day 0). After 2 days, the medium was replaced with DMEM containing 10% FBS and 160 nM insulin for an additional 2 days, after which the medium was replaced every other day until day 14.
2.2 Cell transfection The oligonucleotide sequences of miR-9 mimics (miR-9, sense: 5′-UGC UGC ACC UUA GUC UCU GG-3′, antisense: 5′-GGG UAC UUG CCG CGU UUA AG3′), negative control miRNA mimics (miR-NC, sense: 5′-AGA CUU GGG CAG UUC CUU UGG-3′, antisense: 5′-UUU GCC UGC UAG CUC UAA GUC-3′), miR-9 inhibitor (anti-miR-9, 5′-CUG UCU UAG UGU GUG CGG CUA-3′), negative control miRNA inhibitor (anti-miR-NC, 5′-UGG CUU UAG GGA AGC GUA GUC-3′), shRNA targeting PNPLA3 (shPNPLA3, 5′-CCC GTT CTT CAA CAT TAA CAA-3′), and negative control shRNAs (shRNA-NC, 5′-CAT TTT GCC TGC TAG CTC TAA GT-3′) were designed and synthesized by Genepharma (Shanghai, China). The 3T3-L1 preadipocytes were transfected with 50 nM miR-9, miR-NC, shPNPLA3, or shRNANC using LipofectamineTM 2000 (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Cells were collected 48 h after transfection and used for adipogenic differentiation.
2.3 Plasmid construction The wild-type 3′-UTR of PNPLA3 was amplified from genomic DNA of 3T3-L1 cells by PCR using the following primers: PNPLA3 sense, 5′-GGG TGG TGA CAG CCT TAA CA-3′; antisense, 5′- CTG TCA GCA TCG AAG GGG TT-3′. The mutant PNPLA3
3′-UTR
was
generated
using
the QuikChange site-directed
mutagenesis kit (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions. The wild-type and mutant PNPLA3 3′-UTRs were inserted downstream of the luciferase reporter gene in the psiCHECK-2 dual-luciferase reporter plasmid (Promega, Madison, USA) between XhoI and NotI restriction sites, and the constructs were designated PNPLA3-wt and PNPLA3-mut, respectively.
2.4 Luciferase reporter assay Luciferase reporter assays were performed as previously described (Shen et al., 2018a). Briefly, 3T3-L1 cells were co-transfected with 50 nM miR-9 mimics or miRNC and 100 ng recombinant PNPLA3-wt or PNPLA3-mut psiCHECK-2 vector using LipofectamineTM 2000 (Invitrogen). Luciferase activity was measured at 48 h after transfection using the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s instructions.
2.5 Oil red O staining Lipid droplets in cells were stained with Oil Red O as previously described with minor modifications (Liu et al., 2017). Briefly, 3T3-L1 cells transfected with miR-9 or shPNPLA3 were induced to differentiate into mature adipocytes in the presence or absence of 5 μM AMPK inhibitor compound C (Sigma, Louis, Mo, USA) for 14 days. Differentiated 3T3-L1 cells were washed twice with phosphate buffered saline (PBS), fixed in 4% formaldehyde for 30 min, and washed three times with PBS. The fixed cells were stained with 5% Oil red O (Sigma Aldrich, St. Louis, MO, USA) for 15 min and washed with PBS three times. The lipid droplets were visualized and photographed using a light phase contrast microscope (Nikon, Japan).
2.6 Triglyceride content assays Differentiated 3T3-L1 cells were washed with PBS and sonicated for 1 min to obtain homogenous samples. The triglyceride content in the samples was determined with a commercial kit (Biosino Bio-Technology and Science Inc., Beijing, China).
2.7 Quantitative real time PCR (qRT-PCR) Total RNA was extracted from cells using the Trizol reagent (Invitrogen) according to the manufacturer’s instructions. cDNA synthesis was performed using Reverse Transcription Kits (Beyotime, China). qRT-PCR was performed using a SYBR Green PCR Master Mix (TaKaRa, Japan) on the Bio-RAD IQTM5 system (Bio-Rad,
Hercules, CA, USA). The PCR primer sequences were as follows: miR-9 sense, 5′TCT GCG CCT AGC ATT GGA AA-3′, antisense, 5′-AGT ATG GTG TGT GCG CTT GA-3′; U6 sense, 5′-TTG TTC CCT TCC CTT GTC GC-3′, antisense, 5′-AGA ACT TGT CAC CAT CCC GC-3′; PNPLA3 sense, 5′-CCT CCC AGA CAA TGT CCA CC3′, antisense, 5′-GGT TTT GGC ATC CAG CAC AG-3′; GAPDH sense, 5′-GCC TCG TCC CGT AGA CAA AA-3′, antisense, 5′-GCA ACA ATC TCC ACT TTG CCA-3′. The relative expression level of miR-9 was normalized to that of U6, and the relative expression level of PNPLA3 mRNA was normalized to that of the internal control GAPDH using the 2-ΔΔCt method (Cikos et al., 2007).
2.8 Western blot analysis Protein was extracted from 3T3-L1 cells using lysis buffer (Sigma), and protein concentration was determined with the BCA protein assay kit (Beyotime). For western blotting, 40 μg total cellular protein was separated by 10% SDS-PAGE and transferred to PVDF membranes (Roche, Germany). The membranes were incubated overnight at 4°C with primary antibodies against PNPLA3 (1:1500; Abcam, Cambridge, MA, USA), phospho-AMPK (Thr172) (1:2000; Cell Signaling Technology, USA), AMPK (1:2000; Cell Signaling Technology), PPARγ (1:1000; Abcam), OXPAT (1:1000; Abcam), and β-actin (1:2000; Abcam). After washing, the membranes were incubated with goat polyclonal anti-rabbit IgG–H&L-pre-adsorbed (HRP; 1:2000; Abcam) for 1 h at room temperature. The bands were developed using the enhanced chemiluminescence (ECL) blotting analysis system (GE Healthcare, USA). The densitometric values were quantified using Image J software (National Institutes of Health, USA) and normalized to β-actin.
2.9 Statistical analysis Data are expressed as the mean ± SD of three replicates in each experiment and analyzed using GraphPad Prism 6.0 software (GraphPad Inc., USA). Student's t-test was used to determine statistical differences between two groups, and comparisons between multiple groups were evaluated using one-way analysis of variance. P < 0.05
was considered statistically significant.
3 Results 3.1 miR-9 is upregulated during adipogenesis in 3T3-L1 cells Oil Red O staining showed that the number and size of lipid droplets gradually increased in a time-dependent manner in 3T3-L1 preadipocytes treated with differentiation medium (Fig. 1A), in parallel with an increase in cellular triglyceride content during differentiation (Fig. 1B). miR-9 expression during 3T3-L1 cell differentiation was examined by extracting total RNA from adipocytes on days 0, 1, 3, 7, and 14 after differentiation and measuring miR-9 levels by qRT-PCR. As shown in Fig. 1C, miR-9 expression was low on day 0 and 1 of differentiation induction, and gradually increased on day 3, peaked on day 7, and slightly decreased on day 14. These results indicated that miR-9 may play an important role during 3T3-L1 cell differentiation.
3.2 Overexpression of miR-9 inhibits 3T3-L1 adipogenesis To investigate the role of miR-9 in cell differentiation, 3T3-L1 cells were transfected with miR-9 mimics or miR-9 inhibitor during the 14 days of the differentiation period. miR-9 levels significantly increased upon miR-9 mimic transfection and decreased in response to miR-9 inhibitor transfection compared with the NC group (p < 0.01; Fig. 2A). Oil Red O staining showed that miR-9 overexpression decreased the formation of lipid droplets, whereas miR-9 knockdown increased the formation of lipid droplets (Fig. 2B). Consistent with these results, the triglyceride content in 3T3-L1 cells decreased significantly in the miR-9 mimics group and increased in the miR-9 inhibitor group compared with the NC group (p < 0.01 and P < 0.05, respectively; Fig. 2C). these results suggested that miR-9 can inhibit 3T3-L1 adipogenesis.
3.3 miR-9 directly targets the 3′-UTR of PNPLA3 To explore the mechanisms underlying the effect of miR-9 on suppressing 3T3-
L1 adipogenesis, bioinformatics analysis (TargetScan, Pic Tar and miRanda) was performed to identify the potential targets of miR-9. The result showed binding sites for miR-9 in the 3′-UTR of PNPLA3 (Fig. 3A). To determine whether PNPLA3 is a direct target of miR-9, the wild type (wt) or mutant (mut) PNPLA3 3′-UTR was subcloned into a luciferase reporter vector and co-transfected with miR-9 mimics or miR-NC into 3T3-L1 cells. The luciferase reporter assays showed that miR-9 decreased the luciferase activity of the wt 3′-UTR, but not that of the mut 3′-UTR, of PNPLA3 compared with the miR-NC group (p < 0.05; Fig. 3B). To determine whether miR-9 can regulate PNPLA3 expression, 3T3-L1 cells were transfected with miR-9 mimics, miR-NC, shRNA-NC, or shPNPLA3 as a positive control. As shown in Fig. 3C and D, PNPLA3 mRNA and protein levels were significantly decreased in the miR-9 mimics and shPNPLA3 groups compared with the NC group (p < 0.01). Taken together, these results indicated that PNPLA3 is a direct target of miR-9.
3.4 miR-9 inhibits 3T3-L1 cell adipogenesis by activating AMPK The molecular mechanism underlying the effect of miR-9 on inhibiting 3T3-L1 cell adipogenesis was further examined by inducing 3T3-L1 cells transfected with miR9 or shPNPLA3 to differentiate into mature adipocytes in the presence or absence of 5 μM of compound C (an inhibitor of AMPK) for 14 days. Oil Red O staining showed that the formation of lipid droplets and triglyceride content decreased in the miR-9 mimics and shPNPLA3 groups. Treatment with compound C abolished the effects of miR-9 mimics and shPNPLA3 (Fig. 4A and B), whereas compound C treatment had no effect on the expression of PNPLA3 (Fig. 4C). Western blot analysis of the protein levels of p-AMPK, AMPK, PPARγ, OXPAT, and LC3 in each group showed that PNPLA3 knockdown or miR-9 overexpression increased AMPK phosphorylation and downregulated PPARγ, OXPAT, and LC3 protein expression, and this effect was blocked by compound C (Fig. 4D–H). Taken together, these results suggest that miR-9 inhibits 3T3-L1 cell adipogenesis by inhibiting PNPLA3 and activating the AMPK signaling pathway.
4 Discussion In recent years, a large number of reports have shown that miRNAs are involved in preadipocyte differentiation, lipid metabolism, and lipogenesis (Jin et al., 2010; Ning et al., 2017; Shen et al., 2018b; Lorente-Cebrian et al., 2019). For example, miR-144, miR-16, and miR-214 promote adipogenesis (Shen et al., 2018b; Xi et al., 2019; Xu et al., 2019), whereas miR-152, miR-26, and miR-27 suppress preadipocyte differentiation and lipid metabolism (Acharya et al., 2019; Fan et al., 2019; Jang et al., 2019). However, the molecular mechanism underlying the role of miRNAs in lipogenesis is not fully understood. In this study, we found that miR-9 was upregulated during the adipogenesis of 3T3-L1 cells in the first 7 days, whereas its expression decreased slightly on day 14 (Fig. 1). This suggests the involvement of other genes in the regulation of miR-9 during adipogenesis, such as monocyte chemoattractant protein-1-induced protein 1 (MCPIP1)(Losko et al., 2018). We also showed that overexpression of miR-9 suppressed, whereas downregulation of miR-9 promoted 3T3L1 preadipocyte differentiation and lipid accumulation (Fig. 2). Ao et al. showed that miR-9 is upregulated during hepatic steatosis progression, and overexpression of miR-9 decreases the formation of intracellular lipid droplets in oleic acid-induced L-02 cells (Ao et al., 2016). These results indicate that miR-9 may serve as an inhibitor of adipogenesis. To screen mir-9 targets and identify binding sites involved in the interaction between miR-9 and its target, we used TargetScan, PicTar, and miRanda bioinformatics target prediction packages to predict miR-9 targets. We found that the 3′-UTR of PNPLA3 contains potential binding sites for miR-9. Luciferase report assays confirmed that miR-9 inhibited the transcriptional activity of wt PNPLA3. In addition, miR-9 overexpression and PNPLA3 knockdown inhibited adipogenesis (Fig. 3). These results suggest that PNPLA3 is the target of miR-9. PNPLA3 is a triglyceride lipase/acyltransferase that mediates triglyceride hydrolysis in adipocytes and hepatocytes (Min et al., 2014), and mutations in the PNPLA3 gene predispose obese adolescents to hepatic steatosis (Santoro et al., 2010; Sookoian and Pirola, 2011; Xu et al., 2015). The family of adipogenic-specific factors
include SREBP-1c and PPARγ(Zhang et al., 2004). AMPK can negatively regulate SREBP-1C to reduce the formation of free fatty acids (FFAs) (Seo et al., 2014). PNPLA3 is a determinant of triglyceride accumulation in the liver and is regulated by SREBP-1c (Ren et al., 2017). OXPAT is a homologous protein of Perilipin, which is mainly distributed in tissues and cells with a fast rate of lipid metabolism (Brasaemle, 2007). OXPAT activates the PPAR pathway and promotes the catabolism of fatty acids in cells (Wolins et al., 2006). Hahm et al. showed that alpha-lipoic acid attenuates adipocyte differentiation and lipid accumulation in 3T3-L1 cells via AMPKdependent autophagy(Hahm et al., 2014). To further determine whether the reduction of PNPLA3 induced by miR-9 induces the AMPK pathway, we used shRNA oligonucleotides against PNPLA3 to simulate the role of miR-9. The results showed that the content of triglycerides in 3T3-L1 cells treated with shPNPLA3 decreased compared with that in the NC group (Fig. 4A-C). In addition, the expression of marker genes for adipogenesis, such as PPARγ and OXPAT, as well as the autophagy-related gene LC3, were significantly inhibited, and these effects were blocked by the AMPK inhibitor compound C (Fig. 4D-H). These results further suggest that the antiadipogenic effect of miR-9 can be attributed to inhibition of PNPLA3 and stimulation of the AMPK signaling pathway during 3T3-L1 adipogenesis. In conclusion, our findings suggest that miR-9 activates the AMPK signaling pathway by targeting PNPLA3 and inhibits the expression of PPARγ and OXPAT as well as autophagy, resulting in blocked 3T3-L1 preadipocytes differentiation and adipogenesis. Therefore, miR-9 may serve as a new negative regulator during preadipocyte differentiation and adipogenesis and is of great significance to adipogenesis.
Acknowledgments This study was supported by The Foundation of the National Natural Science Foundation of China (General program, No. 81570519 for Jianhua Shen).
Authors' contributions
JS and YL conceived the experiments. BX and JS wrote the manuscript. DL, BN, and LG conducted the experiments. BX, HB, and JC analyzed the data. All authors read and approved the final manuscript.
Competing interests The authors declare that they have no competing interests.
References Acharya, A., Berry, D.C., Zhang, H., Jiang, Y., Jones, B.T., Hammer, R.E., Graff, J.M. and Mendell, J.T., 2019. miR-26 suppresses adipocyte progenitor differentiation and fat production by targeting Fbxl19. Genes Dev 33, 1367-1380. Ali, A.T., Hochfeld, W.E., Myburgh, R. and Pepper, M.S., 2013. Adipocyte and adipogenesis. Eur J Cell Biol 92, 229-36. Ao, R., Wang, Y., Tong, J. and Wang, B.F., 2016. Altered microRNA-9 Expression Level is Directly Correlated with Pathogenesis of Nonalcoholic Fatty Liver Disease by Targeting Onecut2 and SIRT1. Med Sci Monit 22, 3804-3819. Brasaemle, D.L., 2007. Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. J Lipid Res 48, 2547-59. Bray, G.A., Kim, K.K., Wilding, J.P.H. and World Obesity, F., 2017. Obesity: a chronic relapsing progressive disease process. A position statement of the World Obesity Federation. Obes Rev 18, 715-723. Cikos, S., Bukovska, A. and Koppel, J., 2007. Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis. BMC Mol Biol 8, 113.
Fan, Y., Gan, M., Tan, Y., Chen, L., Shen, L., Niu, L., Liu, Y., Tang, G., Jiang, Y., Li, X., Zhang, S., Bai, L. and Zhu, L., 2019. Mir-152 Regulates 3T3-L1 Preadipocyte Proliferation and Differentiation. Molecules 24. Hadrich, F. and Sayadi, S., 2018. Apigetrin inhibits adipogenesis in 3T3-L1 cells by downregulating PPARgamma and CEBP-alpha. Lipids Health Dis 17, 95. Hahm, J.R., Noh, H.S., Ha, J.H., Roh, G.S. and Kim, D.R., 2014. Alpha-lipoic acid attenuates adipocyte differentiation and lipid accumulation in 3T3-L1 cells via AMPK-dependent autophagy. Life Sci 100, 125-32. Huang, Y., He, S., Li, J.Z., Seo, Y.K., Osborne, T.F., Cohen, J.C. and Hobbs, H.H., 2010. A feed-forward loop amplifies nutritional regulation of PNPLA3. Proc Natl Acad Sci U S A 107, 7892-7. Hwang, H.W. and Mendell, J.T., 2006. MicroRNAs in cell proliferation, cell death, and tumorigenesis. Br J Cancer 94, 776-80. Jang, S.Y., Chae, M.K., Lee, J.H., Lee, E.J. and Yoon, J.S., 2019. MicroRNA-27 inhibits adipogenic differentiation in orbital fibroblasts from patients with Graves' orbitopathy. PLoS One 14, e0221077. Jeffery, E., Wing, A., Holtrup, B., Sebo, Z., Kaplan, J.L., Saavedra-Pena, R., Church, C.D., Colman, L., Berry, R. and Rodeheffer, M.S., 2016. The Adipose Tissue Microenvironment Regulates Depot-Specific Adipogenesis in Obesity. Cell Metab 24, 142-50. Jin, W., Dodson, M.V., Moore, S.S., Basarab, J.A. and Guan, L.L., 2010. Characterization of microRNA expression in bovine adipose tissues: a potential regulatory mechanism of
subcutaneous adipose tissue development. BMC Mol Biol 11, 29. Li, C., Chen, K., Jia, M., Ding, X., Jiang, Z., Li, L. and Zhang, D., 2018. AMPK promotes survival and adipogenesis of ischemia-challenged ADSCs in an autophagy-dependent manner. Biochim Biophys Acta Mol Cell Biol Lipids 1863, 1498-1510. Liu, Z., Qiao, Q., Sun, Y., Chen, Y., Ren, B. and Liu, X., 2017. Sesamol ameliorates dietinduced obesity in C57BL/6J mice and suppresses adipogenesis in 3T3-L1 cells via regulating mitochondria-lipid metabolism. Mol Nutr Food Res 61. Lorente-Cebrian, S., Gonzalez-Muniesa, P., Milagro, F.I. and Martinez, J.A., 2019. MicroRNAs and other non-coding RNAs in adipose tissue and obesity: emerging roles as biomarkers and therapeutic targets. Clin Sci (Lond) 133, 23-40. Losko, M., Lichawska-Cieslar, A., Kulecka, M., Paziewska, A., Rumienczyk, I., Mikula, M. and Jura, J., 2018. Ectopic overexpression of MCPIP1 impairs adipogenesis by modulating microRNAs. Biochim Biophys Acta Mol Cell Res 1865, 186-195. Min, H.K., Sookoian, S., Pirola, C.J., Cheng, J., Mirshahi, F. and Sanyal, A.J., 2014. Metabolic profiling reveals that PNPLA3 induces widespread effects on metabolism beyond triacylglycerol remodeling in Huh-7 hepatoma cells. Am J Physiol Gastrointest Liver Physiol 307, G66-76. Negoita, F., Blomdahl, J., Wasserstrom, S., Winberg, M.E., Osmark, P., Larsson, S., Stenkula, K.G., Ekstedt, M., Kechagias, S., Holm, C. and Jones, H.A., 2019. PNPLA3 variant M148 causes resistance to starvation-mediated lipid droplet autophagy in human hepatocytes. J Cell Biochem 120, 343-356. Ning, C., Li, G., You, L., Ma, Y., Jin, L., Ma, J., Li, X., Li, M. and Liu, H., 2017. MiR-185 inhibits
3T3-L1 cell differentiation by targeting SREBP-1. Biosci Biotechnol Biochem 81, 17471754. Ren, T., Zhu, J., Zhu, L. and Cheng, M., 2017. The Combination of Blueberry Juice and Probiotics Ameliorate Non-Alcoholic Steatohepatitis (NASH) by Affecting SREBP1c/PNPLA-3 Pathway via PPAR-alpha. Nutrients 9. Santoro, N., Kursawe, R., D'Adamo, E., Dykas, D.J., Zhang, C.K., Bale, A.E., Cali, A.M., Narayan, D., Shaw, M.M., Pierpont, B., Savoye, M., Lartaud, D., Eldrich, S., Cushman, S.W., Zhao, H., Shulman, G.I. and Caprio, S., 2010. A common variant in the patatinlike phospholipase 3 gene (PNPLA3) is associated with fatty liver disease in obese children and adolescents. Hepatology 52, 1281-90. Seo, M.S., Hong, S.W., Yeon, S.H., Kim, Y.M., Um, K.A., Kim, J.H., Kim, H.J., Chang, K.C. and Park, S.W., 2014. Magnolia officinalis attenuates free fatty acid-induced lipogenesis via AMPK phosphorylation in hepatocytes. J Ethnopharmacol 157, 140-8. Shen, L., Gan, M., Li, Q., Wang, J., Li, X., Zhang, S. and Zhu, L., 2018a. MicroRNA-200b regulates preadipocyte proliferation and differentiation by targeting KLF4. Biomed Pharmacother 103, 1538-1544. Shen, L., Li, Q., Wang, J., Zhao, Y., Niu, L., Bai, L., Shuai, S., Li, X., Zhang, S. and Zhu, L., 2018b. miR-144-3p Promotes Adipogenesis Through Releasing C/EBPalpha From Klf3 and CtBP2. Front Genet 9, 677. Shenoy, A. and Blelloch, R.H., 2014. Regulation of microRNA function in somatic stem cell proliferation and differentiation. Nat Rev Mol Cell Biol 15, 565-76. Soares, A.G., de Carvalho, M.H.C. and Akamine, E., 2017. Obesity Induces Artery-Specific
Alterations: Evaluation of Vascular Function and Inflammatory and Smooth Muscle Phenotypic Markers. Biomed Res Int 2017, 5038602. Sookoian, S. and Pirola, C.J., 2011. Meta-analysis of the influence of I148M variant of patatinlike phospholipase domain containing 3 gene (PNPLA3) on the susceptibility and histological severity of nonalcoholic fatty liver disease. Hepatology 53, 1883-94. Sozio, M.S., Lu, C., Zeng, Y., Liangpunsakul, S. and Crabb, D.W., 2011. Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells. Am J Physiol Gastrointest Liver Physiol 301, G739-47. Wolins, N.E., Quaynor, B.K., Skinner, J.R., Tzekov, A., Croce, M.A., Gropler, M.C., Varma, V., Yao-Borengasser, A., Rasouli, N., Kern, P.A., Finck, B.N. and Bickel, P.E., 2006. OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization. Diabetes 55, 3418-28. Xi, F.X., Wei, C.S., Xu, Y.T., Ma, L., He, Y.L., Shi, X.E., Yang, G.S. and Yu, T.Y., 2019. MicroRNA-214-3p Targeting Ctnnb1 Promotes 3T3-L1 Preadipocyte Differentiation by Interfering with the Wnt/beta-Catenin Signaling Pathway. Int J Mol Sci 20. Xu, J., Zhang, L., Shu, G. and Wang, B., 2019. microRNA-16-5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1. Biochem Biophys Res Commun 514, 12511256. Xu, R., Tao, A., Zhang, S., Deng, Y. and Chen, G., 2015. Association between patatin-like phospholipase domain containing 3 gene (PNPLA3) polymorphisms and nonalcoholic fatty liver disease: a HuGE review and meta-analysis. Sci Rep 5, 9284. Zhang, F., Pan, T., Nielsen, L.D. and Mason, R.J., 2004. Lipogenesis in fetal rat lung:
importance of C/EBPalpha, SREBP-1c, and stearoyl-CoA desaturase. Am J Respir Cell Mol Biol 30, 174-83.
Fig. 1. Changes in the expression of miR-9 during the adipogenesis of 3T3-L1 cells. (A) 3T3-L1 cells were cultured in differentiation medium for 1, 3, 7, and 14 days, and lipid droplets in cells were detected by Oil red O staining. Scale bars = 50 μm. (B) Total intracellular triglyceride content at the indicated days was quantified using a kit. *p < 0.05, **p < 0.01 versus 1D group. (C) Changes in miR-9 expression in 3T3-L1 cells were analyzed by qRT-PCR on the indicated days during adipocyte differentiation, and snRNA U6 level was used as the internal control. *p < 0.05, **p < 0.01 versus 0 D group.
Fig. 2. Overexpression of miR-9 inhibits 3T3-L1 cell adipogenesis. (A) 3T3-L1 cells were transfected with 50 nM miR-9 mimics (miR-9), negative control miRNA mimics (miR-NC), miR-9 inhibitor (anti-miR-9), or negative control miRNA inhibitor (antimiR-NC) for 48 h. Untreated cells served as controls (NC). miR-9 expression was determined by qRT-PCR. Data were normalized to U6. **p < 0.01 versus NC. (B-C) Different transfected 3T3-L1 cells were induced to differentiate for 14 days. (B) Lipid droplets were examined using Oil red O staining. Scale bars = 50 μm. (C) Total intracellular triglyceride content was quantified using a kit. *p < 0.05, *p < 0.01 versus NC group.
Fig. 3. MiR-9 directly targets the 3′-UTR of PNPLA3. (A) Biological information predicted that PNPLA3 is a target gene of miR-9. (B) Dual-luciferase reporter gene assay confirmed that PNPLA3 is a target of miR-9 in 3T3-L1 cells. *p < 0.05 versus miRNA mimics negative control group (miR-NC). (C-D) 3T3-L1 cells were transfected with miR-NC, miR-9, shRNA targeting PNPLA3 (shPNPLA3), or negative control
shRNA-NC for 48 h. (C) PNPLA3 mRNA expression was determined by qRT-PCR and normalized to GAPDH. **p < 0.01 versus NC. (D) PNPLA3 protein levels were assessed by western blot analysis and normalized to β-actin. **p < 0.01 versus NC.
Fig. 4. miR-9 inhibits 3T3-L1 cell adipogenesis by activating AMPK. 3T3-L1 cells transfected with miR-9 or shPNPLA3 were cultured with or without 5 μM compound C (an inhibitor of AMPK) for 14 days. (A) Lipid droplets were examined using Oil red O staining. Scale bars = 50 μm. (B) Total intracellular triglyceride content was quantified. *p < 0.05 versus NC group. (C-H) Protein expression of PNPLA3, p-AMPK, AMPK, PPARγ, OXPAT, and LC3 was detected by western blot analysis. *p < 0.05, **p < 0.01 versus NC group.