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Functional study of the upregulation of miRNA-27a and miRNA-27b in 3T3-L1 cells in response to berberine

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Berberine is the major active component of Rhizoma Coptidis derived from a traditional Chinese herbal medicine and is known to regulate micro (mi)RNA levels, although the mechanism for this action remains unknown. The present study confirmed that
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  MOLECULAR MEDICINE REPORTS 14: 2725-2731, 2016 Abstract. Berberine is the major active component of  Rhizoma Coptidis  derived from a traditional Chinese herbal medicine and is known to regulate micro (mi)RNA levels, although the mechanism for this action remains unknown. The present study conrmed that treatment of 3T3‑L1 cells with berberine inhibited cell viability and differentiation in a dose‑ and time‑dependent manner, and signicantly increased the mRNA expression levels of miRNA-27a and miRNA-27b. In addition, in 3T3-L1 cells treated with berberine, over-expression of miRNA-27a and miRNA-27b improved the berberine-mediated inhibition of cell differentiation and reduction of triglyceride contents. By contrast, miRNA-27a and miRNA-27b inhibitors attenuated the berberine-mediated inhibition of cell differentiation and reduction of triglyceride contents. Additionally, peroxisome proliferator-activated receptors (PPAR)- γ  was conrmed to be a target of miRNA‑27a in the 3T3-L1 cells. A dual-luciferase reporter assay indicated that the expression of PPAR- γ  was negatively regulated by miRNA‑27a. These ndings may provide novel mechanistic insight into the antiobesity effects of certain compounds in traditional Chinese herbal medicine. Introduction Obesity is a major health obstacle in the industrialized world, increasing the incidence of several illness, including hyper-tension, diabetes and heart disease, and is characterized by a complex multifactorial chronic disease. An imbalance between energy intake and expenditure contributes to a pathological growth of adipocytes (1). It is known that the quantity of adipose tissue can be regulated by the inhibition of adipogen-esis and by the control of adipocyte size. Obesity is induced by the abnormal proliferation of adipocytes and recruits the new adipocytes from precursor cells, two of which are involved in regulating the differentiation of adipocytes (2).Berberine is an alkaloid isolated from Chinese herbs and is currently used as a traditional medicine for the treatment of bacterial diarrhea, diabetes, hyperlipidemia, cancer, heart and inammatory diseases ( 3-6). Previous studies demon-strate that berberine presents anticancer activities via the inhibition of cell proliferation and reproduction of viruses and certain tumsrcenic microorganisms, and the induction of apoptosis in a variety of cancer cell lines (7-10). Also, it has been reported that berberine exhibits antiadipogenic effects in several adipocytes, although its precise mechanism remains to be elucidated (11,12). Therefore, the present study undertook a detailed study of the effect of berberine on the differentiation of 3T3-L1 cells.Micro (mi)RNAs are non-encoding RNA molecules that regulate gene expression by suppressing the translation of target genes and degrading target mRNAs (13). miRNAs serve a critical role in a wide variety of biology processes, including proliferation, division, survival and apoptosis (14-17). In addi-tion, miRNA-27 is one of the most important miRNAs and is associated with the differentiation of adipocytes. A previous study also suggests that both miRNA-27a and miRNA-27b act as antiadipogenic miRNAs, at least in part, by suppressing the proliferation of human adipose tissue-derived stem cells (18).Previous studies have investigated the effects of natural compounds on the expression of miRNAs in different cancer types. Only a few reports on the effect of berberine and miRNAs have been published, and these effects remain to be fully understood. Berberine downregulates the expres-sion of miRNA-21 in human multiple myeloma and ovarian cells, which in turn leads to apoptosis and inhibition of cell proliferation (19,20).In the present study, the effects of berberine on miRNA-27a and miRNA-27b in 3T3-L1 cells were assessed. It was revealed that berberine increased the levels of miRNA-27a and miRNA-27b, which led to the enhancement of differentia-tion suppression and a reduction in triglyceride contents via Functional study of the upregulation of miRNA-27a and miRNA-27b in 3T3-L1 cells in response to berberine YUE-YUE WU * , XIN-MEI HUANG * , JUN LIU, YING CHA, ZAO-PING CHEN, FANG WANG, JIONG XU, LI SHENG and HE-YUANG DING Department of Endocrinology, The Fifth People's Hospital of Shanghai Afliated to Fudan University,  Shanghai 200240, P.R. ChinaReceived June 12, 2015; Accepted April 18, 2016DOI: 10.3892/mmr.2016.5545 Correspondence to:  Professor Jun Liu, Department of Endocrinology, The Fifth People's Hospital of Shanghai Affiliated to Fudan University, 801 Heqing Road, Minhang, Shanghai 200240, P.R. ChinaE-mail: liujun89189@163.com * Contributed equally Key words:  adipocytes, berberine, miRNA-27a, miRNA-27b, cell differentiation  WU  et al : BERBERINE UPREGULATES miRNA-27a AND miRNA-27b 2726 the targeting of peroxisome proliferator-activated receptors (PPAR)- γ . Materials and methods  Adipocyte differentiation and treatments. THe 3T3-L1 cells were obtained from Shanghai Institute of Cell Biology (Shanghai China). The cells were cultured in Dulbecco's modi- ed Eagle's medium (DMEM; GE Healthcare, Piscataway, NJ, USA) and supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientic, Inc., Waltham, MA, USA) at 37˚C and 5% CO 2 . After allowing 2 days for differentia-tion, the 3T3‑L1 cells were passaged and treated at conuence with medium in the presence of 0.5 mM 3-isobutyl-1-meth-ylxanthine (Sigma-Aldrich, St. Louis, MO, USA), 0.25 µM dexamethasone (Sigma-Aldrich) and 10 µg/ml insulin (Sigma-Aldrich) for 24 h. The 3T3-L1 cells were subsequently treated with berberine (0, 1, 10, 20, 40 and 80 µM) for a further 24 h. The medium was changed to DMEM, supple-mented with 1 µg/ml insulin for 2 days, followed by DMEM with 10% FBS for 10 days. The medium was replaced on the cells with DMEM with 10% FBS every 2 days. Cell viability measurement by cell counting kit (CCK)-8  following treatment with berberine. Each concentration of berberine used was regarded as one treatment group, while no berberine was added in the control group. Each treated or control group contained three parallel wells. The culture plates were incubated for 0, 24, 48 and 72 h. The 3T3-L1 cells were subsequently treated with CCK-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), and the absorbance at 450 nm was measured for the supernatant of each well with a Multiskan EX plate reader (Thermo LabSystems, Helsinki, Finland). Oil-Red O staining. On day 10 of adipocyte differen-tiation induction, the 3T3-L1 cells were stained with 1 mg/ml Oil-Red O dye (Abcam, Cambridge, MA, USA). The cells were xed with 70% ethanol and dehydrated with 100% propylene glycol, and were subsequently stained with Oil-Red O. The cells were observed under a microscope (CX41RF; Olympus Corporation, Tokyo, Japan) and fat droplets in the adipocytes were stained red.  RNA extraction and reverse transcription-quantitative  polymerase chain reaction (RT-qPCR) for miRNA-27a and miRNA-27b. The total RNA was extracted using TRIzol Reagent (Invitrogen; Thermo Fisher Scientic, Inc.), according to the manufacturer's protocol. cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientic, Inc.). RT-qPCR was performed to detect the mRNA expression levels of miRNA-27a and miRNA-27b, using a One-Step SYBR PrimeScript RT-PCR kit II (Takara Biotechnology Co., Ltd., Dalian, China) and data collection was conducted using an ABI 7500 (Thermo Fisher Scientic, Inc.).  The PCR cycling conditions were as follows: 95˚C for 10 min, followed by 40 cycles at 95˚C for 15 sec and 60˚C for 45 sec, and a nal extension step of 95˚C for 15 sec, 60˚C for 1 min, 95˚C for 15 sec and 60˚C for 15 sec.  U6 small nuclear RNA served as an internal control. The gene expression was calculated using the 2 - ΔΔ Cq  method. The primers used were as follows: Forward, 5'-ACA CTC CAG CTG GGA GGG CTT AGC TGC TTG-3' and reverse, 5'-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCA GTT GAG TGC TCA-3' for miRNA-27a; forward, 5'-ACA CTC CAG CTG GGA GAG CTT AGC TGA TTG-3' and reverse, 5'-CTC AAC TGG TGT CGT GGA GTC GGC AAT TCY AGTTGA GGT TCAC-3' for miRNA-27b; forward, 5'-CTC GCT TCG GCA GCACA-3' and reverse, 5'-AAC GCT TCA CGA ATT TGCGT-3' for U6. miRNA transfection. The 3T3-L1 cells were trans-fected with 40 µM negative control (NC), miRNA-27a and miRNA-27b, miRNA-27a and miRNA-27b mimics or miRNA-27a and miRNA-27b inhibitors (anti-miRNA-27a and anti-miRNA-27b) obtained from Beyotime Institute of Biotechnology (Shanghai, China), which knockdown miRNA-27a and miRNA-27b, using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientic, Inc.), according to the manufacturer's instructions. The 3T3-L1 cells were prepared for further analysis 48 h after transfection. Triglyceride assay. The content of triglycerides (TGs) were analyzed using the Triglyceride Quantification assay kit (Abcam), according to the manufacturer's protocol. Briey, the 3T3-L1 cells were collected and resuspended in 0.1 M phos-phate-buffered saline. Following centrifugation for 10 min at 400 x g and 4˚C , the cells were lysed in 1-2% Triton X-100 for 30 min for each assay. The samples were measured at 546 nm in a plate reader (Multiskan EX; LabSystems, Helsinki, Finland).  Luciferase reporter assays. PPAR- γ  was predicted to interact with miRNA 27a by bioinformatics analysis using TargetScan, which  predicts biological targets of miRNAs by searching for the presence of 8mer, 7mer, and 6mer sites that match the seed region of each miRNA (21) The 3'-untranslated region (UTR) of human PPAR- γ  predicted to interact with miRNA-27a was synthesized and immediately inserted downstream of the  Renilla  luciferase reporter gene in the pGL3 vector (Promega Corporation, Madison, WI, USA), yielding pGL3-PPAR- γ . The 3T3-L1 cells were co-transfected with miRNA-27a mimics or NC using the Lipofectamine 2000. After 24 h, the luciferase activities were examined using the Dual-Luciferase Reporter assay system (cat. no. E1960; Promega Corporation). Firefly luciferase activity was normalized against that of  Renilla  luciferase activity. Statistical analysis. The data are presented as the mean ± standard deviation. One-way analysis of variance, followed by Dunnett's test was used for statistical analysis. P<0.05 was considered to indicate a statistically signicant difference. Results  Berberine inhibits the cell viability of 3T3-L1 cells. Following treatment with concentrations of berberine (1, 10, 20, 40 and 80 µM) for 72 h, the growth of the 3T3-L1 cells was signicantly reduced compared with that of the control  MOLECULAR MEDICINE REPORTS 14: 2725-2731, 2016  2727 group (P<0.05). The cell viability of 3T3-L1 cells was reduced by berberine treatment in a dose- and time-dependent manner (Fig. 1A).  Berberine inhibits the differentiation of 3T3-L1 cells. The present study first examined the antiobesity potential of berberine by determining pre-adipocyte differentiation into adipocytes. Cultured 3T3-L1 cells were exposed to berberine at different doses and cell differentiation was induced using the differentiation medium. Following reculturing in DMEM with 10% FBS for 10 days, cell differentiation was terminated and fat droplets were detected using Oil-Red O staining. As shown Fig. 1B, the 3T3-L1 cells in the control group exhib-ited normal differentiation, as indicated by the appearance of numerous intracellular lipid droplets. However, treatment of 3T3-L1 cells with berberine at different concentrations (10 and 20 µM) caused a dramatic reduction in lipid droplet accumulation dose-dependently. These results indicated that berberine efciently inhibited adipocyte differentiation and may exhibit antiobesity effects in 3T3-L1 cells.  Berberine upregulates the expression levels of miRNA-27a and miRNA-27b in 3T3-L1 cells. Based on the observed effect of berberine treatment on cell viability and differentiation, the present study selected the 10 µM berberine conditions for further mechanistic studies on miRNA-27a and miRNA-27b changes. RT-qPCR was used to confirm the expression levels of miRNA-27a and miRNA-27b following treatment with berberine. Treatment with berberine was observed to upregulate the expression levels of miRNA-27a and miRNA-27b (1.44- and 1.87-fold increase; P<0.01; Fig. 1C). Overall, the present ndings provided  evidence suggesting that berberine treatment upregulates the mRNA expression levels of miRNA-27a and miRNA-27b in 3T3-L1 cells. miRNA-27a and miRNA-27b regulate the berberine-mediated inhibition of differentiation in 3T3-L1 cells. After deter-mining that miRNA-27a and miRNA-27b were upregulated by berberine in 3T3-L1 cells, the present study investigated whether the expression levels of miRNA-27a and miRNA-27b regulated the differentiation of 3T3-L1 cells after treatment with berberine. To demonstrate the association between cell differentiation and miRNA-27a, as well as miRNA-27b, miRNA-27a and miRNA-27b mimics were transfected into 3T3-L1 cells. The mRNA expression levels of miRNA-27a and miRNA-27b were subsequently assessed by RT-qPCR. As shown in Fig. 2A and B, transfecting 40 nM miRNA-27a or miRNA-27b mimics into 3T3-L1 cells resulted in a 1.71- and 1.70-fold increase in the mRNA expression levels of miRNA-27a and miRNA-27b, respectively. By contrast, knockdown of miRNA-27a or miRNA-27b by transfecting anti-miRNA-27a and anti-miRNA-27b into the 3T3-L1 cells resulted in a 72.2 and 53.8% decrease in the mRNA expression levels of miRNA-27a and miRNA-27b, respectively (P<0.01; Fig. 2C).Furthermore, 3T3-L1 cell differentiation was signifi-cantly decreased following treatment with miRNA-27a and miRNA-27b mimics, combined with berberine treatment Figure 1. Berberine inhibits cell viability, differentiation and upregulates miRNA-27a and miRNA-27b in 3T3-L1 cells. (A) Berberine (1, 10, 20, 40 and 80 µM) signicantly inhibited the viability of 3T3‑L1 cells in a time‑ and dose‑dependent manner when compared with the control group. (B) miRNA‑27a and miRNA‑27b were upregulated in 3T3‑L1 cells following treatment with berberine. (C) Berberine (1, 10 and 20 µM) signicantly inhibited the differentiation of 3T3‑L1 cells in a dose‑dependent manner when compared with the control group (magnication, x200). The data are presented as the mean ± standard deviation (n=3; * P<0.05 and ** P<0.01 compared with the control). miRNA, microRNA.  A B C  WU  et al : BERBERINE UPREGULATES miRNA-27a AND miRNA-27b 2728 alone (Fig. 3A). However, knockdown of miRNA-27a and miRNA-27b increased the differentiation of 3T3-L1 cells following treatment with berberine compared with NC (Fig. 3B). These results suggested that the overexpression of miRNA-27a and miRNA-27b increased the berberine-medi-ated inhibition of 3T3-L1 cell differentiation, and that knockdown of miRNA-27a and miRNA-27b attenuated the inhibition of cell differentiation. miRNA-27a and miRNA-27b regulate TG contents in 3T3-L1 cells following treatment with berberine. After determining that the differentiation of 3T3-L1 cells was regulated by miRNA-27a and miRNA-27b, the present study analyzed the intracellular TG contents in the 3T3-L1 cells following transfection with miRNA-27a or miRNA-27b mimics, as well as anti-miRNA-27a and anti-miRNA-27b, and NC for 48 h. As shown in Fig. 4A, the intracellular TG contents were decreased by 44.8 and 37.9% by the miRNA-27a and miRNA-27b mimics transfection in berberine treated 3T3-L1 cells, respectively (P<0.01). However, as shown in Fig. 4B, the intracellular TG contents were increased by 27.8 and 19.7% by anti-miRNA-27a and anti-miRNA-27b transfection in berberine treated 3T3-L1 cells, respectively (P<0.01). These results indicated that miRNA-27a and miRNA-27b negatively regulate the intracellular TG contents, which have been demonstrated to impair the differentiation of 3T3-L1 cells. Figure 2. mRNA expression levels of miRNA-27a and miRNA-27b in 3T3-L1 cells. (A) RT-qPCR analysis showing the upregulation of miRNA-27a and miRNA-27b in 3T3-L1 cells transfected miRNA-27a and miRNA-27b mimics, respectively. (B) RT-qPCR analysis showing the downregulation of miRNA-27a and miRNA-27b in 3T3-L1 cells transfected with siRNA targeting anti-miRNA-27a and anti-miRNA-27b. The data are presented as the mean ± standard deviation (n=3; ** P<0.01 vs. NC). NC, negative control; siRNA, small interfering RNA; miRNA, micro RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.Figure 3. miRNA-27a and miRNA-27b regulate the berberine-mediated inhibition of differentiation in 3T3-L1 cells. (A) Overexpression of miRNA-27a and miRNA-27b enhanced berberine-mediated inhibition of differentiation in 3T3-L1 cells. (B) Inhibition of miRNA-27a and miRNA-27b attenuated the berberine-mediated inhibition of differentia- tion in 3T3‑L1 cells. Magnication, x200. NC, negative control; miRNA, microRNA.Figure 4. miRNA-27a and miRNA-27b regulate triglyceride contents in 3T3-L1 cells. (A) Overexpression of miRNA-27a and miRNA-27b reduced triglyceride contents in 3T3-L1 cells in response to treatment with berberine. (B) Inhibition of miRNA-27a and miRNA-27b increased triglyceride con-tents in 3T3-L1 cells in response to treatment with berberine. The data are presented as the mean ± standard deviation (n=3; ** P<0.01 compared with the NC). NC, negative control; miRNA, microRNA.  A B A B A B  MOLECULAR MEDICINE REPORTS 14: 2725-2731, 2016  2729 miRNA-27a directly targets the PPAR- γ  in 3T3-L1 cells. To investigate the regulatory mechanisms of miRNA-27a, bioinformatics analysis (TargetScan) was used. TargetScan identied that the mRNA sequence of PPAR‑ γ  contained a potential binding site for miRNA-27a (Fig. 5 A). To conrm PPAR- γ  as a target and that this was regulated by miRNA-27a in 3T3-L1 cells, the PPAR- γ  3'-UTR was cloned and inserted into a luciferase reporter vector. The luciferase assay revealed that miRNA‑27a signicantly suppressed luciferase activity containing the PPAR- γ  3'-UTR (Fig. 5B). Western blot-ting analysis demonstrated that miRNA-27a overexpression significantly suppressed endogenous PPAR- γ  expression, while inhibition of miRNA‑27a signicantly increased the protein expression of PPAR- γ  in 3T3-L1 cells in the absence and presence of berberine (Fig. 5C and D). Together, these results suggested that PPAR- γ  is a target of miRNA-27a and is downregulated by berberine in 3T3-L1 cells. Discussion Berberine serves an essential role in regulating numerous important cellular processes, including growth, differen-tiation, invasion, migration and apoptosis. Previous studies have suggested that berberine inhibits the proliferation of breast cancer cell by inducing cell cycle arrest (22) and promoted osteoblast differentiation by activating Runx2 and p38 mitogen-activated protein kinase (MAPK) (23). By contrast, berberine was observed to suppress Th17 and Th1 T cell differentiation by modulating the activities of extra-cellular-regulated kinase, p38 MAPK and c-Jun N-terminal kinase (24). In the present study, berberine inhibited the viability (Fig. 1A) and differentiation (Fig. 1B) of 3T3-L1 cells in a dose- and time-dependent manner. Therefore, the effects of berberine on different cell types may not be consistent and comparable.To investigate the mechanisms by which berberine suppressed the viability and differentiation of 3T3-L1 cells, the mRNA expression levels of miRNA-27a and miRNA-27b were also measured by RT-qPCR. The present data showed that miRNA-27a and miRNA-27b were upregulated in 3T3-L1 cells following treatment with 10 µM berberine (Fig. 1C), which is in accordance with a recent report by Lo et al  (25) who demon-strated that berberine treatment upregulated miRNA-21-3p in the HepG2 human hepatoma cell line (25). Potential regula-tory miRNAs, which were upregulated or downregulated in 3T3-L1 cells, were recently reported (26,27). No previous study has experimentally defined the direct association between berberine and miRNA-27a, as well as miRNA-27b, although miRNAs have been predicated to be putative targets of berberine. The present ndings provided the rst evidence, to the best of our knowledge, that berberine directly enhances the expression levels of miRNA-27a and miRNA-27b. Figure 5. Effects of miRNA-27a on the expression of PPAR- γ  in 3T3-L1 cells. (A) The TargetScan predicted base-pairing interaction of miRNA-27a seed sequences and PPAR- γ . (B) PPAR- γ  was demonstrated to be a direct target gene of miRNA-27a by assessing the luciferase activity. The PPAR- γ  protein levels were examined in 3T3-L1 cells transfected with miRNA-27a mimics, inhibitors or NC in the (C) absence and (D) presence of berberine. The protein expression was quantied and normalized against the expression of GAPDH. The data are presented as the mean ± standard deviation (n=3; ** P<0.01 compared with the NC). NC, negative control; miRNA, microRNA; PPAR, peroxisome proliferator-activated receptor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.  A B C D
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