ADSC Isolation and culture
Rat adipose tissue was excised from the inguinal region of male Sprague-Dawley rats, as approved by the Institutional Animal Care and Use Committee. These tissue samples were processed for ADSC isolation as described previously (Ning et al., 2006
; Ning et al., 2008
). Briefly, the tissue was rinsed with phosphate-buffered saline (PBS) containing 1% penicillin and streptomycin, minced into small pieces, and then incubated in a solution containing 0.075% collagenase type IA (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 37°C with vigorous shake. The top lipid layer was removed and the remaining liquid portion was centrifuged at 220×g for 10 minutes at room temperature. The pellet was treated with 160 mM NH4
Cl for 10 minutes to lyse red blood cells. The remaining cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), filtered through a 40-µm cell strainer (BD Biosciences, Bedford, MA, USA), and plated at a density of 1 ×106
cells in a 10-cm dish. After reaching 80% confluence, the cells were harvested and stored in liquid nitrogen at a density of 5 × 105
cells per milliliter of freezing media (DMEM, 20% FBS, and 10% dimethylsulfoxide [DMSO]). Cells were thawed and cultured as needed in DMEM supplemented with 10% FBS, 1% nonessential amino acid, 10,000 units/mL penicillin, 10,000 mcg/mL streptomycin SO4
, 0.025 mg/mL fungizone, and 110 mg/mL sodium pyruvate. Culture incubator was set at 37°C with 5% CO2
. Cells in passages four to eight were used in this study.
Treatment with IBMX, IGF-I, picropodophyllin (PPP), and miRNAs
For treatment with IBMX, ADSC were seeded in 6-well plates or 10-cm dishes at 40–60% confluence in DMEM containing 10% FBS. The next day, the cells were washed 3 times with PBS and the medium changed to DMEM supplemented with 500 µM of IBMX (Sigma-Aldrich, St. Louis, MO). For treatment with IGF-I in the presence or absence of PPP, ADSC were seeded in 6-well plates or 10-cm dishes at 70% confluence in DMEM containing 10% FBS. The next day, the medium was changed to in DMEM containing 1% FBS. Twenty-four h later, the cells were treated with or without 2.5 µM of PPP (cat. # 407247, EMD Biosciences, La Jolla, CA) for 1 h and then 100 ng/ml of IGF-I (cat. # 4121-20, Biovision, Mountain View, CA) for another 3 h. For treatment with miRNAs, ADSC were seeded into 6-well plates or 10-cm dishes at 70% confluence in DMEM containing 10% FBS. The next day, the cells were transfected with 50 nM of miR-133b, miR-124a (cat. AM17100, Ambion Inc, Austin, TX) or miRNA negative control (cat. # AM17120, Ambion) using HiPerfect transfection reagent (cat. # 301705, Qiagen, Valencia, CA). Forty-eight h later, the medium was changed to DMEM containing 1% FBS. Another 24 h later, the cells were treated with or without 2.5 µM of PPP for 1 h and then 100 ng/ml of IGF-I or 500 µM of IBMX for another 3 h. Each experiment was repeated three times.
RT-PCR and Real-time PCR
Primers for these experiments are listed in . RT-PCR was performed for the detection of IGF-IR and β-III-tubulin mRNA as previously described (Lin et al., 2002
). Real-time PCR was performed for the quantification of Pitx3 mRNA as following. The reaction consisted of 4 µl cDNA, 4 µl primers (500 nM), 2 µl H2
O and 10 µl SYBR Green PCR master mixture and was run in ABI7300 PCR System (Applied Biosystems, Foster City, CA) with settings of 1 cycle at 95 °C for 10 min, 1 cycle at 95 °C for 15 s and 60 °C for 1 min, 40 cycles at 95 °C for 15 s, 60 °C for 30 s, and 95 °C for 15 s. Real-time PCR was also performed for the quantification of miR-133b as following. MicroRNAs were isolated with an isolation kit (cat. # AM1561, Ambion) and reverse transcribed into cDNA using a reverse transcription kit (cat. # RA620A-1, System Biosciences Inc. [SBI], Mountain View, CA). The PCR reaction was composed of 1 µl cDNA, 1 µl of forward primer of miR-133b (cat. CSRA640B-1, SBI) or U6 (internal control), 0.5 µl of 3’-Universal reverse primer, 10 µl of H2
O and 12.5 µl of SYBR® Green powerful PCR master mixture. The reaction was run in ABI 7300 PCR System (Applied Biosystems, Foster City, California) with settings of 1 cycle at 95 °C for 10 minutes, 1 cycle at 95 °C for 15 seconds and at 60 °C for 1 minute, and 40 cycles at 95 °C for 15 seconds, 60 °C for 30 seconds and 95 °C for 15 seconds. Each experiment was repeated three times.
Construction of miRNA target reporter vectors
Nucleotide sequence of the 3’-untranslated region (3’-UTR) of rat IGF-IR was retrieved from GenBank (Accession # NM_052807). The sequence was uploaded to the RegRNA website (http://regrna.mbc.nctu.edu.tw/html/prediction.html
) for the identification of potential miRNA target sites. One site was identified and its sequence was used to design oligonucleotides for the construction of reporter vectors. The oligonucleotides of the wildtype version were: sense, 5’-ctagtagatcttgtgtgctcactcggtgggcggaggggggagcaggttgtaacaaa; antisense, 5’-agcttttgttacaacctgctcccccctccgcccaccgagtgagcacacaagatcta. The oligonucleotides of the mutant version (as negative control) were: sense, 5’-ctagtagatcttgtgtgctcactcggtgggcggagggggggttgtaacaaa; antisense, 5’-agcttttgttacaaccccccctccgcccaccgagtgagcacacaagatcta. The complementary oligonucleotides were annealed and inserted between the HindIII and SpeI restriction sites of pMIR-Report plasmid (cat. # AM5795, Ambion). The resultant vectors were designated pMIR-IGF1R-3’-UTR and pMIR-IGF1R-3’-UTR-M for the wildtype and mutant, respectively.
We have previously used luciferase assay to analyze gene promoter activities (Lin et al., 2002
). In the present study we employed the same technique to determine whether miR-133b suppressed IGF-IR expression through binding to the predicted binding site in the 3’-UTR. To accomplish this goal, we conducted two types of experiments, as described in the following. In the first set of experiments, miR-133b was introduced into the system by transfection. Briefly, COS7 cells (Lin et al., 2002
) were seeded into 12-well plates in DMEM at 70% confluence. The next day, cells were transfected with 200 ng of pMIR-IGF1R-3’UTR (or pMIR-IGF1R-3’UTR-M), 30 nM of miR-133b (or miR-negative control), and 100 ng of pRL-TK (internal control for transfection efficiency, Promega, Inc., Madison, WI), using Superfectin transfection reagent (Qiagen). Forty-eight h later, the transfected cells were assayed for firefly luciferase activity (pMIR-IGF1R-3’UTR activity) and Renilla luciferase activity (pRL-TK activity) with a Dual-Luciferase Reporter Assay System and TD-20/20 luminometer (Turner BioSystems, Sunnyvale, CA). In the second set of experiments, miR-133b was introduced into the system by treatment of ADSC with IBMX or IGF-I. Briefly, ADSC were seeded into 12-well plates at 70% confluence. The next day, the cells were transfected with 200 ng of pMIR-IGF1R-3’UTR and 100 ng pRL-TK with Superfectin. Forty-eight h later, the cells were treated with 100 ng/ml of IGF-I or 500 µM of IBMX for 3 h. Thereafter, the cells were assayed for luciferase activity as described above. Each experiment was repeated three times.
ADSC were seeded into 6-well plates at 70% confluence and transfected the next day with precursor molecules of miR-124a, miR-133b (Cat. # AM17100, Ambion) or miR-negative control (Cat. # AM17110, Ambion) using HiPerfect transfection reagent (Cat. #301705, Qiagen). Forty-eight hours later, the cells were induced with 500 µM IBMX for 3 hours. The cells were then fixed with ice-cold methanol for 8 min, permeabilized with 0.05% Triton X-100 for 5 min, and blocked with 5% normal horse serum in PBS for 1 hr at room temperature. The cells were then incubated with an anti-β-III-tubulin antibody (ab14545, Abcam, Cambridge, MA) for 1 hr at room temperature. After three washes with PBS, the cells were incubated with a secondary antibody (FITC-conjugated goat anti-mouse or anti-rabbit IgG) for 1 hr at room temperature. After three washes with PBS, the cells were further stained with 40,6-diamidino-2-phenylindole (DAPI, for nuclear staining) for 5 min, examined under a fluorescence microscope, and photographed.
Western blot analysis
Cells were seeded in 10-cm dishes and transfected and induced as above. The cells were then lysed in a lysis buffer containing 1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% SDS, aprotinin (10 µg/ml), leupeptin (10 µg/ml), and PBS. Cell lysates containing 20 µg of protein were electrophoresed in SDS-PAGE and then transferred onto a PVDF membrane (Millipore Corp., Bedford, MA). The membrane was stained with Ponceau S to verify the integrity of the transferred proteins and to monitor the unbiased transfer of all protein samples. Detection of protein on the membrane was performed with the ECL kit (Amersham Life Sciences Inc., Arlington Heights, IL) using anti-β-III-tubulin, anti-IGF-IR (#3027, Cell signaling, Danvers, MA), anti-pitx3 (ab30734, Abcam, Cambridge, MA), or anti-β-actin antibody (A5441, Sigma-Aldrich). Before re-probing with anti-β-actin antibody, the membrane was stripped in 62.5 mM Tris-HCl, pH 6.7, 2% SDS, 10 mM 2-mercaptoethanol at 56 °C for 30 min and then washed four times in 1×TBST.
All the statistical data were analyzed with Prism 4 (GraphPad Software Inc., San Diego, CA). T-test was used to compare each pair of treatment. P value <0.05 was considered significant.