2.1 Reagents, antibodies, and plasmids
TRIzol reagent (15596026) and SuperScript™ First-Strand Synthesis system (18080,51) were purchased from Invitrogen (Grand Island, NY). Actinomycin D (sc-200906) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). The dual-luciferase assay kit was purchased from Promega (Madison, WI, E1960). The specific antibodies against DNMT3b (GTX129127), TET1 (GTX124207), TET2 (GTX124205), and GAPDH (GTX100118) were purchased from Genetex (Irvine, CA, USA) and antibodies specifically against p-c-Jun Ser73 (3270S), c-JUN (9165S), c-Jun(D) (5000S), PARP (9542P), and Elk-1 (9182S) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific for cyclin D1(sc-20044), DNMT3a (sc-373905), JunB (sc-46), CDK4 (sc-601), CDK6 (sc-7180), c-fos (sc-52), and Ets-1 (sc-55581) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-Actin (66009-1-Ig) were purchased from Proteintech (Rosemont, IL, USA). V5-DEST-MIR190A and its control construct were kindly gifted by Dr. Ping-Yee Law (Department of Pharmacology, University of Minnesota, Minneapolis, MN, USA), and GFP–p27 was likewise a gift from Dr. Gustavo Baldassarre (Division of Experimental Oncology, Centro di Riferimento Oncologico, National Cancer Institute, Aviano, Italy). The human miR-190 inhibitor was purchased from GeneCopoeia™ Inc. (Rockville, MD, USA). The human p27 promoter (-1324 to + 162) was cloned into the pGL3-basic luciferase reporter. Human CDKN1B and TET1 3’UTR were cloned into the pMIR-report plasmid. Plasmids were prepared using the Plasmid Preparation/Extraction Maxi kit (Qiagen, Valencia, CA, USA).
2.2 Target identification
Two different online tools, miRDB (http://www.mirdb.org/) (16) and Target scan (http://www.targetscan.org/vert_72/) (17) were used to identify target genes of miR-190.
2.3 Cell cycle analysis
Constructed transfectants were cultured in each well of six-well plates to 70–80% confluence using normal culture medium. The cell culture medium was replaced with 0.1% FBS DMEM with 2 mmol/L L-glutamine and 25 µg gentamicin, and the constructed transfectants were again cultured for 24 h. Cells were then suspended in 70% ethanol for 24 h at 4 °C, then incubated with RNase A at 37 °C for 30 min, and stained with propidium iodide (PI) at 4 °C for 30 min. DNA content was determined by flow cytometry using the Epics XL flow cytometer (Beckman Coulter Inc., San Diego, CA), as previously described(18).
2.4 Cell culture and transfection
UROtsa was cultured in 1640 with 10% FBS and UMUC3 was cultured in DMEM with 10% FBS. Cells were transfected with plasmid DNA using PolyJet™ DNA In Vitro Transfection Reagent (SignaGen Laboratories, Gaithersburg, MD, USA). Stable transfectants were selected with the corresponding antibiotics for 3–4 weeks, depending on the different transfected antibiotic resistance plasmids.
2.5 Dual-luciferase reporter assay
The dual luciferase assay kit was purchased from Promega (Madison, WI, USA). BC cells were co-transfected with either the TLN2 and CDKN1B promoter-luciferase reporter constructs or CDKN1B and TET1 3’-UTR-luciferase reporter constructs, together with the Renilla luciferase vector pRL-TK. After stabilization, the cells were treated with passive lysis buffer according to the dual-luciferase assay manual and then measured with a luminometer (Lumat LB9507, Berthold Tech., Bad Wildbad, Germany). For each analysis, the firefly luciferase signal was normalized to the Renilla luciferase signal to eliminate the difference in transfection efficiency, as previously described(19).
2.6 Reverse transcription-PCR (RT-PCR)
Total RNA from the cells was isolated using the trizol reagent. Total RNA (5 µg) was then used for reverse transcription with oligo dT primer through the SuperScript™ First-Strand Synthesis system IV (Invitrogen, Grand Island, NY). Specific primer pairs were designed to amplify human CDKN1B (forward: 5’-CAA GTA CGA GTG GCA AGA G-3’, reverse: 5’-ATG CGT GTC CTC AGA GTT AG -3’) and GAPDH (forward: 5’-AGA AGG CTG GGG CTC ATT TG-3’, reverse: 5’-AGG GGC CAT CCA CAG TCT TC-3’). Total miRNAs were extracted using the miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). Total RNA (1.0 µg) was used for reverse transcription following the manufacturer’s instructions, and miRNA expression was determined by the Q6 real-time PCR system (Applied Biosystems, Carlsbad, CA, USA) using the miScript PCR Starter Kit and miScript PCR kit II RT Kit (Qiagen, Valencia, CA, USA). U6 was used as the endogenous normalizer. The primer for miR-190 (5’- TGA TAT GTT TGA TAT ATT AGG T-3’) was synthesized by Genewiz Biotechnology (South Plainfield, USA). The cycle threshold (CT) value was measured, and the relative expression of mRNA was calculated based on the value of 2-ΔΔCT as described in our published studies (20).
2.7 Immunoblotting assay
Whole cells were washed twice with ice-cold PBS and then extracted using cell lysis buffer (10 mM pH7.4 Tris-HCl, 1% SDS, 1 mM Na3VO4, and proteasome inhibitor) on ice. The materials were heated at 100 °C for 10 min and then ultrasonicated to destroy all nucleic acids. The protein concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Holtsville, NY, USA). The cell extracts were subjected to Western blot analysis with each antibody. The protein bands specifically binding to the primary antibodies were detected using an alkaline phosphatase (AP) conjugate secondary antibody and enhanced chemifluorescence (ECF) Western blot analysis system (Amersham Pharmacia Biotech, Piscataway, NJ) as previously described(21). The results shown are from at least three independent experiments.
2.8 Anchorage-independent growth
In brief, 1 × 104 UROtsa (miR-190), UMUC3 (miR-190 inhibitor), UROtsa (miR-190/GFP-p27), and UMUC3 (miR-190 inhibitor/shp27) stable transfectants and control vector transfectants were exposed to Basal Medium Eagle (BME) containing 0.33% agar and seeded on the bottom layer of 0.5% agar in 10% FBS BME in each well of six-well plates. The cultures were maintained at 37 °C in a 5% CO2 incubator for 3–4 weeks, and the cell colonies with > 32 cells were scored. Colonies were observed and counted under a microscope (DMi1, Leica, Germany). The results are presented as the mean ± SD of colony number per 10,000 seeded cells in soft agar as described in a previous paper(22).
2.9 DNA Extraction, Bisulfite DNA Modification, and Methylation-Specific PCR
CpG islands were predicted using MethPrimer 2.0 (http://www.urogene.org/ methprimer2/) for the upstream region of the CDKN1B Promoter. Genomic DNA from UROtsa and UMCU3 cells was extracted using a DNeasy Blood and Tissue Kit (# 69504, Qiagen, Valencia, CA, USA). Sodium bisulfite modification of DNA and subsequent purification was performed according to the manufacturer’s instructions for bisulfite conversion of unmethylated cytosines in DNA (EpiTect Bisulfite kit; #59104, Qiagen, Valencia, CA, USA).
Then, the bisulfite-treated genomic DNA was optimized for a methylation-specific PCR protocol, that is, 20 µL reactions containing 10 ng template, 10 µL 2 × EpiTect Master Mix (Qiagen, Valencia, CA, USA), and 0.4 µM of a given set of methylation primers (Methylation-F, GTA GAT TAC GAG GTG GGG GTC; and Methylation-R, CTA AAA CGA AAC CTA AAA TTC GAA) or 0.4 µM each unmethylation primers (Unmethylation-F, TAG ATT ATG AGG TGG GGG TTG T; and Unmethylation-R, CCT AAA ACA AAA CCT AAA ATT CAA A). Touchdown PCR was then performed as follows: 95 °C for 10 min followed by five cycles of 94 °C for 30 sec, 70 °C for 30 sec, 72 °C for 30 sec; five cycles of 94 °C for 30 sec, 65 °C for 30 sec, 72 °C for 30 sec; and 30 cycles of 94 °C for 30 sec, 60 °C for 30 sec, 72 °C for 30 sec. The final extension was performed at 72 °C for 7 min. All products were then separated on 2% high-resolution agarose gels and visualized by ethidium bromide staining. All PCR products were run in duplicate using EpiTect PCR Control DNA Set (#59695, Qiagen, Valencia, CA, USA).
2.10 Statistical analyses
Student’s t-test was used to determine significant differences between the treated and untreated groups. Results are expressed as the mean ± SD from at least three independent experiments. P < 0.05 was considered to be a significant difference between the compared groups. All data were analyzed using GraphPad Software 6.0 (La Jolla, CA, USA).