Cell Lines and Cell Culture
SV-HUC-1, T24 and J82 cells were purchased from the American Type Culture Collection (ATCC) and grown according to ATCC protocols. SV-HUC-1 cells were cultured in F-12K Medium (ATCC) with 10% FBS. T24 cells were cultured in McCoy’s 5A medium supplemented with 10% FBS and J82 cells were cultured in Minimum Essential Media (MEM) supplemented with 10% FBS.
Plasmids, Precursors and Transfection
TaqMan probes and precursors for hsa-miR-1280 and negative control pre-miR were purchased from Applied Biosystems (Foster City, CA). pmir-GLO Dual-Luciferase miRNA Target Expression Vector was purchased from Promega. Lipofectamine 2000 (Invitrogen) was used for all transfections.
miRNA and total RNA were extracted from cell lines using a miRNeasy Mini Kit and an RNeasy Mini Kit (Qiagen). miRNAs from clinical samples were extracted using laser capture microdissection techniques with a miRNeasy FFPE kit (Qiagen).
Human Clinical Samples
Clinical samples were obtained from the San Francisco Veterans Affairs (VA) Medical Center. Written informed consent was obtained from all patients and the study was approved by the UCSF Committee on Human Research (Approval number: H9058-35751-01).
Quantitative Real-time PCR
Mature miRNAs were assayed using the TaqMan MicroRNA Assays in accordance with the manufacturer’s instructions (Applied Biosystems). All RT reactions, including no-template controls and RT minus controls, were run in a 7500 Fast Real Time PCR System (Applied Biosystems). RNA concentrations were determined with a NanoDrop (Thermo Scientific, Rockford, IL). Samples were normalized to RNU48 (Applied Biosystems). Gene expression levels were quantified using the 7500 Fast Real Time Sequence detection system Software (Applied Biosystems). Comparative real-time PCR was performed in triplicate, including no-template controls. Relative expression was calculated using the comparative Ct.
In Situ Hybridization
hybridization was performed as described previously 
. Briefly cell lines were stained using DIG-labeled locked nucleic acid (LNA)-based probes specific for mir-1280 following the manufacturer’s protocol (Exiqon,Inc Woburn, MA) and detected using anti-DIG-Fluorescein, Fab Fragments (Roche Applied Science, Indianapolis, IN).
Cell Viability and Clonability Assay
Cell viability was determined at 24, 48 and 72 h by using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) according to the manufacturer’s protocol. Absorbance was measured at 490 nm using SpectraMAX 190 (Molecular Devices). Data are presented as the mean value for triplicate experiments compared to the negative control. For colony formation assay, cells were seeded at low density (1000 cells/plate) and allowed to grow untill visible colonies appeared. Then, cells were stained with Giemsa and colonies were counted.
Migration and Invasion Assays
Cytoselect 24-well cell migration and invasion assay kits (Cell Biolabs, Inc) were used for migration and invasion assays according to the manufacturer’s protocol. Briefly, T24 and J82 cells transfected with Pre-miR miRNA precursor or negative control were harvested 72 hours after transfection and resuspended in serum-free Opti-MEM. Cells (10×104 per 300 µl media without serum) were added to the upper chamber, and the lower chamber was filled with 500 µl of media containing 10% FBS. Cells were incubated for 16 hours at 37°C in a 5% CO2 tissue culture incubator. After 16 hours, non-migrated/non-invading cells were removed from upper side of transwel membrane filter inserts using a cotton-tipped swab. Migrated/invaded cells on the lower side were stained and the absorbance was read at 560 nm according to the manufacturer’s protocol.
Protein was isolated from 70–80% confluent plates of cultured cells using the M-PER Mammalian Protein Extraction Reagent (Pierce Biotechnology, Rockfield, IL) following the manufacturer’s directions. Protein concentrations were determined by the Bradford method. Equal amounts of protein were resolved on 4–20% sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to a nitrocellulose membrane by voltage gradient transfer. The resulting blots were blocked with 5% non-fat dry milk and probed with specific antibodies. Blots were then incubated with appropriate peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence (Pierce Biotechnology, Rockford, IL).
Luciferase Reporter Assay
A pmirGLO Dual-Luciferase miRNA target expression vector was used for 3′-UTR luciferase assays (Promega, Madison, WI). The target oncogene of miRNA-1280 was selected on the basis of online microRNA target database http://www.microrna.org/microrna/home.do
. The primer sequences for the wild type 3′UTR were: Forward 5′ CGCGGCCGCTAGTCTGTGGAATCGTGTGGGAT
3′ and Reverse 5′ ctagatcccacacgattccacagactagcggccgcgagct 3′. For the mutant 3′UTR, the primer sequences were: Forward 5′ CGCGGCCGCTAGTCTGTGGAATCGTTCATACT
3′ and reverse 5′ ctagagtatgaacgattccacagactagcggccgcgagct 3′. For lucifease assay, T24 and J82 cells were cotransfected with hsa-miR-1280 and pmirGLO Dual-Luciferase miRNA target expression vectors with wild-type or mutant target sequence using Lipofectamine 2000. Firefly luciferase activities were measured using the Dual Luciferase Assay (Promega, Madison, WI) 18 hr after transfection and the results were normalized with Renilla luciferase. Each reporter plasmid was transfected at least three times (on different days) and each sample was assayed in triplicate.
Statistical analyses were performed with GraphPad Prism 5 and MedCalc version 10.3.2. All quantified data represents an average of at least triplicate samples or as indicated. Error bars represent standard deviation of the mean. All tests were performed two tailed and p-values <0.05 were considered statistically significant. Receiver operating curves (ROC) were calculated to determine the potential of miR-1280 to discriminate between malignant and non-malignant samples. For disease progression, Kaplan-Meier (log-rank test) analysis was performed.