br PI K Akt signaling pathways In light of the
PI3K/Akt-signaling pathways. In light of the therapeutic effect of systemic or intravesical administration seen in vivo, nucleic Puromycin medi-cine using miR-143#12 would be a novel strat-egy for treating RAS-driven BC.
MATERIALS AND METHODS
Patients and Their Samples
All human samples were obtained from patients who had undergone biopsy or surgery for resec-tion at Osaka Medical College Hospital (Takat-suki, Osaka, Japan). Informed consent in writing was obtained from each patient.
The consent and this study were reviewed and approved by the University Hospital Medical Information Network Center (approval R000027312), in accordance with the tenets of the Declaration of Helsinki. 20 patients with previously untreated BC or with recurrence after curative treatment were selected. The distribu-tion according to other clinical parameters is
shown in Table 1. Under a pathologist’s supervision, all tissue sample pairs were collected from surgically resected tissues, with these paired samples being from the primary tumor and its adjacent non-tumor tissue in the same patient. These paired samples were examined by western blot analysis and real-time RT-PCR.
Cell Culture and Cell Viability
Human BC 253J-BV cell line was obtained from the JCRB (Japanese Collection of Research Bioresources) Cell Bank. Cell line authentica-tion was done by short-tandem-repeat (STR) analysis, which was per-formed by using primers for TH01, TPOX, vWA, amelogenin, CSF1PO, D16S539, D7S820, D13S317, D5S818, and D21S11 (GenePrint 10 System; Promega, Madison, WI, USA). The cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO, USA) and 2 mM L-glutamine, under an atmosphere of 95%
Molecular Therapy: Methods & Clinical Development
air and 5% CO2 at 37 C. The number of viable cells was determined by performing the trypan blue dye exclusion test.
253J-BV cells were seeded into 6-well plates at a concentration of 0.5 105/well (10% 30% confluence) on the day before the transfec-tion. Three types of miRNA were used: the mature type of miR-143, which was a commercially available miR-143 from Ambion (mirVana miRNA mimic; Ambion, Foster City, CA, USA) (miR-143Am), and 2 types of synthetic miR-143s (Syn-miR-143s: miR-143#1 and miR-143#12). Basically, miR-143 was chemically modified in the guide strand of miR-143#1, as shown in Figure 2A. All types of miR-143s were used for the transfection of the cells, which were achieved by using cationic liposomes, Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer’s lipofection protocol. The nonspecific control miRNA (HSS, Hokkaido, Japan) sequence was 50-GUA GGA GUA GUG AAA GGC C-30, which miRNA was used as a control for nonspecific effects.37
In treatment experiments, the sequence of the siR-KRAS was 50-CGG UCA UCC AGU GUU GUC AUG CAU U-30 and that of siR-SOS1 was 50-CGG CAU GUA CUA CAG GCC UGU UUA-30. The effects manifested by the introduction of all types of miR-143, siR-KRAS, siR-HRAS, and siR-SOS1 into the cells were assessed at 72 h after the transfection. We used the same dose of Lipofectamine RNAiMAX in all transfection experiments.
Western Blot Analysis
Protein extraction and western blotting analysis were performed as described in previous reports.38,39 The following primary antibodies were used: antibodies against c-Myc, p-AKT, AKT, p-ERK, ERK, PARP, and GAPDH (Cell Signaling Technology, Danvers, MA, USA); Total RAS (Abcam, Cambridge, UK); and K-RAS and H-RAS (Santa Cruz Biotechnology). Anti-rat, anti-rabbit, and horse anti-mouse immunoglobulin G (IgG) (Cell Signaling Technology) were used as secondary antibodies. GAPDH served as an internal control.
253J-BV cells were seeded into 6-well plates at a concentration of 0.5 105/well (10% 30% confluence) on the day before the transfec-tion. AKT inhibitor (Calbiochem, USA) and MEK inhibitor PD 98059 (Calbiochem, USA) were used for the transfection of the cells without a drug delivery system. The effects manifested by the introduction of the AKT inhibitor and MEK inhibitor into the cells were assessed at 72 h after the transfection. AntagomiR from Ambion (Anti-miR miRNA Inhibitors, Foster City, CA, USA) was used for the transfec-tion of the cells, which was achieved by using cationic liposomes, Lip-ofectamine RNAi MAX (Invitrogen), according to the manufacturer’s lipofection protocol.
Total RNA was isolated from cultured cells or tumor tissues by using a NucleoSpin miRNA isolation kit (TaKaRa, Otsu, Japan). RNA con-centrations and purity were assessed by UV spectrophotometry.