br RNA integrity was checked by formaldehyde gel electrophor
RNA integrity was checked by formaldehyde gel electrophoresis. To determine the expression levels of miR-143, we conducted qRT-PCR by using TaqMan MicroRNA Assays (Applied Biosystems) and THUNDERBIRD Probe qPCR Mix (TOYOBO,Osaka, Japan), according to the manufacturer’s protocol. RNU6B was used as an in-ternal control. For determination of the expression levels of K-RAS, H-RAS, N- RAS, SOS1, and GAPDH mRNAs, total RNA was reverse transcribed with a PrimeScript H RT reagent Kit (TaKaRa). RT-PCR was then performed with primers specific for them by using THUNDERBIRD SYBR qPCR Mix (TOYOBO). The primers for K-RAS, H- RAS, N-RAS, SOS1, and GAPDH were the following: K-RAS-sense, 50-CCT GCT CCA TGC AGA CTG TTA-30, and K-RAS-antisense, 50-TGG GGA GAG TGA CCA TGA CT-30; H-RAS-sense, 50- TCA AAC GGG TGA AGG ACY CG-30, and H-RAS-antisense, 50-CTT CCT CCT CCT TCC GTC TG-30; N-RAS-sense, 50-GAA CCA AAC CGC AAA CGT GA-30, and N-RAS-antisense, 50-TCA AGC CCC TAT TGC TGT GG-30; SOS1-sense, 50-GGA GGA GTG TCC CAA TTT ATT AG-30, and SOS1-antisense, 50-TTT CAT TGG CTC ATG TAT AAG GG-30; and GAPDH-sense, 50-CCA CCC ATG GCA AAT TCC ATG GCA-30, and GAPDH-antisense, 50- TCT AGA CGG CAG GTC AGG TCC ACC-30. GAPDH and RNU6B were used as internal controls. The relative expression levels were calculated by use of the DDCt method.
253J-BV cells collected at 72 h after transfection were stained with Hoechst 33342. The details of the experimental protocol were given in a previous report.40 The apoptotic cells were observed.
Luciferase Reporter Assay
Searching the TargetScan 7.1 database (http://www.targetscan.org/) to find algorithm-based 58-60-6 of miR-143, we found the predicted binding sites to be at positions 3,438 3,444 in the 30 UTR of SOS1 mRNA. The sequence region, containing the putative binding sequence of miR–143, was inserted into a pMIR-REPORT Luciferase miRNA Expression Reporter Vector (Applied Biosystems), according to the manufacturer’s protocol. Moreover, we made another pMIRconstruct encompassing a mutated seed sequence for miR-143 (wild-type, CATCTCA; mutant, CAGACCA) by using a PrimeSTARH Mutagen-esis Basal Kit (TaKaRa). The mutation of the vector was confirmed by sequence analysis. pRL-TK Renilla Luciferase Reporter vector (Prom-ega, Madison WI, USA) was used as an internal control vector. 253J-BV cells were seeded into 96-well plates at a concentration of 0. 4 104/well at 2 days before the transfection. 253J-BV cells were co-trans-fected with either reporter vector (0.01 mg/well each) or 20 nM miR-143#12, co-transfection of which was achieved by using Lipofectamine RNAi MAX. Luciferase activities were measured at 48 h after co-trans-fection by using a Dual-Glo Luciferase Assay System (Promega), according to the manufacturer’s protocol. Luciferase activities were reported as the firefly luciferase:Renilla luciferase ratio.
In Vivo Xenograft Model
Animal experimental protocols were approved by the Committee for Animal Research and Welfare of Gifu University (28-87, January
31, 2017). BALB/cSLC-nu/nu (nude) mice were obtained from Japan (Hamamatsu, Japan). Human BC 253J-BV cells, which have many gene mutations, were inoculated at 2.0 106 cells/50 mL with Matrigel (Corning)/50 mL, the mixture being transplanted into the back of each mouse. The inoculation day was set as day
0. At 14 days after inoculation, we confirmed the engraftment of the tumors. miR-143s was administered in 2 formulations, except the control miRNA. In one formulation, Syn-miR-143s (#1 and
#12; 250 mg/kg/1 administration) dissolved in 10 mL HEPES (1 mM) and 10 mL Opti-MEM was mixed with 1 mL Lipofectamine RNAi MAX (Invitrogen) and 80 mL saline and then injected 4 times every 72 h into a large vein. In the other formulation, Syn-miR-143s (#1 and #12; 250 mg/kg/1 administration) dissolved in 10 mL HEPES (1 mM) was mixed with 10 mL PIC comprising poly (ethylene gly-col) and cationic poly (ornithine) dissolved in HEPES (25 mg/mL) and 80 mL NS (not significant) to prepare the PIC nanocarrier32 and then generally administered 4 times, once every 72 h. The tumor volume was calculated by the following formula: 0.5236 L1 (L2),2 where L1 is the long axis and L2 is the short axis of the tumor. An-imal experiments in this study were performed in compliance with the guidelines of the Institute for Laboratory Animal Research of Gifu University and the UKCCCR Guidelines for the Welfare of Animals Used for Experimental Neoplasia. Y.A. was named in the approved experiment. r> In Vivo Orthotopic Model
Human BC 253J-BV cells were implanted at 2.0 106 cells/50 mL with Matrigel (Corning)/50 mL, with the mixture being transplanted into the bladder wall of female nude mice. Once tumors had devel-oped (average weight 0.035 g after about 28 days), they were treated with 8 intravesical injections of miR-143. miR-143 was administered in 2 ways, except the control miRNA. One way was that Syn-miR-143s (#12; 83 mg/kg/1 administration) complexed with 10 mL HEPES (1 mM) and 80 mL saline was delivered intravesically every other day. For the other administration, Syn-miR-143s (#12; 83 mg/kg/1 admin-istration) was mixed with the block copolymer to formulate the PIC nanocarriers as described above and then delivered 8 times intravesi-cally every other day. After the administration, some mice were sacrificed at around 30 days after the inoculation of the cells, and the tumor weight was measured. Others were observed until they died from the disease after the treatments.