br To confirm the level of miR in each
To confirm the level of miR-143 in each organ, including tumor, we performed RT-PCR (Figure 5B). As a result, PIC groups showed the highest blood levels of miR-143 among the groups tested. The levels of miR-143#12 in the xenografted tumors were elevated, which would reflect the levels in the blood samples (Figure 5C). Especially, the miR-143#12/PIC group showed more accumulation of miR-143 in the tumors compared with the other groups. To understand whether miR-143 delivered by PIC inhibited tumor growth through RNAi, we performed an Argonaute2 (Ago2) loading assay (Figure 5D). The result suggested that miR-143 was included into the RNA-induced silencing complex (RISC) at least in part by binding with Ago2 pro-tein in the cells. Also, western blot analysis of the possible target pro-teins of miR-143 in samples from the treated tumors gave results similar to those obtained in vitro (Figures 5E and 2D). The protein levels of T-RAS and RAS-related proteins were downregulated in all treated groups compared with those in the control group. Patho-logically, lipofection caused hepatocyte toxicity, which was not observed in the case of PIC (Figure S4B).
Effect of Intravesical Administration of miR-143#12/PIC on Orthotopic Mouse Model
To examine the growth-inhibitory effect of miR-143#12 on the growth of Puromycin tumors in vivo, we used a mouse model bearing 253J-BV cell-xenografted tumors that was established by transplant-ing these cancer cells into the bladder wall of nude mice. miR-143#12/PIC, which was more effective in intravenous adminis-tration experiments, was used. The mice were treated with miR-143#12/Lipo or -143#12/PIC, and the level of miR-143 was compared in some organs (Figure S4C). Based on the results, miR-143#12 was delivered into the bladder cavity via the PIC nano-carrier, which proved to be safe and effective for general administra-tion. As a result, miR-143#12/PIC significantly inhibited the tumor growth, when compared with that for the control-miR/PIC group, and the mice treated with miR-143#12/PIC showed almost 60% in-hibition of tumor growth (Figure 6A; Figure S4D). Kaplan-Meier plots showed that the treatment group showed a significantly pro-longed animal survival when compared with the control-miR/PIC group (Figure 6B). The latter group showed remarkable weight loss compared with the former one (Figure 6C). There was an in-verse correlation between the bladder weight and body weight (data not shown).
We also performed the Ago2 loading assay to examine whether miR-143/PIC suppressed the tumor growth through RNAi in the tu-mor cells. As shown in Figure 6D, a significant amount of miR-143
Figure 2. Anti-proliferative Effects of Syn-miR-143s on 253J-BV Bladder Cancer Cells
Molecular Therapy: Methods & Clinical Development
Figure 3. Effects of AKT Inhibitor or MEK Inhibitor on the Proliferation and Expression of RAS and RAS-Related Genes
was included into RISC, as observed in the case of general injection. Western blot analysis of protein samples from the treated tumors gave results similar to those of in vitro experiments. Namely, the levels of RAS and RAS-related proteins in the miR-143#12/PIC group were decreased compared with those in the control-miR/ PIC group, except for PI3K/AKT-signaling proteins (Figure 6E). To confirm the distributed levels of miR-143 in general organs as well as in tumors, we performed RT-PCR (Figure 6F). As expected,
Figure 4. Effects of Silencing K-RAS and H-RAS on
Cell Growth and the Ras Networks and Targeting of
(F) Active K-RAS-GTP is downregulated by treatment with miR-143#12 through the silencing of SOS1. (G) Luciferase activities after co-transfection of 253J-BV cells with miR-143#12 and pMIR vectors having the wild-type or mutant-type miR-143-binding site in the 30 UTR of SOS1 mRNA.
the treatment group of miR-143#12/PIC showed the increased levels of miR-143 mainly in the bladder tumors and blood samples. Our findings suggested that our intravesical infu-sion of chemically modified miR-143#12 would be a candidate of novel miRNA medicine for early BC.
In this study, we demonstrated that miR-143 was downregulated in the clinical BC samples and that there was a potent anti-cancer activity of the novel synthetic miR-143#12 toward BC cell proliferation by systemic or intravesical infusion in 253J-BV cell-xenografted mice. The synthetic miR-143#12 was selected among more than 100 kinds of miR-143 derivatives. cDNA microarray analysis indicated the similar profiles of the silenced genes after the transfection with each synthetic miR-143 used in this study (data not shown). Previously, we reported that miR-143#12 downregulated K-RAS-signaling networks through silencing K-RAS, its effector-signaling molecules Erk and Akt, and K-RAS GDP/GTP transducer SOS1.20 In 253J-BV BC cells, both H-RAS and K-RAS are overexpressed and oncogenic. miR-143 silenced K-RAS directly and impaired the positive circuit of the RAS expression sys-