Supplementary Figure S5. Evaluation of the toxicity of super carbonate apatite-MIRTX conjugate and effect of systemic administration of formulated MIRTX on tumor growth in vivo. The DLD1 xenograft mouse models received intravenous administrations of MIRTX or control-miR using super carbonate apatite (sCA) as a vehicle on days 0, 2, 4, 7, 9, 12, 14, and 16 via a tail vein injection. Each injection contained 40 μg of formulated oligo with a 40% conjugation rate. Tumors were resected on day 18. (A) Blood chemistry tests were performed on day 18 for the parent, treated with sCA-control-miR, and treated with sCA-MIRTX. NS: not significant. (B) Hematoxylin and eosin-stained section of each organ tissue (brain, heart, lung, liver, spleen, kidney, colon) revealed no particular histological damage in formulated sCA-MIRTX-treated mice. (C) Tumor weights were evaluated for the parent (n = 8), treated with sCA-control-miR (n = 8), and treated with sCA-MIRTX (n = 8). Injection of sCA-MIRTX significantly decreased tumor weights. (D) Another cohort using HT29. All data represent mean {plus minus} SD. *p < 0.01.
ARTICLE ABSTRACT
We previously demonstrated that miR-29b-3p is a hopeful miRNA-based therapy against colorectal cancer. In this study, we aimed to clarify a value of miR-29b-1-5p as a next-generation treatment, especially for KRAS-mutant colorectal cancer. RT-PCR assay showed that the expression of miR-29b-3p was high, and its partner strand, miR-29b-1-5p, level was only negligible in clinical colorectal cancer samples. Mimic-miR-29b-1-5p significantly inhibited proliferation of KRAS-mutant colorectal cancer cell lines DLD1 and SW480 and KRAS wild-type HT29 cells. Proliferative activity was further examined by either miR-29b-1-5p strand or its opposite complementary sequence because miR-29b-1-5p is a passenger miRNA and may have no physiologic function. We found that completely opposite complementary strand to miR-29b-1-5p, but not miR-29b-1-5p, possessed a potent antitumor effect and named this byproduct miRNA sequence “MIRTX.” MIRTX directly targeted the 3′-UTR of CXCR2 and PIK3R1 mRNA and suppressed the NF-κB signaling pathway in KRAS-mutated colorectal cancer cells. MIRTX induced apoptosis in DLD1 with downregulation of antiapoptotic BCL2, BCL-xL, and MCL1 and upregulation of cleaved caspase-3 and cleaved PARP. In mouse xenograft models, systemic administration of MIRTX using a super carbonate apatite as a delivery vehicle significantly inhibited tumor growth of DLD1 and HT29 cells without any particular toxicities. In conclusion, these findings indicate that inhibition of NF-κB signaling by this novel miRNA-based therapeutic could be a promising treatment against refractory KRAS-mutant colorectal cancer and KRAS wild-type colorectal cancer. Mol Cancer Ther; 17(5); 977–87. ©2018 AACR.
