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  • br RESULTS br Expression of

    2020-08-12


    RESULTS
    Expression of miR-143 Was Extremely Downregulated in Clinical Tumor Samples from BC Patients
    We first examined the expression levels of miR-143 in BC tumor and adjacent normal tissue samples from the same patient (Table 1). Totally, 20 cases were examined. The expression levels of miR-143 in the clinical tumor samples examined by real-time PCR were extremely downregulated compared with those in the adjacent normal tissues (Figure 1A). Since miR-143 silences K-RAS,24 the 
    The Expression Levels of K-RAS and H-RAS Were Upregulated in miR-143-Downregulated Human BC 253J-BV Cells
    We examined the levels of miR-143 and RAS isoforms in human normal transitional human urothelial Puromycin (HUCs) and in the BC 253J-BV cell line used in this study. As shown in Figure 1D, K-RAS was the major isomer (approximately 70%) as judged from the mRNA levels in 253J-BV cells. On the other hand, the expression level of miR-143 in 253J-BV cells was extremely downregulated compared
    Molecular Therapy: Methods & Clinical Development
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    with that in HUCs (Figure 1E). To the contrary, the expression levels of K-RAS and H-RAS were upregulated in 253J-BV cells compared with those in HUCs (Figure 1E), which was remarkable in the case of K-RAS.
    Synthetic miR-143s Showed a Potent Growth-Suppressive Effect on BC Cells
    The structures of synthetic miR-143s (Syn-miR-143s) used in this study are shown in Figure 2A. Among them, miR-143#12 that was chemically modified with fluorine, methoxy group, phosphorylation, de-oxythymidine, and phosphorothioate in the guide strand of miR-143#1 was markedly stable in RNase-rich 10% fetal calf serum solu-tion20 (Figure S2A). First, we examined the growth-suppressive effect of Syn-miR-143s on 253J-BV cells to determine their anti-prolifera-tive activity. Among them, miR-143#12 was the most effective; the half-maximum inhibitory concentration (IC50) values of miR-143Am (as a standard miR-143), miR-143#1, and miR-143#12 were >40, 16.1, and 7.9 nM, respectively (Figure 2B). The protein expression levels of total RAS (T-RAS) and RAS-related Erk and Akt were downregulated in the cells transfected with either miR-143Am or Syn-miR-143s at the concentrations of IC50 values (Figure 2C). Also, we found that the Syn-miR-143s decreased the mRNA and protein expression levels of the target genes in a dose-dependent manner (Figure 2D). These results revealed that Syn-miR-143s worked like miR-143 and that their growth-inhibitory effects were more potent than that effect of miR-143Am. Therefore, we focused on the Syn-miR-143s in subsequent experiments.
    Previously, we reported that the ectopic expression of Syn-miR-143s induced apoptosis.17 To confirm whether apoptosis was induced in miR-143-transfected BC cells, we performed Hoechst 33342 staining of the transfected 253J-BV cells. Morphologically, the apoptotic cells estimated by the characteristic findings of apoptosis, such as nuclear fragmentation and chromatin condensation, were frequently observed, especially in miR-143#1- and #12-transfected cells, as compared with their number among the control cells (Figure 2E). Also, the levels of cleaved PARP were increased in typical miR-143#12-treated cells (Figure 2F). Next, we validated the target genes of Syn-miR-143s by using antagomiR-143. Treatment with the antagomiR-143 reversed the growth suppression of BC cells and the decrease in the levels of T-RAS, K-RAS, H-RAS, and RAS-related Erk and Akt elicited by the transfection with Syn-miR-143s (Fig-ure 2G), which reversal was significant in the case of miR-143#12/ antagomiR-143 (Figure 2H). The treatment with antagomiR-143 alone did not affect the cell growth or the expression profiles exam-ined (data not shown). These results altogether showed that Syn- r> miR-143s suppressed the proliferation of 253J-BV BC cells through the decreased expression of RAS isoform proteins (K-RAS and H-RAS), Erk, and Akt by RNAi.
    To confirm how Syn-miR-143s affected the expression of RAS iso-form genes in the cells, we examined the expression levels of each RAS isoform mRNA after the transfection. Importantly, the levels of RAS isoform mRNAs, such as those of K-RAS, H-RAS, and N-RAS, were markedly downregulated by the transfection with Syn-miR-143s (Figure 3A). Guanosine triphosphate (GTP)-RAS ac-tivates the protein kinase Raf, which then activates MEK (MEK1 and MEK2), a mitogen-activated protein kinase (MAPK)/extracel-lular signal-regulated kinase (ERK) Ras-Raf-MEK-ERK in BC
    cells,25 and GTP-RAS also activates the PI3K/AKT pathway, which was also proven to play a major role in bladder carcinogenesis.26,27