br To examine the activation of RAS related signaling pathwa
To examine the activation of RAS-related-signaling pathways PI3K/ AKT and MAPK/Erk, we conducted signaling inhibitor experiments in which 253J-BV cells were incubated with an AKT inhibitor or MEK inhibitor to see their effect on the proliferation of the cells. As a result, cell proliferation was significantly suppressed by either inhibitor (Figure 3B). The levels of T-RAS, K-RAS, and H-RAS pro-teins and the activation of PI3K/AKT- and MAPK/Erk-signaling pathways were also downregulated in the case of the AKT or MEK inhibitor (Figure 3C). Interestingly, the expression of each RAS mRNA was markedly downregulated in either AKT or MEK inhibitor-treated cells (Figure 3D), as in the case of the transfection with Syn-miR-143s (Figure 3A). These results altogether showed that the effector-signaling pathways of PI3K/AKT and Raf/MEK/ Erk positively regulated the expression of RAS isoform genes, which indicated the establishment of a positive circuit for RAS expression (Figure 3E).
Next, we examined the contribution of RAS isoforms to the prolifer-ation of 253J-BV cells by using small interfering RNAs (siRNAs) for K-RAS (siR-KRAS) and H-RAS (siR-HRAS). When 253J-BV cells were transfected with either siRNA, the ratio of cell viability was significantly decreased (Figures 4A and 4C), and the levels of RAS and RAS-related proteins were downregulated (Figures 4B and 4D). However, H-RAS protein levels were decreased even in the siR-KRAS-transfected cells (Figure 4B), whereas K-RAS protein levels were not decreased in the siR–HRAS-transfected ones (Figure 4D). Thus, both K-RAS and H-RAS would positively contribute to 253J-BV cell proliferation, but they independently affected it. Importantly, silencing of K-RAS significantly suppressed its effector-signaling mol-ecules Akt and Erk and c-myc expression compared with the case of siR-HRAS.
Figure 1. Downregulated miR-143 in Clinical Bladder Cancer Samples and the Relationship between Expression of miR-143 and that of K-RAS and H-RAS in Bladder Cancer Cell Line 253J-BV
(A) Relative expression levels of miR-143 in 20 clinical GSK1838705A cancer samples. (B) Total RAS, K-RAS, and H-RAS expressions in 20 clinical bladder cancer samples as determined by western blot analysis. Densitometric values of total RAS, K-RAS, and H-RAS with respect to GAPDH of each sample were calculated, and the values are shown as each normal sample was 1.0. (C) Correlation between miR-143 and K-RAS, miR-143 and H-RAS, and K-RAS and H-RAS in clinical bladder cancer samples. (D) The mRNA expression levels of each RAS isomer in 253J-BV cells. (E) Expression levels of miR-143, K-RAS, and H-RAS in 253J-BV and normal bladder HUCs. *p < 0.05, **p < 0.01, ***p < 0.001.
Molecular Therapy: Methods & Clinical Development
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Syn-miR-143s Silences SOS1 in BC Cells
SOS1 is a guanine nucleotide exchange factor (GEF) that facilitates RAS activation by catalyzing the release of guanosine diphosphate (GDP) from RAS.28,29 Previous reports showed a positive correlation between SOS1 high expression and several cancers, such as ovarian cancer30 and hepatocellular carcinoma.31 According to in silico pre-diction tool TargetScan, SOS1 has a miR-143-binding site in the 30 UTR. Expectedly, the ectopic expression of Syn-miR-143s reduced the expression levels of SOS1; and, further, co-transfection with antagomiR-143 partially canceled the decrease elicited by each Syn-miR-143 (Figure 4E). Even when knocking down SOS1, mRNA expression levels of K-RAS, H-RAS, and N-RAS were downregulated in 253J-BV cells (Figures S3A and S3B). Also, each RAS and RAS-related proteins were decreased in these cells (Figure S3C). These re-sults are similar to miR-143 administration (shown as in Figure 2D). When 253J-BV cells were transfected with siR-SOS1, the ratio of cell viability was significantly inhibited (Figure S3D). Western blot anal-ysis of the clinical samples showed that, in 60% of cases tested, SOS1 expression levels were upregulated in the BC tumors compared with the normal tissue samples (Figure S3E). Also, the ratio of GTP-K-RAS:Total K-RAS was significantly decreased in a dose-dependent manner by the transfection with miR-143#12 (5 and 10 nM) (Fig-ure 4F). Therefore, the 30 UTR of SOS1 mRNA containing the miR-–143-binding site was cloned into the downstream of the firefly luciferase gene in a reporter plasmid for use in the luciferase reporter assay. The activity of wild-type pMIR-SOS1 was significantly reduced after the introduction of miR-143#12 into 253J-BV cells. In the case of the mutated SOS1 30 UTR-binding site, the decrease in the luciferase activity in the wild-type was considerably abolished (Figure 4G).
Effect of miR-143/PIC Nanocarrier on Bladder Tumor Growth in Xenografted Mouse Model
Polyionic copolymer (PIC) was prepared by mixing miR-143s (aniomer) with poly (ethylene glycol)-b-poly (ornithine) (block catiomer) (Figure S4A)32. To evaluate the effect of Syn-miR-143s delivered by the PIC nanocarrier (Syn-miR-143s/PIC) on bladder tu-mor growth in a xenografted mouse model, we transplanted 253J-BV cells into the back of each mouse. We injected Syn-miR-143s/Lipo or PIC intravenously into a mouse 4 times every 72 h (Figure 5A). A sig-nificant suppression of tumor growth was observed in both groups at a low dose of total, 210 mg/kg. miR-143#1 seemed to be more potent in anti-tumor activity than did miR-143#12. The PIC group was more effective at tumor suppression than the lipofectamine group in the case of miR-143#12 (Figure 5A). No body weight loss was observed in any of the groups (Figure 5A).