Fig 2: Prediction and confirmation of miR-193a-3p/KRAS's binding. A, Predicted binding sites in KRAS 3'-UTR and miR-193a-3p, with free energy values of each hybrid indicated. B, Recognition of the KRAS 3'-UTR by miR-193a-3p. Firefly luciferase reporters containing either wild-type (WT) or mutant (MUT) miR-193a-3p binding sites in the KRAS 3'-UTR were co-transfected into 293T cells with equal doses of the miR-193a-3p mimics, inhibitors and negative control. Forty-eight hours post-transfection, the cells were assayed using a luciferase assay kit. C,D, Western blotting and quantitative analysis (KRAS protein vs GAPDH protein) of the expression levels of the KRAS protein in aforementioned 12 pairs of CRC and NAT samples. E,F, H&E-stained sections and immunohistochemical staining for KRAS in the one pair of aforementioned CRC samples followed by quantitative analysis for KRAS-positive cells. G, Pearson's correlation scatter plot of the fold change of miR-193a-3p and KRAS in the aforementioned specimens. The results are presented as the mean ± SD of three independent experiments. (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig 3: Effect of miR-193a-3p and KRAS plasmid on the development of CRC cell xenografts in nude mice. A, SW480 cells co-infected with negative control, KRAS plasmid, miR-193a-3p mimic lentivirus, KRAS plasmid plus miR-193a-3p mimic lentivirus were transplanted subcutaneously into nude mice and harvested. B, Tumor size of subcutaneous implantation models of SW480 cells. C, Quantitative comparison of tumor weight between the four groups. D, Quantitative RT-PCR analysis of the expression levels (miR-193a-3p vs U6) of miR-193a-3p in the tumors from the four groups. E, Quantitative RT-PCR analysis of the expression levels (KRAS mRNA vs GAPDH mRNA) of KRAS in the tumors from the four groups. F,G, Western blotting and quantitative analysis of KRAS protein levels in the tumors from the four groups. H, H&E-stained sections and immunohistochemical staining for KRAS in the tumors from the four groups. I, Quantitative comparison of KRAS-positive cells between the four groups. The results are presented as the mean ± SD of three independent experiments. (*P < 0.05; **P < 0.01; ***P < 0.001).

Fig 4: (A) Histograms of cell cycle distribution after the treatment with control, siR-93C@Lipo2000, siR-93C@PAMAM, and HA-SeSe-COOH/siR-93C@PAMAM. (B) Relative distribution of cell population in the cell cycle phase. (C) Cell apoptosis and (D) Cell apoptosis rate of A549 cells after the treatment with control, siR-93C@Lipo2000, siR-93C@PAMAM, and HA-SeSe-COOH/siR-93C@PAMAM. (E) Western blotting analysis. (F) Semi-quantitative western blot analysis for the expression level of KRAS.

Fig 5: Glutamine and leucine uptake in KRAS mutant CRC cells associated with the expression of selected AATs: (A, B) l-glutamine and l-leucine uptake levels in KRAS mutant or wt CRC cell lines at 20 min, determined by radiolabeled amino acid transport assay. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical comparisons were computed by one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Heat map shows mRNA expression levels (fold change) of SLC-amino acid transporter genes in different CRC cell lines from three independent biological replicates (n = 3). (D) Western blot images show the protein expression of AATs (SLC38A2, SLC7A5, and SLC1A5), total-MEK1/2, phospho-MEK1/2, total ribosomal S6, and phospho-ribosomal S6 protein in different CRC cell lines. ß-actin was used as protein loading control. All the experiments were performed three times independently, and the blots shown are representative of three independent replicates.