Fig 2: Nanoparticles loaded with siRNA mixture targeting AFAP1-AS1 and MLK7-AS1 exhibited inhibitory effects on tumor formation and liver metastasis in vivo(A) Injection schedule of nanoparticles in the subcutaneous xenograft CRC model. (B) Tumor volume was calculated as described in materials and methods at intervals of 3 days for 4 weeks after injection of nanoparticles. (C) Tumor weight was measured after the mice were sacrificed. (D and E) The levels of asRNA (D) (AFAP1-AS1 and MLK7-AS1) and miRNAs (E) (miR-149-5p and miR-485-5p) in the enucleated tumor were examined by qRT-PCR. (F) The mRNA and protein levels of SHMT2 and IGFBP5 were determined by qRT-PCR and western blotting analyses, respectively. (G) Injection schedule of nanoparticles in the intrasplenic liver metastasis CRC model. (H) The degree of liver metastasis was assessed by counting the number of nodules. (I and J) The levels of asRNA (I) (AFAP1-AS1 and MLK7-AS1) and miRNAs (J) (miR-149-5p and miR-485-5p) in the enucleated tumor were examined by qRT-PCR.Data are expressed as mean ± standard deviation. Statistical significance was set at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001).

Fig 3: miR-149-5p and miR-485-5p were required for the downregulation of SHMT2 and IGFBP5 by knockdown of AFAP1-AS1 and MLK7-AS1(A) DLD1 and HT29 cells were transfected with indicated siRNA or miRNA and the levels of asRNA (AFAP1-AS1 and MLK7-AS1), miRNAs (miR-149-5p and miR-485-5p), and common targets (SHMT2 and IGFBP5), were assessed by qRT-PCR. (B and C) DLD1 cells were transfected with siRNA targeting AFAP1-AS1 (B) or MLK7-AS1 (C), and the enrichment of SHMT2 and IGFBP5 mRNA was examined using AGO2 IP. (D) The protein expression of SHMT2 and IGFBP5 in AFAP1-AS1- and MLK7-AS1-silenced DLD1 cells was determined by Western blot analysis. ß-Actin was used as a loading control.Data are expressed as mean ± standard deviation. Statistical significance was set at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001).

Fig 4: SHMT2 and IGFBP5 were common targets of the AFAP1-AS1/MLK7-AS1-miR-149-5p/miR-485-5p regulatory axis(A) Venn diagram and schematic workflow for selecting SHMT2 and IGFBP5 as common target genes. (B) Kaplan-Meier overall survival curves of patients with CRC with low and high level of SHMT2 and/or IGFBP5. More than 2-fold high expression was classified as “High,” and all others were classified as “Others” to analyze the prognosis. (C) RNA sequencing data representing higher expression of SHMT2 and IGFBP5 in CRC compared with that in adjacent normal tissues. (D) The correlation between the expression levels of miRNAs (miR-149-5p and miR-485-5p) and target genes (SHMT2 and/or IGFBP5) was examined by analyzing RNA sequencing data. (E) The enrichment of SHMT2 and IGFBP5 mRNA in AGO2 IP was examined using PEB cell lysates of DLD1 cells transfected with miR-149-5p and/or miR-485-5p mimics. (F and G) To verify the direct interaction of miR-149-5p and miR-485-5p with the 3' UTR of SHMT2 (F) and IGFBP5 (G) mRNA, reporter vectors were constructed, and luciferase activity was examined. Detailed information on MREs and reporter vectors (WT and MT) is shown in Figure S7. (H) After DLD1 cells were transfected with either miR-149-5p or miR-485-5p mimics and both, the protein expression levels of SHMT2 and IGFBP5 were assessed by western blotting analysis.Data are expressed as mean ± standard deviation. Statistical significance was set at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001).