Fig 2: Low expression of proliferation and angiogenic proteins in tumours originating from miR-485-5p@SPIONs-HuGSCs. The proliferation, neurotrophic factors, and angiogenesis of glioma cells is directly proportional to Tie1 expression level and the number of cancer stem cells. When the number of cancer stem cells in tumour tissues is decreased and Tie1 expression is reduced, cell division and angiogenesis rates will decrease. **p<0.01 vs. miR-mut@SPIONs-HuGSCs group. t test. n=3. Scale bar = 30μm.

Fig 3: miR-485-5p negatively regulates Tie1 expression in human glioma stem cells (HuGSCs). (A) Flow cytometry results showed that the proportion of CD44+/CD133+ HuGSCs is significantly lower than that of CD44-/CD133- HuGs. ** p<0.01 vs. CD44-/CD133- HuGs group. t test. n=6. (B) qPCR results showed that Tie1 is significantly expressed in CD44+/CD133+ HuGSCs. **p<0.01 vs. CD44-/CD133- HuGs group. t test. n=6. (C) Western blot results showed that Tie1 is significantly expressed in CD44+/CD133+ HuGSCs. **p<0.01 vs. CD44-/CD133- HuGs group. t test. n=6. (D) Bioinformatics software predicted that 9 microRNAs target and regulate Tie1. (E) qPCR results showed that 4 microRNAs are significantly expressed in CD44+/CD133+ HuGSCs. **p<0.01 vs. CD44-/CD133- HuGs group. t test. n=3. (F) Northern blot results showed that the miR-485-5p hybridisation signal in CD44+/CD133+ HuGSCs is significantly weakened. (G) Bioinformatics software predicted that miR-485-5p is complementary to the 3′-UTR of Tie1 mRNA. (H) Reported luciferase assay results showed that miR-485-5p can significantly inhibit the activity of luciferase containing specific loci of the Tie1 3′-UTR region. *p<0.05 vs. miR-mut group. t test. n=3.

Fig 4: miR-485-5p@SPIONs downregulate Tie1 expression in human glioma stem cells HuGSCs. (A) SPION particles under the transmission electron microscope. (B)Particle size analysis found that the size of the inner core of SPIONs ranged from 2-13 nm. (C) Agarose gel electrophoresis showed that miR-485-5p can bind to SPIONs. (D) qPCR results showed that Tie1 expression is significantly lower in miR-485-5p@SPIONs-HuGSCs than in miR-mut@SPIONs-HuGSCs. **p<0.01 vs. miR-mut@SPIONs-HuGSCs group. t test. n=3. (E) Western blot results showed that Tie1 expression level is significantly lower in miR-485-5p@SPIONs-HuGSCs than in miR-mut@SPIONs-HuGSCs. **p<0.01 vs. miR-mut@SPIONs-HuGSCs group. t test. n=3. (F) Transmission electron microscopy results showed that a large number of highly dense electron cloud substances (SPIONs) aggregated in the microvesicles in the cytoplasm of HuGSCs.

Fig 5: Tie1 activates FGF2 to promote the protein expression of GDNF/GFAP/BDNF. (A) Protein-protein interaction prediction software showed that FGF2 protein is downstream of Tie1 protein and FGF2 directly interacts with GDNF/GFAP/BDNF proteins. (B) qPCR results showed that the expression levels of stem cell markers and FGF2, GDNF, GFAP, and BDNF were significantly lower in miR-485-5p@SPIONs-HuGSCs. **p<0.01 vs. miR-mut@SPIONs-HuGSCs group. t test. n=3. (C) Western blot results showed that the protein expression levels of stem cell markers and FGF2, GDNF, GFAP, and BDNF were significantly lower in miR-485-5p@SPIONs-HuGSCs than in miR-mut@SPIONs-HuGSCs. **p<0.01 vs. miR-mut@SPIONs-HuGSCs group. t test. n=3.