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. 2019 Dec 4;11:10229–10238. doi: 10.2147/CMAR.S221150

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© 2019 Sun et al.

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miR-194 inhibits cell proliferation, blocks the G1/S transition, and promotes apoptosis in colorectal cancer (CRC) stem cells by directly targeting the slingshot 2 (SSH2) gene. (A) The 3′-UTR of SSH2 contains three binding sites for miR-194. (B) NC represents CRC stem cells transfected with miR-194-NC; miR-194 represents CRC stem cells transfected with miR-194. The SSH2 mRNA expression levels, determined via quantitative RT-PCR, were reduced in CRC stem cells transfected with miR-194 compared with those in CRC stem cells transfected with miR-194-NC (*P<0.05). (C) The SSH2 protein expression level, determined via Western blot, was reduced in CRC stem cells transfected with miR-194 compared with that in CRC stem cells transfected with miRNA-194-NC. (D) Schematic presentation of luciferase reporter vector, this containing either the wild-type or mutant 3′-UTR of SSH2 were co-transfected into HEK 293T cells with miR-194-NC or miR-194. (E) Compared with co-transfection with miR-194-NC, co-transfection with miR-194 led to a significant decrease in the luciferase activity of the pGL3-SSH2-3′ UTR-Wt reporter (P<0.05 according to the two-tailed t test), but not in that of the pGL3-SSH2-3′-UTR-Mut reporter. (F) Colony formation assay demonstrating that CRC stem cells transfected with miR-194-NC formed a substantially higher number of colonies than CRC stem cells transfected with miR-194(P<0.05 according to the two-tailed t test). (G) MTT assay showing that CRC stem cell proliferation was significantly decreased in miR-194 transfected cells compared with that in cells transfected with miRNA-194-NC (P<0.05 according to ANOVA). (H) miR-194 blocks the G1/S transition in CRC stem cells. Cells were transfected with miR-194 or miR-194-NC and subjected to flow cytometry for cell-cycle analysis. The results showed that, compared with CRC stem cells transfected with miRNA-194-NC, the proportion of miRNA-194-transfected CRC stem cells at the G1 phase was increased, whereas the proportion at the S phase was markedly reduced(P<0.05 according to the two-tailed t test). (I) The effect of miR-194 and miR-194-NC on CRC stem cell apoptosis was investigated by Annexin V–FITC/PI staining. A greater percentage of apoptotic cells were observed among miR-194-transfected cells than among those transfected with miRNA-194-NC(P<0.05 according to the two-tailed t-test).