Fig. 4. miR-708 promotes malignant proliferation of colonic cells via targeting of SOCS3 expression.

a Predicted base-pairing between SOCS3 3′-UTR and miR-708. The mutant type (MUT) of the SOCS3 3′-UTR was generated by site-specific mutation of the paired bases. b The relative luciferase activities in DLD1 and RKO cells that were transfected with miR-708 and SOCS3 WT 3′UTR or SOCS3 MUT 3′UTR. c RT-qPCR analyses were performed to measure SOCS3 mRNA levels in DLD1 (left) and RKO (right) cells constitutively expressing miR-708, or in DLD1 and RKO cells transfected with a miR-708 antagomir. d The statistics of miR-708 levels and SOCS3 protein levels in eight colorectal cancer cell lines. e Western blotting was performed to detect levels of SOCS3 and activated STAT3 in RKO cells with knockdown of miR-708 and in DLD1 cells with forced expression of miR-708. f Western blotting was performed in colonic tumor tissues from Apc^min WT mice and from Apc^min KO mice. g Western blotting was performed in organoids. Organoids derived from Apc^min mice were treated with control agomir (CT) or miR-708 agomir (miR-708) and cultured for 6 days; organoids from Apc^min WT mice (+/+) and Apc^min miR-708 KO mice (−/−) were cultured for 6 days. h Representative images (scale bar: 100 μm) and quantification of the size of acinar sphere after 6 days of 3D-culture. Cells constitutively expressing miR-708 had been transfected with pCMV-SOCS3. i Representative images (scale bar: 100 μm) and quantification of the size of organoids from Apc^min mice. Organoids, derived from Apc^min WT mice and from Apc^min miR-708 KO mice, were infected with Adv-control or Adv-SOCS3 and cultured for 7 days; organoids derived from Apc^min mice were treated with miR-708 agomir or control agomir and Adv-control or Adv-SOCS3 and were cultured for 7 days. The results are presented as the mean ± S.D. in panels (b, c, h, i). Statistical analyses were conducted by non-paired two-tailed Student’s t tests.