Fig 2: miR-141-3p inhibits NF-?B signaling pathway. a and b NF-?B transcriptional activity was assessed by luciferase reporter constructs in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. c and d Western blotting of nuclear NF-?B/p65 expression in the indicated cells. The nuclear protein p84 was used as the nuclear protein marker. e-g Real-time PCR analysis of Vimentin, TWIST1, SNAIL2, MMP13 and IL11 in the indicated cells. Transcript levels were normalized to U6 expression. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05

Fig 3: miR-196b-5p activates STAT3 signaling via targeting multiple negative regulators of STAT3 signaling(A) Real-time PCR analysis of miR-196b-5p expression in the indicated cells. Transcript levels were normalized by U6 expression. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (B) Western blotting of SOCS1, SOCS2, SOCS3, SOCS4 and SOCS5 expression in the indicated cells. a-Tubulin served as the loading control. (C and D) Luciferase assay of cells transfected with pmirGLO-3'UTR reporter of SOCS1 and SOCS3 in miR-196b-5p overexpressing and silencing HCT116 and SW480 cells, respectively. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (E and F) MiRNP IP assay showing the association between miR-196b-5p and SOCS1,SOCS3 transcripts in HCT116 and SW480 cells. Pulldown of IgGantibody served as the negative control. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (G) STAT3 transcriptional activity was assessed by luciferase reporter constructs in the indicated cells. Error bars represent the mean ± SD of three independent experiments. *P < 0.05. (H) Western blotting of nuclear STAT3 expression. The nuclear protein p84 was used as the nuclear protein marker.

Fig 4: miR-210-3p activates NF-?B signaling pathway. a NF-?B transcriptional activity was assessed by luciferase reporter constructs in the indicated cells. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. b Western blotting of nuclear NF-?B/p65 expression. The nuclear protein p84 was used as the nuclear protein marker. c and d Real-time PCR analysis of TWIST1, MMP13 and IL11 in the indicated cells. Transcript levels were normalized to U6 expression. Error bars represent the mean ± S.D. of three independent experiments. *P < 0.05. e NF-?B signaling inhibitors LY2409881 (10 µM) and JSH-23 (10 µM) attenuated the stimulatory effect of miR-210-3p on NF-?B transcriptional activity in the indicated cells respectively. Error bars represent the mean ± s.d. of three independent experiments. *P < 0.05 and **P < 0.01. f NF-?B signaling inhibitors LY2409881 (10 µM) and JSH-23 (10 µM) attenuated the stimulatory effect of miR-210-3p on invasion ability in the indicated cells respectively. Error bars represent the mean ± s.d. of three independent experiments. *P < 0.05. g NF-?B signaling inhibitors LY2409881 (10 µM) and JSH-23 (10 µM) attenuated the stimulatory effect of miR-210-3p on migration ability in the indicated cells respectively. Error bars represent the mean ± s.d. of three independent experiments. *P < 0.05 and **P < 0.01

Fig 5: miR-505-3p inhibits the activation of TGF-ß signaling by targeting SMAD2 and SMAD3 in PCa cells. (A-C) GSEA revealed that miR-505-3p downregulation was positively associated with TGF-ß signaling activity. (D) Transcriptional activity based on a TGF-ß/Smad-responsive luciferase reporter as assessed in the indicated cells. *P<0.05. (E) Western blotting of nuclear pSMAD2/3 expression. The nuclear protein p84 was used as a nuclear protein marker. (F-H) Real-time PCR analysis of downstream target gene expression of TGF-ß signaling, including IL11, PTHRP and CTGF in the indicated cells. *P<0.05. PCa, prostate cancer; GSEA, gene set enrichment analysis.