Fig 2: LINC01123 positively regulated TUFT1 by targeting miR-34a-5p in HCC cells. (A) The expression of TUFT1 mRNA was negatively correlated with miR-34a-5p level in HCC tissues. (B) miR-34a-5p overexpression clearly decreased the luciferase activity of vector containing 3'UTR of TUFT1. However, MT-miR-34a-5p did not affect the luciferase activity of vector carrying 3'UTR of TUFT1. (C) Rescue experiment of TUFT1 protein expression by cotransfection of corresponding vectors in Hep3B cells. (D) The expression of TUFT1 mRNA was positively correlated with LINC01123 level in HCC tissues. *P<0.05.

Fig 3: BACE1-AS promotes TUFT1 dependent Wnt signaling activation. A and B GSEA plots (upper panel) and heatmap (lower panel) depicting microarray analysis of mRNAs encoding proteins involved in signaling pathways regulating pluripotency of stem cells (A) and Wnt signaling pathway (B) in liver metastatic or primary CRC tissues. LM: liver metastasis; CP: CRC primary. C Confirmation of the top microarray targets WNT3A, WNT7B, CTNNB1, MYC, SOX2 and FOSL1 by RT-qPCR in collected liver metastatic and non-metastatic CRC tissues. *p < 0.05, **p < 0.01. D Protein levels of β-catenin, Myc, SOX2 and FOSL1 in collected liver metastatic and non-metastatic CRC tissues. E Knockout of BACE1-AS inactivated Wnt signaling pathway could be rescued by exogenous expression of BACE1-AS. F TUFT1 depletion decreased GSK3β phosphorylation and the protein levels of β-catenin, SOX2 and c-Myc. G TUFT1 over-expression increased GSK3β phosphorylation and the protein levels of β-catenin, SOX2 and c-Myc. H TUFT1 depletion reversed BACE1-AS over-expression induced Wnt signaling pathway hyper-activation

Fig 4: BACE1-AS enhances TUFT1 expression through sponging miR-214-3p. A Schematic diagram of miR-214-3p potential binding sites on BACE1-AS and TUFT1. B BACE1-AS knockout significantly increased miR-214-3p expression. **p < 0.01. C Over-expression of miR-214-3p suppressed BACE1-AS expression. **p < 0.01, ***p < 0.001. D Relative enrichments of BACE1-AS and miR-214-3p in Ago2 immunoprecipitated pellets were examined by RIP assay using anti-Ago2 antibody followed by qRT-PCR. **p < 0.01, ***p < 0.001. E MiR-214-3p mimics inhibited luciferase activity in wild-type BACE1-AS groups rather than in mutant BACE1-AS groups. **p < 0.01. F MiR-214-3p suppressed TUFT1 expression. Representative blots were shown (upper panel). Actin served as a loading control. Ratios of levels of TUFT1 vs. Actin was calculated using NIH Image J 1.61 (right panel). **p < 0.01. G MiR-214-3p mimics inhibited luciferase activity in wild-type TUFT1 groups rather than in mutant TUFT1 groups. ***p < 0.001. H MiR-214-3p inhibitor suppressed TUFT1 expression in BACE1-AS knockout cells. Representative blots are shown (upper panel). Actin served as a loading control. Ratios of levels of TUFT1 vs. Actin was calculated using NIH Image J 1.61 (right panel). *p < 0.05. I over-expression of miR-214-3p reversed BACE1-AS induced TUFT1 up-regulation. Representative blots were shown (upper panel). Actin served as a loading control. Ratios of levels of TUFT1 vs. Actin was calculated using NIH Image J 1.61 (right panel). *p < 0.05, **p < 0.01. J MiR-214-3p mimics reversed BACE1-AS over-expression-induced luciferase activity increasing in wild-type TUFT1 groups rather than in TUFT1 mutant groups. **p < 0.01

Fig 5: BACE1-AS promotes CRC metastasis through TUFT1. A CRC patients with BACE1-AS/TUFT1 ceRNA network suffered shorter overall survival. B TUFT1 expression was decreased in BACE1-AS knockout cells. Actin served as a loading control. Representative blots from three experiments were shown (left panel). Ratios of the level of TUFT1 vs. Actin were calculated using NIH Image J 1.61 (right panel). **p < 0.01. C Ectopic expression of TUFT1 restored TUFT1 level in BACE1-AS knock-out cells. Actin served as a loading control. Representative blots from three experiments are shown (upper panel). Ratios of the level of TUFT1 vs. Actin were calculated using NIH Image J 1.61 (lower panel). **p < 0.01. D Ectopic expression of TUFT1 restored metastasis abilities in BACE1-AS knockout cells determined by trans-well assay. E Exogenous expression of TUFT1 rescued tumor sphere formation ability in BACE1-AS knockout cells. Representative images are shown (left panel). Bar graphs show the mean numbers (middle panel) and diameters (right panel) of tumorspheres. F Exogenous expression of TUFT1 restored liver metastasis capability in BACE1-AS knockout cell