Fig 1: SNHG1 knockdown reduced FRAT1 expression by targeting miR‐361‐4p. (a and b) FRAT1 mRNA expression was detected by qRT‐PCR assay in H23 and H1299 cells transfected with si‐NC, si‐SNHG1, si‐SNHG1 + anti‐miR‐NC or si‐SNHG1 + anti‐miR‐361‐3p. (c and d) FRAT1 protein expression was measured by western blot assay in transfected H23 and H1299 cells. *P < 0.05.
Fig 2: FRAT1 was a target of miR‐361‐3p. (a) The putative binding sequences between miR‐361‐3p and FRAT1 and the mutant sequences of FRAT1 were exhibited. (b and c) Dual‐luciferase reporter assay was conducted to detect the luciferase activities of H23 and H1299 cells transfected with miR‐NC or miR‐361‐3p and FRAT1 3′‐UTR‐WT or FRAT1 3′‐UTR‐MUT. (d) The correlation between miR‐361‐3p expression and FRAT1 expression was determined by Pearson analysis. (e and f) The expression levels of miR‐361‐3p and FRAT1 were measured by qRT‐PCR assay in H23 and H1299 cells transfected with miR‐NC mimics or miR‐361‐3p mimics. (g) FRAT1 protein expression was examined by western blot assay in transfected H23 and H1299 cells. *P < 0.05.
Fig 3: FRAT1 overexpression in NIH3T3 cells induces tumor formation in nude mice. (A) Western blot analysis of HA-FRAT1 expression in NIH3T3 parental, NIH3T3/control or NIH3T3/FRAT1 cells. B-actin was used as a loading control. (B) An image showing tumor growth in nude mice at 8 weeks following injection with NIH3T3/FRAT1 (right lateral) or NIH3T3/control cells (left lateral). HA-FRAT1, hemagglutinin-tagged frequently rearranged in advanced T-cell lymphomas-1.
Fig 4: SNHG1 and FRAT1 expression were upregulated in NSCLC tissues and cells. (a) SNHG1 expression was detected by qRT‐PCR assay in NSCLC tissues (n = 40) and normal tissues (n = 40). (b) SNHG1 expression was measured by qRT‐PCR assay in BEAS‐2B, H23 and H1299 cells. (c) FRAT1 mRNA expression was examined by qRT‐PCR assay in NSCLC tissues and normal tissues. (d) FRAT1 protein level was detected by western blot assay in NSCLC tissues (n = 40) and normal tissues (n = 40). (e) FRAT1 expression was measured by qRT‐PCR assay in BEAS‐2B, H23 and H1299 cells. (f) FRAT1 protein expression was examined by western blot assay in BEAS‐2B, H23 and H1299 cells. *P < 0.05.
Fig 5: Effects of FRAT1 on β-catenin/TCF activity. TOPFLASH β-catenin/TCF complex binding site reporter assay analysis and western blot analysis of β-catenin, p-β-catenin (Ser33/37/Thr41) and HA-FRAT1/FRAT1 expression in (A) DU-145 cells following transfection with HA-FRAT1 or control expression vectors, and (B) PC-3 cells following transfection with pSilencer-FRAT1 or pSilencer-control shRNA. B-tubulin was used as a loading control for western blot analysis. To normalize transfection efficiency, pRL-TK Renilla luciferase reporter constructs were cotransfected in each sample. Luciferase reporter activity in cells transfected with control vectors was set as 1.0. Experiments were performed in triplicate and data are presented as the mean ± standard deviation. TCF, T-cell factor; p-β-catenin, phosphorylated β-catenin; HA-FRAT1, hemagglutinin-tagged frequently rearranged in advanced T-cell lymphomas-1; shRNA, short hairpin RNA. ***P<0.001 vs. control.
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