Fig. 8

Let-7c participated in the regulation of LHX9 and metastasis-related proteins by repressing TGF-beta R1 on transcriptional level. a U2OS cells were transfected with shNC or shFRS2, then they were cultured in the absence or presence of transcription inhibitor (ActD) for 24 h. The relative expression of TGF-beta R1 was assessed by RT-qPCR every 6 h during the culturing time. The TGF-beta R1 mRNAs were normalized to GAPDH mRNA (n = 3). b U2OS cells were transfected with shFRS2, then they were cultured for 0 h, 6 h, 24 h and 48 h, respectively. The expression of FRS2 protein was assessed by Western blot, β-actin was used as the loading control. The normalized protein expression levels against β-actin were shown in the right histograms. c U2OS or Saos-2 cells were co-transfected with let-7c mimic control/let-7c mimic and Wt-TGF-βR1 3′UTR/Mut-TGF-βR1 3′UTR. Dual luciferase assay was performed to measure the luciferase activity. d U2OS or Saos-2 cells were un-transfected or transfected with shFRS2, then they were cultured without or with let-7c mimic or inhibitor for 48 h, then the expression of LHX9 and TGF-βR1 was assessed by Western blot. β-actin was used as the loading control. The normalized protein expression levels against β-actin were shown in the below histograms. e U2OS or Saos-2 cells were transfected with shNC or shFRS2, then they were cultured in the absence or presence of let-7c mimic for 48 h. The expression of LHX9, beta-catenin, TGF-βR1, p-Smad2, Smad2, COL1A1, MMP-9, MMP-1 and FRS2 was measured by Western blot. β-actin was used as the loading control. The normalized protein expression levels against β-actin were shown in the below histograms. a–e The result was a representative of three independent experiments. Error bars represented mean ± SD. p values were determined by two-way analysis of variance (ANOVA) (a) or one-way analysis of variance (ANOVA) followed by Tukey post hoc test (b–e). **p < 0.01, *p < 0.05, ns not significant