Research Paper Volume 13, Issue 10 pp 14198—14218

Figure 5. FSCN1 is a direct target of miR-328-5p and enhances the progression of ESCC cells. (A) TCGA, miRDB and miRWalk database analyses show that FSCN1 is a potential miR-328-5p target gene (FGF19, EN1, ADAM8, ELFN2, FSCN1, CLDN6, IFIT1). (B) The predicted miR-328-5p seed region in the wild-type (WT) and mutated (Mut) 3′UTR of FSCN1 is shown in the top panel. Dual luciferase reporter assay results (bottom panel) show relative luciferase activity of Kyse30 cells co-transfected with luciferase reporter plasmids containing wild-type or mutated 3′UTR of FSCN1 (WT or Mut) and miR-328-5p mimics, miR-NC act as control. (C) Dual luciferase reporter assay results show luciferase activity in control or LOC146880-silenced Kyse30 cells transfected with luciferase reporter vector with wild-type or mutated 3′UTR of FSCN1 plus miR-NC or miR-328-5p inhibitors. (D) IHC assay shows higher FSCN1 expression level in tumor tissues than in adjacent tissues. (E) ICC assay shows localization of FSCN1 in the cytoplasm and nucleus of ESCC cells. (F–H) QRT-PCR analysis shows expression levels of FSCN1 transcripts in (F) 21 pairs of esophageal cancer and adjacent normal esophageal tissues, (G) ESCC cells transfected with miR-NC, miR-328-5p mimics, and miR-328-5p inhibitors, and (H) control and LOC146880-silenced ESCC cells. (I) Colony formation assay results show the viability of si-NC and si-FCSN1-transfected Kyse30 and TE-1 cells. (J–K) Flow cytometry analysis shows (J) apoptotic rate and (K) cell cycle distribution of si-NC and si-FCSN1-transfected Kyse30 and TE-1 cells. (L) Western blot analysis shows the levels of apoptosis-related proteins (cleaved caspase 3 and Bax) and cell cycle proteins (cyclinD1 and CDK4) in si-NC and si-FCSN1-transfected Kyse30 and TE-1 cells. (M–N) Wound-healing and Transwell invasion assay results show (M) migration and (N) invasiveness of si-NC and si-FCSN1-transfected Kyse30 and TE-1 cells. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.