Fig 1.
Position of the miR-203 promoter and transcription factor binding sites in the genome.
(A) MiR-203 expression levels in normal human cervical tissues, cervical cancer tissues and cervical cancer cell lines (CaSki and HeLa) were measured by qRT-PCR using RNU6B as an internal control. (B) 5’RACE PCR results show the transcription start sites of human (HeLa cells) pri-miR-203. (C) Schematic of the predicted binding sites of IRF1 in the miR-203 gene promoter. The number indicates the relative distance from the 5’ end of the primary miR-203 transcript (indicated as +1). (D) Alignment results show the conservation of miR-203 promoter sequences between various animals. (E) MiR-203 promoter activity analysis by dual-luciferase assays. The predicted miR-203 promoter was cloned into the pGL3-basic vector upstream of the firefly luciferase gene. Firefly luciferase activity was normalized to Renilla luciferase activity and plotted relative to the control (*P<0.05). The results are representative of three independent experiments.
Fig 2.
IRF1 is involved in the transcriptional regulation of miR-203.
(A) Dual-luciferase assays show the effect of several transcription factors (IRF1, MZF1, AP-1, HSF1, and Sp1) on miR-203 promoter activity. The constructs were co-transfected into HeLa cells with vectors expressing transcription factors and the pRL-TK vector as a transfection control. The wild type pCDNA3.1 plasmid served as a negative control. The relative luciferase activities are shown as the ratio of firefly/Renilla luciferase activity. (B) QRT-PCR assays show the effects of IRF1 overexpression on miR-203 expression. (C) Chromatin immunoprecipitation assays show the in vivo interaction between IRF1 and the miR-203 promoter. HeLa cell chromatin fragments were immunoprecipitated with an antibody for IRF1 or negative control antibody (normal IgG). The DNA samples from immunoprecipitates were analyzed by qRT-PCR using primers specific for the miR-203 promoter. (D) Luciferase assays using the mutant promoter constructs. The results are representative of three independent experiments. *P<0.05.
Fig 3.
MiR-203 suppresses BANF1 expression by targeting the BANF1 3’UTR.
(A) Mature miR-203 sequences and recognition sites within the 3’UTR of BANF1. The seed sequence of miR-203 is underlined. The wild type (WT), mutated (Mut) and deleted (Del) BANF1 3’UTR recognition sites are also shown. (B) The relative luciferase activity of constructs containing the BANF1 WT, Mut or Del 3’UTRs. Luciferase constructs were co-transfected with miRNA mimic negative control (NC, 30 nM) or miR-203 mimic (30 nM) into HeLa cells. Renilla luciferase activity was measured 36 h after incubation and normalized to firefly luciferase activity. An asterisk indicates significant downregulation of pre-miR-203 compared with the expression of the WT BANF1 3’UTR construct. Data are representative of three independent experiments. (C) QRT-PCR analysis of BANF1 mRNA from CaSki and HeLa cells transfected with miRNA mimic NC (30 nM) or miR-203 mimic (30 nM). Data were normalized to the level of GAPDH mRNA, and the ratio of BANF1/GAPDH in the negative control was set to 1. Data are representative of three independent experiments. (D) Western blot analysis of endogenous BANF1 expression in CaSki and HeLa cells 48 h after transfection with miR-203 mimic or inhibitor. Tubulin served as a loading control. The results are representative of three independent experiments. (E) BANF1 expression levels in human normal cervical tissues, cervical cancer tissues and cervical cancer cell lines (CaSki and HeLa) were measured by qRT-PCR, using GAPDH as an internal control. (F) Western blot analysis of BANF1 levels in 20 paired tumor tissues (T) and adjacent normal cervical tissues (N). Tubulin served as a loading control. The five paired samples that exhibited no differences in BANF1 expression are indicated by boxes. *P<0.05, **P<0.01.
Fig 4.
IRF1 indirectly regulates BANF1 expression.
(A, B) QRT-PCR and western blot analysis of IRF1 mRNA and protein levels in CaSki and HeLa cells transfected with pcDNA-IRF1. IRF1 mRNA and protein levels were significantly increased when transfected with pcDNA-IRF1. (C) QRT-PCR analysis of BANF1 mRNA expression in HeLa cells transiently transfected with control vector or pcDNA-IRF1. The data were normalized to GAPDH expression. (D) Western blot analysis of BANF1 expression in CaSki and HeLa cells transiently transfected with pcDNA-IRF1. The results are representative of three independent experiments. *P<0.05.
Fig 5.
MiR-203 suppresses cervical cancer cell proliferation and colony formation.
(A, B) Cell proliferation assays of CaSki and HeLa cells transfected with miR-203 mimic or negative control. (C, D) Colony formation assays of CaSki and HeLa cells transfected with miR-203 mimic or negative control. All data are representative of three independent experiments. *P<0.05.
Fig 6.
MiR-203 is involved in cell cycle regulation and suppresses cervical cancer cell migration and invasion.
(A) Flow cytometry analysis showing the effect of miR-203 overexpression on the cell cycle of CaSki and HeLa cells. Cells were transfected with 30 nM miR-203 mimic or negative control (NC) and collected, stained and analyzed 48 h after transfection. (B, C) Transwell migration assays of CaSki and HeLa cells transfected with miR-203 mimic or NC. The migrated cells were quantified and representative images are shown at the bottom. (D, E) Transwell invasion assays of CaSki and HeLa cells transfected with miR-203 mimic or NC. The invaded cells were quantified and representative images are shown. The results are representative of three independent experiments. *P<0.05.
Fig 7.
BANF1 knockdown suppresses cell colony formation, migration and invasion.
(A, B) QRT-PCR and western blot analysis of BANF1 levels in BANF1-knockdown CaSki and HeLa cells. BANF1 mRNA and protein levels were significantly reduced when transfected with siRNA-BANF1. (C, D) Colony formation assays revealed that the average colony formation number was reduced in BANF1-knockdown CaSki and HeLa cells. (E, F) Cell migration assays revealing that the migrated cells were decreased by knocking down BANF1. (G, H) Cell invasion assays revealing that the invaded cells were decreased by knocking down BANF1. All data are representative of three independent experiments. *P<0.05.
Fig 8.
Model for the potential roles of IRF1 and miR-203 in diverse biological processes.