Figure 1.
NFIB was down-regulated in CSCC cell lines and primary tumors.
(A) The expression of miR-365 and NFIB in CSCC cell lines (Tca8113, HSC-1 and A431) compared with normal HaCaT cells was detected by qRT-PCR and/or western blotting. The expression of NFIB is inversely correlated with miR-365 levels. (B) The expression of miR-365 in CSCC primary tumors was detected by microRNA-FISH using normal skin tissue as control. Bars = 50 µm. (C) Correlation between miR-365 expression and NFIB RNA/protein expression in CSCC primary tumors. The expression of NFIB is inversely correlated with miR-365 levels. In this figure, the expression of miR-365 was examined by qRT-PCR and normalized to U6 snRNA expression. The expression of NFIB protein or mRNA in CSCC tumors compared with normal skin tissue was detected by western blot using as a loading control or qRT-PCR normalized to GAPDH expression. The P value (<0.01) shows significant inverse correlation between the levels of miR-365 and NFIB.
Figure 2.
NFIB is targeted by miR-365 in both normal and CSCC cells.
(A) Two WT luciferase reporter plasmids were generated by fusing miR-365 binding sites of the NFIB 3′UTR downstream of the luciferase reporter gene. Two mutant plasmids were generated by mutating the binding sites. The mutated sequences were underlined. WT or mutant reporter constructs were then transfected into HaCaT cells with NC or miR-365 mimics. Dual luciferase assay were performed 48 h post transfection and normalized to Renila luciferase activities. Data represent the average of three independent experiments ± SD. (B) NFIB mRNA (Left panel) and miR-365 (Right panel) expression was measured in NC, miR-365, NC inhibitor or miR-365 inhibitor transfected HaCaT cells respectively by qRT-PCR normalized to GAPDH for NFIB or to U6 for miR-365. Expression folds are shown with respect to NC mimic or NC inhibitor transfected cells where normalized copy number was set to 1. (C) qRT-PCR and western blot showing NFIB mRNA and NFIB protein expressed after hsa-miR-365 inhibitors were transferred into A431 and HSC-1 cells. Representative experiments are shown. Means ± SD, n = 4.
Figure 3.
AntagomiR-365 inhibits tumorgenesis and restores NFIB expression both in vitro and in vivo.
(A) The expression of NFIB, p53, CDK6 and Bcl-2 proteins in CSCC cells transfected with antagomiR NC and antagomiR-365 was detected by western blot using GAPDH as a loading control. A representative result is shown here. (B) A representative picture shows the change in tumor volume in xenograft model of BALB/c-nu mice after antagomir-365 treatment 3 weeks with intratumoural injection. The right back flank of BALB/c-nu mice was injected subcutaneously with A431 cells in vivo with a volume of more than 150 mm3 (n = 5) in comparison with PBS treatment (n = 5). Red arrow head shows the tumor formation from representative mice 21 days after treatment (controls were treated with PBS). (C) Tumor volumes (mm3) were recorded in time points as indicated in the growth curve. Relative tumor volumes are shown with respect to day 7. Data are plotted as mean ± S.E. (D) AntagomiR-365 injection drastically decreased the expression levels of the miR-365 in xenografts and thus led to the up-regulation of NFIB and p53 and down-regulation of CDK6 and Bcl-2. Each bar represents the average expression from 5 individual xenografts. Data are plotted as mean ± S.E. (E) IHC staining of NFIB, p53, CDK6 and Bcl-2 on sections of xenograft tumors. Representative fields are shown here and index of positive signal was calculated (n = 10).
Figure 4.
Knockdown of NFIB by siRNA oligos mimics the pro-carcinogenic effects of downregulation of NFIB by miR-365.
(A) NFIB protein levels were detected in A431 cells after siRNA oligos of NFIB was transferred and incubated for 24 h, 48 h, 72 h. (B) The expression levels of NFIB, p53, CDK6 and Bcl-2 proteins and mRNA in CSCC cells transfected with two distinct siRNA oligos against NFIB were detected by western blot using GAPDH as a loading control or by qRT-PCR normalized to GAPDH expression. (C) The expression miR-365 examined by qRT-PCR and normalized using U6 snRNA after siRNA NFIB_2, siRNA NFIB_3 and siRNA negative control were transfected into A431 and HSC-1 cells and incubated for 72 h. (D) A mechanism model of miR-365 in CSCC shows that the pro-carcinogenic role of miR-365 is functionally performed through targeting NFIB.