MicroRNA-28-5p Regulates Liver Cancer Stem Cell Expansion via IGF-1 Pathway

Figure 4

IGF-1 is required for miR-28-5p-mediated liver CSC expansion. (a) The mRNA expression of IGF-1 and IL-34 in the miR-28-5p sponge and control liver CSCs was determined by RT-PCR assay (). (b) The mRNA expression of IGF-1 and IL-34 in the miR-28-5p mimic and control liver CSCs was determined by RT-PCR assay (). (c) The protein expression of IGF-1 in the miR-28-5p sponge and control liver CSCs was checked by western blot assay. GAPDH acted as a loading control. (d) The protein expression of IGF-1 in the miR-28-5p mimic and control liver CSCs was checked by western blot assay. GAPDH acted as a loading control. (e) The miR-28-5p potential binding sites at the 3-UTR of IGF-1 and the nucleotides mutated in the IGF-1-3-UTR mutant. (f) Luciferase reporter assay was performed to detect the effect of the miR-28-5p sponge on the luciferase intensity controlled by 3-UTR of IGF-1 ().(g) The correlation between the transcription level of miR-28-5p and IGF-1 in forty HCC tissues was determined by RT-PCR analysis. Data were normalized to U6 or β-actin as ΔCt and analyzed by Spearman’s correlation analysis. (h) Huh7 and HepG2 cells were transfected with IGF-1 siRNA or negative control and then subjected to western blot assay. GAPDH acted as a loading control. (i) The EpCAM-positive cells in siIGF-1 and control HCC cells were determined by flow cytometry (). (j) The self-renewal ability of siIGF-1 and control HCC cells was compared by spheroid formation assay (). (k) The miR-28-5p sponge and control HCC cells were transfected with siIGF-1 and siNC followed by flow-cytometric assay (). (l) The miR-28-5p sponge and control HCC cells were transfected with siIGF-1 and siNC followed by spheroid formation assay (). (m) The Huh7 miR-28-5p sponge and its control cells were transfected with siIGF-1 and siNC and then subjected to in vivo limiting dilution assay. Tumors were observed over 2 months; for each group. Data are represented as ; ; two-tailed Student’s -test.

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