Research Paper Volume 13, Issue 8 pp 12207—12223

Anti-oncogenic effects of SOX2 silencing on hepatocellular carcinoma achieved by upregulating miR-222-5p-dependent CYLD via the long noncoding RNA CCAT1

miR-222-5p binds to and downregulates CYLD in HepG2 cells. (A) Binding relationship determined by interrogation of online bioinformatics website. (B) Binding relationship determined by dual luciferase reporter gene assay. (C) miR-222-5p expression and CYLD mRNA expression in normal liver (n = 28), para-cancerous (n = 68) and HCC tissues (n = 68) detected by RT-qPCR normalized to U6 and β-actin. (D) miR-222-5p mRNA expression in HCC cell lines detected by RT-qPCR normalized to U6. (E) CYLD mRNA expression in HCC cell lines detected by RT-qPCR normalized to β-actin. (F) Representative micrographs showing CYLD expression in normal liver (n = 28), para-cancerous (n = 68) and HCC tissues (n = 68) determined by immunohistochemistry (400 ×). (G) CYLD positive staining in normal liver (n = 28), para-cancerous (n = 68) and HCC tissues (n = 68). (H) miR-222-5p expression in HepG2 cells after alteration of miR-222-5p detected by RT-qPCR normalized to U6. (I) CYLD protein expression in HepG2 cells after alteration of miR-222-5p determined by western blot analysis normalized to GAPDH. (J) CYLD mRNA expression in HepG2 cells after alteration of miR-222-5p determined by RT-qPCR normalized to β-actin. * p vs. normal liver cell, LO2 or HepG2 treated with mimic-NC; # p vs. HepG2 treated with inhibitor-NC. Data are expressed as mean ± standard deviation. Data between two groups were compared by independent sample t-test, and data from multiple groups were compared by one-way ANOVA and Tukey’s post hoc testing.

Figure 4. miR-222-5p binds to and downregulates CYLD in HepG2 cells. (A) Binding relationship determined by interrogation of online bioinformatics website. (B) Binding relationship determined by dual luciferase reporter gene assay. (C) miR-222-5p expression and CYLD mRNA expression in normal liver (n = 28), para-cancerous (n = 68) and HCC tissues (n = 68) detected by RT-qPCR normalized to U6 and β-actin. (D) miR-222-5p mRNA expression in HCC cell lines detected by RT-qPCR normalized to U6. (E) CYLD mRNA expression in HCC cell lines detected by RT-qPCR normalized to β-actin. (F) Representative micrographs showing CYLD expression in normal liver (n = 28), para-cancerous (n = 68) and HCC tissues (n = 68) determined by immunohistochemistry (400 ×). (G) CYLD positive staining in normal liver (n = 28), para-cancerous (n = 68) and HCC tissues (n = 68). (H) miR-222-5p expression in HepG2 cells after alteration of miR-222-5p detected by RT-qPCR normalized to U6. (I) CYLD protein expression in HepG2 cells after alteration of miR-222-5p determined by western blot analysis normalized to GAPDH. (J) CYLD mRNA expression in HepG2 cells after alteration of miR-222-5p determined by RT-qPCR normalized to β-actin. * p < 0.05 vs. normal liver cell, LO2 or HepG2 treated with mimic-NC; # p < 0.05 vs. HepG2 treated with inhibitor-NC. Data are expressed as mean ± standard deviation. Data between two groups were compared by independent sample t-test, and data from multiple groups were compared by one-way ANOVA and Tukey’s post hoc testing.