Fig. 6.

ZCCHC11 is regulated by miR-622 in HCC. (A) Immunohistochemical analysis and correlation of ZCCHC11 and LIN28A expression levels applying tissue micro arrays (staining intensity and percentage of positive cells for both ZCCHC11 and LIN28A were incorporated into a semi-quantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression)) (n = 102). (B) MicroRNA recognition elements (MRE) in the ZCCHC11 3’UTR represent potential miR-622-binding sites and were identified applying the "TargetScan 7.2" database. (C) Quantitative RT-PCR analysis of ZCCHC11 mRNA levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC (n = 2); Hep3B (n = 3)). (D) Representative Western blot images and (densitometric) quantification of ZCCHC11 protein levels in control-transfected as compared to miR-622-transfected different HCC cell lines (PLC (n = 2); Hep3B (n = 2)). (E) Luciferase ZCCHC11 3′UTR-reporter (containing a conserved miR-622 MRE) activity in control-miR (CTR) as compared with miR-622-mimic (622)-transfected HCC cells (PLC) (n = 3). (F,G) TCGA-derived datasets and the Gene Expression Profiling Interactive Analysis (GEPIA) database were used for overall survival analysis comparing high and low ZCCHC11/miR-622-ratios (F) or high and low miR-622/ZCCHC11-ratios (G), respectively in HCC patients. "HR": Hazard ratio. Data are presented as the mean ± SEM. Statistical significance was determined by two-sided Fisher's exact test together with Spearman correlation analysis (A), and 2-tailed, unpaired t-test (C,D,E). Survival analysis was performed computationally applying log-rank testing and hazard ratio estimates (F,G). *P < 0.05, **P < 0.01, ****P< 0.0001.