Fig. 2.

LIN28A is overexpressed in human liver cancer. (A) Quantitative RT-PCR analysis of LIN28A mRNA levels in primary human hepatocytes (PHH) (n = 10) as compared with human HCC cell lines (PLC (n = 9), Hep3B (n = 10), HepG2 (n = 11)). (B,C) Quantitative RT-PCR analysis (A) and representative Western blot images depicting LIN28A mRNA and recombinant protein expression, respectively, after transfection of HepG2 cells (low endogenous LIN28A expression as compared with PLC and Hep33B) applying a LIN28A-overexpression (OE) plasmid vector (provided by G. Meister and described in detail elsewhere [29] (n = 4). (D) Immunofluorescence analysis of LIN28A expression and localization in two different HCC cell lines (PLC (n = 2), Hep3B (n = 2)) (representative images, 40-fold magnification). (E) LIN28A mRNA levels (qRT-PCR analysis) of paired human HCC and corresponding nontumorous liver tissue (CNTLT) samples (n = 10pairs). (F-I) Immunohistochemical analysis of LIN28A protein expression and cellular localization patterns in patients applying tissue micro array derived paired HCC tissues and CNTLT. Staining intensity and percentage of positive cells were incorporated into a semiquantitative score describing "0" (very low/no expression), "1" (low/moderate expression) and "2" (strong expression). (F) depicts representative immunohistochemical images. (G) LIN28A protein expression score in human HCC tissues and CNTLT applying a tissue micro array (paired analyses). (H) LIN28A expression in HCC (n = 103) as compared with CNTLT (n = 115) (semiquantitative analysis applying Fisher's exact test). (I) Spearman correlation analysis of LIN28A expression levels in HCC patients with early (stage I & II) (n = 71) compared with late (stage III and IV) tumor stages (UICC 2010). Data are presented as the mean ± SEM. Statistical significance was determined by 2-tailed, unpaired t-test (A,B), paired t-test (E,G), by two-sided Fisher's exact test (H), and Spearman correlation (I). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.