Materials and methods Patients and tissue samples A total of 100 patients undergoing LT for HCC and the follow-up data about the patients in this study were obtained from Liver Transplantation Surgery, Shanghai First People’s Hospital, Shanghai, China, from 2002 to 2007. All the patients were followed until December 2010. The median recurrence-free period was 12 months for patients with HCC recurrence and 64 months for patients without HCC recurrence. All of these 100 patients fulfilled the Up-To-Seven transplantation criteria Alpelisib datasheet for HCC [14] and none of them had macro-vascular invasion. HCC samples were from the FFPE tissue blocks and the normal liver tissues were from the

liver hemangioma resection. The clinicopathological features of patients were summarized in Table 1. Pre-LT serum AFP level stratification was according to the previous study [15]. All patients provided informed consent according to the protocols approved by the Institutional Review Boards of Shanghai First People’s Hospital. Table 1 Clinical characteristics of the 100 HCC patients according to high- or low miR-20a expression level Parameter N Patients with low miR-20a expression Patients with high miR-20a expression P-value Age 100 57.820 ± 7.330 53.64 ± 8.341 0.212† Sex           Male 84 44 40 0.585‡   Female 16 6 10   Underlying

liver disease           HBV 95 47 48 1.000§   others 5 3 2   Gemcitabine research buy Cirrhosis           Yes 95 47 48 1.000 §   No 5 3 2   Tumor stage           I + II 66 32 34 0.673‡   III 34 18 16   Histologic grade           Differentiated 88 41 47 0.065§   Undifferentiated 12 9 3   Milan criteria           In 55 24 31 0.159‡   Out 45 26 19   Tumor size (cm)           ≤5 Tolmetin 60 24 36 0.014‡   >5 40 26 14   Multinodular           Yes 43 25 18 0.034 ‡   No 57 25 32   Micro-vascular invasion           Yes 22 16 6 0.016 ‡   No 78 34 44   pre-LT serum AFP level           ≤400

(ng/ml) 63 30 33 0.534 ‡   >400 (ng/ml) 37 20 17     Overall survival 42/100 11/50 31/50 –   HCC recurrence 58/100 37/50 21/50 – NOTE: AFP, alpha-fetoprotein. †Unpaired student t test; ‡chi-square test; §Fisher’s exact test. Cell culture and transfection All the cell lines used in this study were KU55933 purchased from the cell bank of the Chinese Academy of Sciences and grown in DMEM (GIBCO, Grand Island, NY), supplemented with 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO), 2 mM glutamine, 100 U of penicillin/ml and 100 μg of streptomycin/ml (Cambrex, Verviers, Belgium). All cells were incubated at 37°C in a humidified chamber supplemented with 5% CO2. Control negative oligonucleotide, and double-stranded RNAs that mimic endogenous precursor miR-20a were purchased from Ambion (Ambion, Austin, TX) were transfected into cells using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s instruction.