WT1-AS promotes cell apoptosis in hepatocellular carcinoma through down-regulating of WT1

Study data were obtained from 90 patients who presented between September 2012 and October 2013 at The First Affiliated Hospital of Nanjing Medical University (Nanjing, China). Informed consent for tissue analysis was obtained prior to surgery; the study was approved by our Institutional Ethics Committee. All research was performed in compliance with government policies and the Helsinki Declaration. Experiments were undertaken with the understanding and written consent of each subject. The clinical information was analyzed in Table 1.

Quantitative real time polymerase chain reaction (qRT-PCR) was performed to determine the expression levels of WT1-AS of all related genes. Total RNA was obtained from tissues using TRIzol reagent as described by the manufacturer (Invitrogen Life Technologies Co, CA, USA). For mRNA detection, total RNAs (500 ng) were reverse transcribed using the reverse transcription kit (Takara, Tokyo, Japan) at 37 °C for 15 min and 85 °C for 30 s. QRT-PCR was performed using ABI Prism 7900HT (Applied Biosystems, CA, USA). β-actin was used as an internal control. PCR was performed for 5 s at 95 °C and for 30 s at 60 °C for 40 cycles. The detailed sequence information was presented in additional file 1 online.

For Western-blots, total proteins were extracted from tissues or cultured cells using RIPA buffer containing protease inhibitors cOmplete, ULTRA, Mini, EDTA-free, EASYpack (Roche, Basel, Switzerland), while the membrane proteins were extracted from tissues by Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Scientific, Rockford, USA). Protein concentrations were determined by the BCA method. Equal amount of proteins (100ug) were separated with 7.5 %/12.5 % sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane. Membrane was blocked using 5 % skimmed milk and incubated with respective antibodies. Primary polyclonal antibodies were purchased from Santa Cruz Biotechnology (CA, USA) and Abcam (CA, USA) including WT1 (ab96792), p-STAT3 (ab30647), STAT3 (ab68153), p-MAPK (ab63378), MAPK (ab197348), GAPDH (sc-48166), β-actin (sc-47778), Caspase 3 (ab2302), MDR1 (sc-13131). The secondary antibodies were anti-rabbit or anti-mouse HRP-linked were purchased from Santa Cruz Biotechnology (CA, USA). The blots were developed using ECL reagent (Millpore, MASS, USA). Equal amount of protein loading in each lane was confirmed using GAPDH antibody. All experiments were repeated at least three times.

Human HCC cell lines were obtained from American Type Culture Collection (ATCC, Manassas VA, USA), which were cultured in complete growth medium DMEM (Hyclone, UT, USA), supplemented with 10 % fetal bovine serum (10 % FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C, 5 % CO₂ Cells were cultured to about 50 % confluence and transfection was carried out using Lipofectamine 2000 (Invitrogen Corp, CA, USA).

For the apoptosis analysis, cells were washed in PBS, and then processed with Annexin V-FITC Apoptosis Detection Kit (BD Biopharmingen, NJ, and USA) for 15 min in the dark. All experiments were analyzed by BD Biasciences FACS Calibur Flow Cytometry (BD Biasciences, NJ, and USA). The tests were repeated for three times with triplicate per experiment. Cell proliferation was assayed by using EDU (5-ethynyl-2′-deoxyuridine). (Roche, Basel, Switzerland) as described previously [15].

Cells were briefly rinsed in PBS and fixed in 4 % formaldehyde in PBS (pH 7.4) for 15 min at room temperature. Then the cells were permeabilized in PBS containing 0.5 % Triton X-100 on ice for 10 min; washed with PBS and rinsed once in 2 × SSC prior to hybridization. Using an anti-WT1-AS oligodeoxynucleotide probe which was conjugated with Alexa Fluor 488 (Invitrogen, Carlsbad, CA), hybridization was performed in hybridization solution (probe dilution 1:1250) (Boster, China) for 16 h at 50 °C in a moist chamber. For FISH, cells were washed for 30 min in 25 % deionized formamide/2 × SSC at 50 °C and then at 50 °C, 30 min in 2 × SSC.

The promoter sequence of WT1 predicted to interact with WT1-AS or a mutated sequence with the predicted target sites were inserted into the KpnI and SacI sites of pGL3 promoter vector (Genscript, Nanjing, China). These constructs were named pGL3-WT1, and pGL3-WT1 mut. For reporter assay, cells were plated onto 24-well plates and transfected with 100 ng of pGL3-WT1, pGL3-WT1-mut, respectively, using Lipofectamine 2000 (Invitrogen Corp, CA, and USA). A Renilla luciferase vector pRL-SV40 (5 ng) was also co-transfected to normalize the differences in transfection efficiency. After transfection for 48 h, cells were harvested and assayed with the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer’s instructions. Transfection was repeated three times in triplicate.

All experiments were independently repeated at least triplicate. Data were expressed as mean ± SD. Chi-square tests was used to evaluate statistical differences in demographic and clinical characteristics in HCC patients. Differences between two independent groups including clinical samples and in vitro study were tested with the student t test. Pearson correlation was applied to analyze the correlation between WT1 and WT1-AS. All statistical analyses were carried out using SPSS version 18.0 and presented with Graphpad prism software. Kaplan-Meier survival curves were plotted and log rank test was done. The significance of various variables for survival was analyzed by Cox proportional hazards model in a multivariate analysis. The results were considered to be statistically significant at P < 0.05.