Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers

Objective Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/β-catenin pathway in liver cancer. Design Hypomorphic Apc mice (Apcfl/fl) and thiocetamide (TAA)-treated rats developed Wnt/β-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation. Results Overexpression of the T-UCR uc.158− could differentiate Wnt/β-catenin dependent HCC from normal liver and from β-catenin negative diethylnitrosamine (DEN)-induced HCC. uc.158− was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of β-catenin altered uc.158− expression in human malignant hepatocytes. uc.158− expression was increased in CTNNB1-mutated human HCCs compared with non-mutated human HCCs, and in human HCC with nuclear localisation of β-catenin. uc.158− was increased in TAA rat CCA and reduced after treatment with Wnt/β-catenin inhibitors. uc.158− expression was negative in human normal liver and biliary epithelia, while it was increased in human CCA in two different cohorts. Locked nucleic acid-mediated inhibition of uc.158− reduced anchorage cell growth, 3D-spheroid formation and spheroid-based cell migration, and increased apoptosis in HepG2 and SW1 cells. miR-193b was predicted to have binding sites within the uc.158− sequence. Modulation of uc.158− changed miR-193b expression in human malignant hepatocytes. Co-transfection of uc.158− inhibitor and anti-miR-193b rescued the effect of uc.158− inhibition on cell viability. Conclusions We showed that uc.158− is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics.

NanoString. miRNA expression profiling was analysed with nCounter from NanoString Technologies (Seattle, WA, USA), using the nCounter Mouse miRNA Expression assay kit, as previously described. [2] Technical normalization was performed using the synthetic positive controls to adjust the counts for each miRNA target in that assay. Then biological normalization was performed to correct for differences in sample abundances. Each sample was normalized to the geometric mean of the top 50 most highly expressed miRNAs.
Student's t test was used on normalized counts to calculate statistical significances of pairwise comparisons.
Rapid Amplification of cDNA ends (RACE). RNA from the liver of Apcfl/fl mouse was used to generate RACE-ready cDNA using the SMARTer RACE cDNA amplification Kit (Clontech, Mountain View, CA, USA) following the manufacturer's protocol. Amplification of cDNA ends was performed by using Universal Primer mix (UPM) and gene-specific primers. Heart mouse RNA and Transferrin-Receptor-specific primers provided by the manufacturer were used as controls. In case the primary PCR reaction failed to give distinct bands, we performed a "nested" PCR using the nested universal primer. PCR products were separated in a 1.5% agarose gel, and DNA extracted, cloned in the TOPO.TA.2 plasmid and sequenced using the 48-capillary Applied Biosystems 3730 DNA Analyzer (Applied Biosystem, Foster City, CA, USA).
Transfection. Cells were transfected using HiPerfect (Qiagen, Hilden, Germany) according to the manufacturer's protocol. siRNA against CTNNB1 (Thermo-scientific, Hemel, UK) was used at 50 nM for 72 hours. For uc.158-silencing LNA probes control, and LNA anti-uc.158-(GAPMERs, Exiqon, Vedbaek, Denmark) were used at a final concentration of 25nM. Western Blotting. Cells were collected and protein extracted. Immunoblot analysis was performed as previously described. [3] The primary antibodies used were as follows: mouse monoclonal actin (MP Biomedical, Santa Ana, CA, USA), rabbit polyconal PARP (9542 Cell Signalling, Danvers, MA, USA), rabbit monoclonal cleaved-PARP (D64E10 Cell Signalling), and mouse monoclonal caspase 9 and cleaved caspase 3 (7237 and 9664 Cell Signalling), and mouse monoclonal β-catenin (D-10, Santa Cruz Biotechnology, Santa Cruz, CA, USA).The latter recognizes the C-terminus of β-catenin and therefore identifies two bands in HepG2 cells that harbour also a truncated form of β-catenin as result of their mutation. Immunofluorescence Microscopy. Cell monolayers were fixed in 4% formaldehyde, washed with PBS three times and blocked in 5% BSA for 1 hour at room temperature, then incubated with mouse monoclonal β-catenin antibody (1:100; Santa Cruz Biotechnology).
After 1 hour incubation, cells were washed serially with PBS and incubated for another 1 hour with fluorochrome-conjugated secondary antibodies at room temperature. After washing with PBS, cells were incubated for 10 minutes with nuclear dye (Hoechst 1ug/ml; Pierce, Rockford, IL, USA). Cells were then mounted in a coverslip glass and fluorescence visualized and captured using a Carl Zeiss confocal imaging system (Carl Zeiss, Heidenheim, Germany).
In Situ RNA hybridization. A locked nucleic acid (LNA) probe with complementarity to a 21-bp section of uc.158was labeled with 5′-digoxigenin and synthesized by Exiqon. Tissue sections on the tissue microarray were digested with ISH protease 1 (Ventana Medical Systems) and in situ hybridization performed as described. [3] Negative controls included omission of the probe and the use of a scrambled LNA probe. Each sample was classified by two independent reviewers according to a 4-tiered scoring system based on the intensity of uc.158-expression as follows: 0: indicates no stain or stain in less than 10% of tumor cells; 1+: faint/weak cytoplasm/nuclear stain in 10% or more of cells; 2+: moderate cytoplasm/nuclear stain in 10% or more of tumor cells; and 3+: strong cytoplasm/nuclear stain in 10% or more of tumor cells. In all the considered tissue samples, fibroblasts, lymphocytes, and endothelia featured uc.158-cytoplasm/nuclear expression and were assumed as positive internal control (not considered in ISH score). 3D-spheroid formation analysed with Celigo S (Nexcelom, Manchester, UK) as previously described. [4] After 7 days, spheroids were transferred to a flat bottom plate previously coated with 0.1% gelatin and cell migration analysed as previously described. [5] Caspase 3/7 activation. Cytotoxicity and caspase 3/7 activation were measured by using the ApoTox-Glo™ Triplex Assay and following manufacturers' instruction.