The knockdown of H19lncRNA reveals its regulatory role in pluripotency and tumorigenesis of human embryonic carcinoma cells

The function of imprinted H19 long non-coding RNA is still controversial. It is highly expressed in early embryogenesis and decreases after birth and re-expressed in cancer. To study the role of H19 in oncogenesis and pluripotency, we down-regulated H19 expression in vitro and in vivo in pluripotent human embryonic carcinoma (hEC) and embryonic stem (hES) cells. H19 knockdown resulted in a decrease in the expression of the pluripotency markers Oct4, Nanog, TRA-1-60 and TRA-1-81, and in the up-regulation of SSEA1; it further attenuated cell proliferation, decreased cell-matrix attachment, and up-regulated E-Cadherin expression. SCID-Beige mice transplanted with H19 down-regulated hEC cells exhibited slower kinetics of tumor formation, resulting in an increased animal survival. Tumors derived from H19 down-regulated cells showed a decrease in the expression of pluripotency markers and up-regulation of SSEA-1 and E-cadherin. Our results suggest that H19 oncogenicity in hEC cells is mediated through the regulation of the pluripotency state.

2 listed in Table S1. The sequences were generated by annealing of the sense and antisense oligonucleotide: For the H19-shRNA: 5'GATCCCTAAGTCATTTGCACTGGTTTTCAAGAGAAACCAGTGCAAATGACTTATTTTTGGAAA 3'
Each annealed oligonucleotide was inserted into the BglII and HindIII sites of pTER+ plasmid, containing the H1 promoter and Tetracycline Operator, to produce plasmid pTER/shLuc and pTER/shH19. The complete shH19/shLuc expression cassettes were excised from the pTERshH19/shLuc as an EcoRI fragment (Blunt ends) and inserted into the EcoRV site of a modified pRRL.sin.PPT lentiviral vector (3) containing the hEF1alpha promoter driving the expression of the tet-repressor fused to EGFP (generously given by Prof. Yinon Ben-Neriah and his team -The Lautenberg Center for Immunology, The Hebrew University, Jerusalem, Israel). The final modified lentiviral vector pRRL.sin.cPPT H1/TO-shRNA hEF1-TetR-EGFP harbors an HI promoter, tet-operator driving the induced expression of the shRNAsequence and hEF1alpha promoter driving the expression of the tetrepressor fused to EGFP. Cloning strategy is depicted in

Production of lentiviruses and transduction of hES and hEC cells
Production of lentiviral particles and transduction of HES-1, NCCIT and NT2 cells were performed as described (4,5). Briefly, high-titer lentiviral particles (titers of 3.2x10E 8 transduction units/ml) were generated by transfecting HEK 293T cells cultured on 10-cm plates, with 10 g of the lentiviral plasmid encoding the silencing cassettes (shH19/shLUC), 6.5 g of the packaging plasmid pCMV∆R8.91, and 3.5 g of the envelope protein (pMD.G) (a total of 20 g plasmid DNA) as described previously (5)(6)(7). The supernatant containing the viral particles was collected, concentrated, using an ultracentrifuge (Sorvall ultracentrifuge model Discovery 100, with a Surespin 630 swinging bucket rotor) and used to transduce hES and hEC cells. Transduction was carried out as previously described, but without the presence of polybrene. (8). Briefly, prior to transduction, hES cells were dissociated into a single cell suspension with 0.05% EDTA solution (biological Industries). 1 x 10 5 cells were incubated with the concentrated viral supernatant and plated on feeders in a well of six-well dish. For transduction of NCCIT and NT2 lines, 2.5x10 5 cells/well of six-well dish were plated 24 hours before the transduction and then incubated with the concentrated viral supernatant.

RNA Extraction, Reverse Transcription-PCR and PCR
Total RNA was extracted using the Quick-RNA MiniPrep Kit (Zymo Research, CA, USA).
Integrity of RNA was checked by agarose gel electrophoresis and ethidium bromide staining.
Subsequently, cDNA was generated from 1 g of total RNA using Quanta bioSciences cdna synthesis kit according to the manufacturer's instructions (Quanta Bioscience, Gaithersburg, USA). miR cDNA was generated from 500ng of total RNA using Quanta Bioscience microRNA cDNA Synthesis Kit according to the manufacturer's instruction (Quanta Bioscience). cDNA was amplified by PCR using Ready mix for PCR (Bio-Lab, Jerusalem, Israel) and specific primers (Table S2). Cycle parameters for all genes were 30 s at 94 °C, 30 s at 55-60°C, and 30 s at 72°C for 29 cycles, and a final extension of 5 min at 72°C. PCR products were separated on 1% agarose gel.

Quantitative PCR (qPCR)
qPCR reactions were performed with the 7900HT Fast Real-Time PCR System (Applied Biosystems, Carlsbad, USA) in a total reaction volume of 20 μl using 100ng cDNA for H19 expression and 30ng cDNA for the other genes, PerfeCTa® SYBR® Green FastMix® (Quanta BioSciences, Inc.), and 500 nM of forward and reverse primers. All qPCR assays were performed in triplicates and the relative gene expression was determined after normalization with beta-Actin or GUSB transcript levels using the ΔΔCT method, displayed as mRNA fold change or Relative quantitation. The primers used for SYBR Green detection were designed by Quanta biosciences (Primer sequences are shown in Table S3).
miRNA expression was quantified in real-time SYBR Green qrt-PCR amplification reaction with the desired PerfeCta microRNA assay and the PerfeCta Universal PCR Primer following the manufacture's instruction (Quanta BioSciences, Inc.)
The gels were then electroblotted onto PVDF membranes. After blocking with 1% milk, membranes were incubated with specific primary anti-human antibodies against OCT4 (mouse anti-

FACS analysis
hES and hEC cells were dissociated to a single-cell suspension. The cells were then washed with FACS buffer (PBS containing 1% BSA and 0.05% sodium azide-both from Sigma). The cells were incubated for 30 minutes with primary antibodies (Table S4). Control cells were stained with respective isotype control antibodies. Primary antibodies were detected using APC-labeled goat anti-mouse immunoglobulin M (1:100, Bioscience San Diego CA USA). PI was added to a final concentration of 4μg/ml for gating of viable cells. FACS analysis was performed using the FACSCalibur system (Becton Dickinson).

Annexin V assay for detection of apoptotic cells
The percentage of apoptotic cells was measured using Annexin V conjugated to APC apoptosis For negative controls, primary antibody was omitted.   Figure S2. Design of a lentivirus vector for the expression of H19 and Luc specific siRNA.