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  • Total RNA was isolated from cells and tissues using

    2019-10-22

    Total RNA was isolated from Meropenem and tissues using Trizol reagent (Invitrogen, USA) according to the manufacturer's instructions. NanoDrop ND2000 (Thermo Scientific Inc., USA) was used to determine the purity and quantify the concentration of RNA. A First Strand cDNA Synthesis Kit (TakaRa, China) was used to perform reverse transcrip-tion. Primers used for real-time quantitative polymerase chain reaction (RT-qPCR) were obtained from GenScript (Nanjing, China). According to the manufacturer's instructions, RT-qPCR was performed using the SYBR Prime-Script RT-PCR kit (Takara, China) in a Roche lightcycler 96 fluorescent quantitative PCR system (Roche, Germany). The reaction started at 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 30 s. Actin was used as the internal control to normalize qPCR re-sults. Relative gene expression levels were measured using cycle threshold (CT) in the ΔΔCT calculation. The qRT-PCR primers of TONSL-AS1 are Forward: 5′-AGCCCTCTCTCCCTGCACCAC-3'; Reverse: 5′-AGCTGAGCACCTTCCTGAAACCC-3'. The qRT-PCR primers of TONSL are Forward: 5′-CCTGGAGCTGGCACATTCCCT-3'; Reverse: 5′-CCCCTC CAGCTCCTCATCCAC-3'.
    2.4. Cell transfection and viral infection
    Plasmid and siRNA transfections were performed using Lipofectamine 2000 (Life Technologies). Lentiviral Expression System psPAX2, and pMD2.G were used to transduce target cells. For transient knockdown experiments, the small interfering RNAs (siRNAs) were purchased from GenePharma Co. (Shanghai, China). The sequences of TONSL siRNAs are
    #3: 5′- AGGCCCCUGAGACCGAAACCAG-3′ 2.5. Tumorigenesis assay The animal care and experimental protocols were approved by the Nanjing Medical University Experimental Animal Welfare Ethics Committee. Male BALB/c nude mice of 4-week-old were purchased from the Charles River laboratories and maintained under pathogen-free conditions. Mice (six in each group) were injected subcutaneously with 0.2 ml of cell suspension containing 1 × 107 cells in the back flank. When a tumor was visible, it was measured every week and its volume was calculated according to the formula vo-lume = 0.5 × length × width2.
    2.6. Western blot assay
    Protein extracts from cells or immunoprecipitation samples were prepared using detergent-containing lysis buffer. Total protein (60 μg) was subjected to SDS-PAGE and transferred to 0.45 μm PVDF mem-brane (Millipore, Germany). Antibodies were as follows: anti-TONSL (abcam, USA), anti-β-Actin (Santa Cruz, USA). Proteins were detected with a peroxide lumiglo reagent (Cell Signaling Technology, USA).
    2.7. Luciferase reporter assay
    HEK-293T cells were cultured in 48-well plates and co-transfected with 50 ng of TONSL promoter luciferase reporter vector or control vector, and 500 ng of TONSL-AS1 vector by using Lipofectamine 2000. Forty-eight hours after transfection, the luciferase activities were as-sayed using the Dual-Luciferase® Reporter Assay System (Promega, USA). The TONSL promoter was amplified followed the PCR primers: Forward: 5′-GCCATTGCGCCCGGCCCTCT-3’; Reverse: 5′-ATCGCCGCG CCATCCGGACT-3’.
    2.8. Fluorescence in situ hybridization (FISH)
    The 5′-Cy3-labeled TONSL-AS1 probe were designed and synthe-sized by GenePharma Co. (Shanghai, China). The sequences of the probes were: #1, 5′-TGGCGACATCAGCTGCACGGAGGG-3’; #2, 5′-GGACACGTGGGGCGAAGAGTCAGT-3’; #3, 5′-GTGTTTGCATGTCA GGGTGGGGCG-3’. The signals of the probe were detected according to the manufacturer's protocols by the Fluorescent In Situ Hybridization Kit (Ribobio, China). All images were acquired on ZEISS LSM800 Confocal Microscope system (Carl Zeiss AG, Germany).
    2.9. Statistical analysis
    Values are presented as the Mean ± SD. Statistical analysis was evaluated by the unpaired student's t-test. Values of P < 0.05 were considered to be statistically significant.