Hek293t

The human colorectal HCT 116 (CVCL_0291), Caco-2(CVCL_0025), HT-29 (CVCL_0320), SW480 (CVCL_0546), SW620 (CVCL_0547), breast MCF-7(CVCL_0031), T-47D (CVCL_0553), MDA-MB-231(CVCL_0062), ovarian Caov-3(CVCL_0231), SK-OV-3(CVCL_0532) cancer cell lines and HEK293T(CVCL_0063) were obtained from the GeneChem Corporation (Shanghai, China). OVCAR-3(CVCL_0465) was obtained from the Nanjing KeyGen Biology (Nanjing, China). All human cell lines have been authenticated using STR profiling. OVCAR-3, Caco-2, MCF-7, Caov-3 and HEK293T cells were cultured in DMEM (Gibco, Carlsbad, CA, USA). T-47D and OVCAR-3 cells were cultured with RPMI 1640 (Gibco, Carlsbad, CA, USA). HCT 116 and HT-29 cells were cultured with McCoy’s 5a (Gibco, Carlsbad, CA, USA). SW480, SW620, and MDA-MB-231 cells were cultured with Leibovitz’s L-15 (Gibco, Carlsbad, CA, USA) medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin. To inhibit NF-κB activities, BAY 117082 (Selleckchem, Houston, TX, USA) was used.

HEK293T was obtained directly by Shanghai GeneChem (Lot number: GCPC53508) and cultured in DMEM medium (Hyclone) containing 10% heat-inactivated fetal bovine serum (Hyclone) in an incubator (5%CO₂) at 37°C (Thermo). According to the prediction of miR-7 target using TargetScan (http://www.targetscan.org/) and miRanda-mirSVR (http://www.microrna.org/), two regions, position 86–92 and 1111–1118 of COX-2 were combined with miR-7 seed region possibly. Then both regions of COX-2 3’-UTR were cloned and constructed independently into pGL3 vector. In addition, the miR-7 up eukaryotic expression plasmid and the mutants of COX-2 3’-UTR plasmids were also supplied by Shanghai GeneChem.

The human liver cancer cell lines HepG2, Huh7 and HCCLM3 were purchased from the China Center for Type Culture Collection (CCTCC, Wuhan, China) and were identified by short tandem repeat (STR). L02 hepatocytes were gift from Zhongshan Hospital of Fudan University (Shanghai, China). The embryonic kidney cell line HEK-293 T was a gift from Shanghai GeneChem Co., Ltd. (Shanghai, China). All cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; pH = 7.2, Gibco Company, Grand Island, NY, USA) containing 10% (v/v) foetal bovine serum (FBS, HyClone, Logan, UT, USA). All cells were cultured in a humidified incubator (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO2. All cells were tested for mycoplasma contamination.

Human embryonic kidney 293T cells (HEK293T) were used for lentiviral packing. HEK293T cells was provided by Genechem Co., Ltd. (Shanghai, China) and originally purchased from American Type Culture Collection (ATCC, Manassas, VA, CRL-3216). The cells were cultured in 10 cm dishes for 2–3 days until they reached 90–95% confluency. The recombinant virus plasmid pLV-nol3-EGFP, which encodes the full-length nol3, and the control vector pLV-EGFP, together with packaging plasmids (pLP1, pLP2, and pLP/VSVG), were cotransfected into HEK293T cells using Lipofectamine™ 2000 (all from GeneChem Co., Ltd., Shanghai, China). After 48 h of transduction, the lentiviral particles contained in the supernatant of 293T cells were harvested and then concentrated by passing through a 0.45-μm filter. The concentrated solutions were then added to cultured BMSCs at a variety of multiplicities of infections ranging from 0 to 100. After 72 h, the transduction efficiency was assessed via fluorescence microscopy and qRT-PCR.