Sh con

Small-interfering RNA (siRNA) against p53 and scrambled siRNA were synthesized by GenePharma (China), and their sequences were as follows: the p53-specific siRNA (si-p53) 5′-GACTCCAGTGGTAATCTACTT-3′ and scrambled control siRNA (si-con): 5′-UUCUCCGAACGUGUCACGUTT-3′[16] (link). To silence the expression of the p53 gene, LX-2 cells were transfected with si-p53 using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's instructions. After transfected for 30 h, the cells were treated with or without the SEA for another 18 h and then harvested for western blot and quantitative real-time polymerase chain reaction (qRT-PCR) assays.
To silence DR5 expression, we transfected shRNA-control (sh-con, Genechem, China) or shRNA-DR5 (sh-DR5, Genechem, China) in LX-2 cells using FuGENE 6 (Promega, USA) according to the manufacturer's instructions. After transfection for 30 h, the cells were treated with or without the SEA for another 18 h and then harvested for western blot and qRT-PCR assays.

Cells were seeded into six-well plates for 24 h. To knock down p53 expression, ShRNA-p53 (Sh-p53; Genechem, Shanghai, China) or ShRNA-control (Sh-con; Genechem) were transfected into LX-2 cells. Cells were treated with or without Sjp40 (20 μg/ml) 24 h after transfection. Human small-interfering RNAs against STAT3 (Si-STAT3) and a scrambled sequence control (Si-con) were purchased from GenePharma (Shanghai, China). LX-2 cells were transfected with Si-STAT3 or Si-con for 6 h using Lipofectamine 2000 (Invitrogen) and then were treated with or without stimulus for 48 h.

The GV248 lentiviral vector sh-TAK1 containing short hairpin RNA targeting sequence (5′- GTGTGTCTTGTGATGGAAT-3′) and the negative control lentiviral vector sh-Con were obtained from Genechem Company Ltd (Shanghai, China) and were used to knock down TAK1 expression. To generate clones stably knock down TAK1, the HepG2-NTCP cells first were infected at a multiplicity of infection of 20 with sh-TAK1 or sh-Con. Stable clones were selected after 2 weeks using puromycin and the expression level of TAK1 was determined by Western blot. HBV infection of HepG2-NTCP cells was performed according to Prof. Wenhui Li’s protocol4 (link). Briefly, HepG2-NTCP cells were seeded in 6-well plates and infected with HBV at 100 genome equivalent (GEq)/cell in the present of 4% polyethylene glycol (PEG 8000; Sigma) for 16 h, and then rinsed three times with phosphate-buffered saline and maintained in the maintenance medium containing 2.5% dimethyl sulfoxide. The supernatant samples were collected at 3, 6 and 9 days post infection and HBsAg and HBeAg were assessed.

SNHG12 small interfering RNA and overexpression plasmid (si-SNHG12 and SNHG12) or their negative controls (si-con and pcDNA), miR-148a mimic and inhibitor (miR-148a and anti-miR-148a) or their negative controls (miR-con and anti-miR-con), CDK1 overexpression plasmid (CDK1) and its negative control (pcDNA) were designed and synthesized by GenePharma (Shanghai, China). Lentiviral short hairpin RNA targeting SNHG12 (sh-SNHG12) and its negative control (sh-con) were constructed by Genechem (Shanghai, China). Cells were transfected with indicated plasmids using Lipofectamine 3000 (Invitrogen).

To obtain cell lines stably overexpressing DBH-AS1, HepG2 and SMMC7721 cells were infected with the Lv-DBH-AS1 and Lv-control viruses (GeneChem, Shanghai, China). To observe the knockdown effects of DBH-AS1 in vitro, Hep3B and SK-Hep1 cells were transfected with the shRNA-DBH-AS1 (Sh-DBH-AS1) or control (Sh-con) viruses (GeneChem). Recombinant lentiviruses containing HBx (Lv-HBx) or control (Lv-con) purchased from LAND (Guangzhou, China) were used to infect HepG2 and LO2 cell lines. The infection efficiency was confirmed by qRT-PCR or western blot.

This animal experiment was approved by the Animal Care and Use Committee of Jining NO. 1 People’s Hospital. First of all, the lentiviral vector (lenti-short hairpin, sh-KCNQ1OT1) for stable expression ofKCNQ1OT1 knockdown and its control (sh-con) were provided by Genechem (Shanghai, China). Moreover, BALB/c nude mice (4–5 weeks old) were purchased from Shanghai Animal Laboratory Center (Shanghai, China), and then mice were randomly divided into two groups (n = 5 per group). Subsequently, 1 × 10⁷ SW480 cells transfected with sh-KCNQ1OT1 or sh-con were subcutaneously injected the left flank of the nude mice. After 7 days of injection, tumor volume was tested every 7 days. Meanwhile, tumors were excised and weighed on day 35 after inoculation. Besides, qRT-PCR and western blot assays were applied to detect the levels of KCNQ1OT1, miR-329-3p, CTNND1, CyclinD1, Cleaved-casp-3, and Bcl-2 in xenograft.