Sh snhg1

Lentiviruses expressing specific targeted knockdown SNHG1 [short hairpin-SNHG1 (sh-SNHG1)] and MDM2 (sh-MDM2) sequences and a scramble shRNA (sh-NC; control shRNA) were constructed by GenePharma (Shanghai, China) (Table 2). Lentiviruses overexpressing SNHG1 (oe-SNHG1), MDM2 (oe-MDM2), and PPARγ (oe-PPARγ), oe-NC, Inhibitor NC, and miR-9-3p inhibitor were obtained from GenePharma. Transfection was implemented using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA).

Short-hairpin RNA directed against human SNHG1, FOXP2 and KDM5B was constructed in pGPU6/GFP/Neo vector (sh-SNHG1, sh-FOXP2 and sh-KDM5B), their respective non-targeting sequence (negative control, NC) were also synthesized (GenePharma, Shanghai, China). FOXP2 full length with and without 3’UTR plasmids and their non-targeting sequence (negative control, NC) were synthesized (GenScript, Piscataway, NJ, USA). U87 and U251 cells was transfected at about 70–80% confluency in 24-wells plates using Lipofectamine 3000 reagents (Invitrogen, CA, USA) according to the manufacturer’s instructions. Stable transfected cells were obtained by continuous application of Geneticin (G418, Invitrogen, CA, USA) and evaluated for their over-expression and silence efficiencies by qRT-PCR.
The oligonucleotide sequences of human miR-154-5p and miR-376b-3p mimics, as well as miR-154-5p and miR-376b-3p inhibitors were synthesized (GenePharma, Shanghai, China). A scrambled sequence was used as the negative control. Furthermore, miR-376b-3p + 44 A to I editing mimics were synthesized (GenePharma, Shanghai, China). Cells were transfected using Lipofectamine 3000 reagent (Invitrogen, CA, USA). The transfected efficacy was evaluated by qRT-PCR, and the high transfection efficacy of these could sustain 7 days from 48 h post-transfection.

The lentivirus vectors (sh-SNHG1, sh-LMNB2) and small interfering RNAs (siRNAs) against human SNHG1 or LMNB2 were synthesized by GenePharma Co. Ltd. The stable Huh7 cells with SNHG1 and LMNB2 knocked down were generated using lentiviral vectors. Infected cells were then treated with puromycin (2 µg/ml) for 2 days, and surviving cells were maintained in complete medium with puromycin (0.5 µg/ml). The siRNAs were transfected into hepatocellular cancer cells using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to their instructions. Besides, miR-326 mimics, miR-326 inhibitors, and negative controls were purchased from GenePharma Co. Ltd. When the cell confluence reached 50%, oligonucleotide transfection was performed using Lipofectamine 2000 according to the manufacturer’s protocol.