Nc oligonucleotides

In the RNA interference-mediated gene knockdown assays, the specific siRNA sequence against C. idellus PKZ, IRF9, and the negative control RNA (N.C.) oligonucleotides were prepared (Shanghai GenePharma Co., Ltd.) (Table 1). The siRNA sequence against IRF3 was used as in the previous study (35 (link)). The transfection reagent, Hiperfect® (Qiagen, Germany), has been widely used in our previous study (35 (link)). The transfection was carried out according to the manufacturer's protocol and instructions.

Lentivirus-containing short hairpin RNA (shRNA) targeting SNHG12 was purchased from OBiO (Shanghai, China), the sequences are as follows: sh-SNHG12-1: CcggGCTGTCCTCATTTGTGACTTTCAAGAGAAGTCACAAATGAGGACAGCTTTTTTg, sh-SNHG12-2: CcggCCTATGGAGTTGGGACAATTTCAAGAGAATTGTCCCAACTCCATAGGTTTTTTg. And the pCDH-CMV-Human vector for SNHG12 overexpression was purchased from Allwin (Shanghai, China). miR-218-5p mimics, miR-218-5p inhibitors, and negative control (NC) oligonucleotides were obtained from GenePharma (Shanghai, China). SiRNAs for YY1, YWHAZ and HuR were obtained from Sangon Biotech (Shanghai, China), sequences are listed as follows: si-YY1 sense (5'-3'): CCAAACAACUGGCAGAAUUTT, antisense (5'-3'): AUUCUGCCAGUUGUUUGGTT; si-HuR sense (5'-3'): GCGUUUAUCCGGUUUGACAtt, antisense (5'-3'): UGUCAAACCGGAUAAACGCtt; si-YWHAZ sense (5'-3'): GATGACATGGCAGCCTGCATGAAGT. GC cells were transfected with the abovementioned oligonucleotides and plasmids using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol.

EZH2 siRNA (siEZH2) and negative control (NC) oligonucleotides were purchased from GenePharma (Shanghai, China, see Additional file 1). Oligonucleotides were transected into U87, U251, and GL261 cells using the transfection reagent Lipofectamine2000 (Invitrogen) following the manufacturer’s instructions with oligonucleotides at a working concentration of 100 nM. The transfected cells were incubated for 24 or 48 h before subsequent experiments.
The EZH2 functional inhibitor, DZNep, was purchased from Sigma-Aldrich. DZNep was added to U87 and U251 cell medium at a concentration of 5 μM for indicated time duration.

Chi-miR-200a mimics, inhibitor and its negative control (NC) oligonucleotides were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The oligonucleotide sequences were listed in Additional file 1.

MiR-23b mimics, miR-23b inhibitor, ST7L siRNA and NC oligonucleotides were obtained from GenePharma. ST7L expression plasmids and flag-tagged AKT plasmids were obtained from Biogot Technology (Nanjing, China). To generate reporter construct, one fragment of ST7L 3′-UTR including two putative miR-23b complementary sites was fused to a modified pcDNA3.1 vector containing a luciferase gene, which was inserted upstream of multiple cloning sites. Reporter plasmids with mutant sites were prepared by Mutagenesis Kit (Stratagene, La Jolla, CA, USA). TOP/FLASH reporter gene including β-catenin binding sites was obtained from Millipore (Billerica, MA, USA). Oligonucleotides were transfected by Hiperfect transfection reagent (Qiagen, Valencia, CA, USA) and plasmids were transfected by Lipofectamine 3000 (Invitrogen) into cells. Luciferase activity assay was conducted using Dual Luciferase Assay System (Promega, Madison, WI, USA).

miRNA mimics and the negative control (NC) oligonucleotides were synthesized by Shanghai GenePharma Co., Ltd (Shanghai, China). The viability of 32D-BA cells was assayed using the CCK-8 Kit (Dojindo, Japan). Cells were plated in triplicate at a density of 1×10⁴/well in a 96-well plate at 24h hours after transfection with miRNA mimics. CCK-8 reagent (10μL) was added to each well at 24, 48, and 72h, respectively. The optical density at 450nm was measured after one hour of incubation. Apoptosis ability of murine BM cells was assessed using annexin V/PI (BD Pharmingen, USA) staining.