Smart silencer

Super-Fidelity DNA Polymerase (Cat# P501-d1, Vazyme) was used to amplify the cDNAs of CTD-3252C9.4, IFI6 and IRF1, which were cloned into the expression vector pcDNA3.1 (Cat# V79020, Invitrogen). All PCR products were verified by DNA sequencing. In Additional file 3: Table S3, primers used for plasmid construction are listed. Cells were transfected with the above plasmids or purchased lentivirus pLVX-CTD-3252C9.4 (General Biosystems) for overexpressing the corresponding genes, and pcDNA3.1 (pcDNA-Ctrl) or pLVX-Ctrl were used as control. The plasmid construct map can be found in Additional file 4: Fig. S1. For knocking down, cells were transfected with Smart Silencers (RiboBio) that contains 3 siRNAs, coincident with si-Ctrl as negative control.

smart silencers targeting the lncRNA SNHG26 and siRNAs targeting NCL were synthesized by RiboBio (Guangzhou, China). GC cells were seeded in 6-well plates, and after cell adherence, the smart silencer and siRNAs were transfected at a final concentration of 50 nM using Lipofectamine RNAiMAX (Invitrogen). The indicated cells were harvested for protein extraction and proliferation, migration, and invasion assays after transfection for 48 h.

The Smart Silencer and siRNA used in our study were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China), and the sequences were listed in Additional file 3: Table S3. The plasmids were constructed by HanYin Biotechnology Co., Ltd. (Shanghai, China). Transfection was performed using Lipofectamine 3000 reagent (Invitrogen) following the manufacturer’s instructions.

MEG3 overexpression plasmids were constructed in the pcDH vector by Genomeditech (Shanghai, China). Lentivirus packaging was in 293T cells with l-pei. Following viral infection and puromycin screening, stably transfected cells were acquired. The Smart Silencer (mix of siRNA and ASO) of MEG3 was purchased from RiboBio (Guangzhou, China). Smart Silencer was transiently transfected with Lipofectamine 3000 (Invitrogen, USA) for 48 h and then was used for subsequent assays.

Dendritic cells were treated with TNFα (25 ng/ml, PharMingen), TLR3 ligands (polyinosinic-polycytidylic acid, 2 µg/ml, Sigma-Aldrich), and TLR5 ligand (flagellin, 0.1 µg/ml, InvivoGen). For MALAT1 upregulation, cDNA encoding lncRNA MALAT1 (position: 3201–5600, length 2,400 bp) was PCR-amplified and subcloned into the pcDNA3.1 vector. Interfering RNAs (siRNA) that specifically target mouse MALAT1 were purchased from RiboBio Smart Silencer™. The mouse miR-155 mimic and inhibitor were purchased from GenePharma (Shanghai, China). DCs were transfected with the MALAT1 pcDNA3.1 vector (pMALAT1, 2.5 µg/ml), control vector (Vector, 0.625 µg/ml), MALAT1 siRNA (siMALAT1, 100 nM), or siRNA control (siNC, 25 nM) using Lipofectamine 2000 (Invitrogen) for 6 h on day 6 before LPS stimulation, according to the manufacturer’s protocol. To inhibit NF-κB activity in BMDCs, at day 6, PDTC (50 µM, 30 min, Abcam) or SC-514 (100 mM; Sigma-Aldrich) was used before the LPS treatment. In several experiments, DCs were conditioned with siRNA targeting DC-SIGN (25 nM; GenePharma, China).

Motor neuron progenitors were transfected with lncRNA Smart Silencer (RiboBio Co., Guangzhou, China) to knock down the expression of NCM1-AS. The lncRNA Smart Silencer contained a mixture of three siRNAs and three antisense oligonucleotides. The target siRNA sequences were as followed: 5′-ACAACCCGATGACAGCAGA-3′, 5′-CCAAATGGAGAACGTGCAA-3′and 5′-GTACTCGGTCTTTGCTGGC-3′. The target antisense oligonucleotides sequence were as followed: 5′-ATGAAAGGAAAGGCACCAGC-3′, 5′-ACATCTAACAAGGAGGACAC-3′, 5′-AGGTTGACCGCAATGCACAT-3′. The negative control (NC) Smart Silencer did not contain domains sequences homologous to those of humans, rats, or mice. The cells were transfected with the lncRNA Smart Silencer using Lipofectamine 3000 (Invitrogen, USA) and collected 48 h after transfection for RNA isolation.