Pcdna3

The pcDNA3.1 (His tag in-frame with the 3ʹ-end of the cDNA; CMV promoter; neomycin resistance), pcDNA-SNHG7 (containing full-length rat SNHG7), short hairpin (sh)-negative control (NC), sh-SNHG7, miR-NC, miR-9 mimics, mimics NC, miR-9 inhibitor, inhibitor NC and sh-SIRT1 were bought from RiboBio Company (Beijing, China). Additionally, we have listed the sequences of shRNAs or miRs as follows: sh-SNHG7 (5ʹ-GCCTGGGTGTTGCTGTGTATT-3ʹ), sh-SIRT1 (5ʹ-CCATTCTTCAAGTTTGCAA-3ʹ), miR-9 mimics (5ʹ-UCAUACAGCUAGAUAACCAAAGA-3ʹ), mimics NC (5ʹ-UCACAGUGAACCGGUCUCUUU-3ʹ), miR-9 inhibitor (5ʹ-GCTAGATAACCAAAG-3ʹ) and inhibitor NC (5ʹ-ACGTCTATACGCCA-3ʹ). When cells grew 80–90% confluence, the above transcripts were transfected into PC12 cells with Lipofectamine3000 (Invitrogen, Carlsbad, CA, USA) for 48 h.

Short interfering RNA (siRNA) for H19 (si-H19; 50 nM), negative control siRNA (si-NC; 50 nM), H19-overexpression plasmid (pcDNA3.1-H19; 2 µg), pcDNA3.1 (2 µg), miR-29b-3p mimic (50 nM), miR-29b-3p inhibitor (50 nM), STAT3-overexpression plasmid (pcDNA3.1-STAT3; 2 µg), STAT3 siRNA (si-STAT3, 2 µg) and negative control (NC) were all designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The sequences of H19-siRNA1#, H19-siRNA2# and H19-siRNA3# were 5′-CCGUAAUUC ACUUAGAAGAdTdT-3′, 5′-CACAUAGAAAGGCAGGA UAdTdT-3′ and 5′-CCUUCUAAACGAAGGUUUAdTdT-3′, respectively. The sequence of si-NC was 5′-UUCUCCGAAC GUGUCACGUTT-3′. The sequences for miR-29b-3p mimic, miR-29b-3p inhibitor and miR-NC were 5′-UAGCACCAUUU GAAAUCAGUGUU-3′, 5′-AACACUGAUUUCAAAUGGUG CUA-3′ and 5′-UUCUCCGAACGUGUCACGUTT-3′, respectively. The forward and reverse primers for si-STAT3 were 5′-AAGCAGCAGCTGAACAACATGTTCAAGAGACATGT TGTTCAGCTGCTGCTT-3′ and 5′-AAGCAGCAGCTGAAC AACATGTCTCTTGAACATGTTGTTCAGCTGCTGCTT-3′, respectively. Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for cell transfection, according to the manufacturer′s protocol. After 48 h of transfection, Calu-3 and NCI-H1975 cell lines were used for subsequent experiments.

The interaction between SNHG12 and miR-603 was assessed using an RNA pull-down assay. Biotinylated miR-603 mimics and biotinylated NC mimics were synthesized by Guangzhou RiboBio Co., Ltd. The plasmid backbone employed was pcDNA3.1 (Guangzhou RiboBio Co., Ltd.) used at a concentration of 100 ng/µl. HUVECs were lysed and incubated (4˚C) with the biotinylated RNA probes for 2 h. Following this, 50 µl streptavidin agarose beads (Thermo Fisher Scientific, Inc.) was added to the mixture and incubation continued for an additional 2 h at 4˚C. The centrifugation steps were conducted at 1,000 x g for 5 min at 4˚C, both after the initial incubation and following the addition of the beads. After these steps, the beads were washed and the bound RNAs were extracted using the PureLink RNA Mini Kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The beads were washed and the bound RNAs were extracted using TRIzol (Thermo Fisher Scientific, Inc.) for RT-qPCR analysis.