Rna oligoribonucleotides

The RNA oligoribonucleotides targeting human HOTAIR, EZH2 and miR-122 were synthesized and purchased from Genepharma (Shanghai, China), and the sequences were listed in Supplementary Table SII. Human DNMT1-siRNA, DNMT3A-siRNA and DNMT3B-siRNA were purchased from Origene.
Lipofectamine 2000 (Invitrogen) was used for transfection of RNA oligoribonucleotides [30 (link)], and X-tremeGENE (Roche) was used for transfection of plasmid DNA, according to their manufacturer's instructions, respectively.

MALAT1 and all RNA oligoribonucleotides for in vitro studies were purchased from GenePharma (Shanghai, People’s Republic of China). Oligoribonucleotides were performed using Lipofectamine 2000 (Thermo Fisher Scientific). Unless otherwise indicated, 100 nM of RNA duplex or 80 nM of MALAT1-siRNA was used for each transfection, and all the experiments were repeated in triplicate.

For transfection experiments, miRNA duplexes corresponding to the indicated miRNAs were designed as previously described (Lim et al, 2005 (link)). The negative control (NC) RNA duplexes for the miRNA mimics and the small-interfering RNAs (siRNAs) were not homologous to any known human gene sequences. The siRNAs were designed to target the human Smad7 transcript (GenBank Access No: NM_001190821). All RNA oligoribonucleotides were purchased from Genepharma (Shanghai, China). The sequences were as follows: miR-367 mimics, 5′-UAGCUUAUCAGACUGAUGUUGA-3′ and 5′-AACAUCAGUCUGAUAAGCUAUU-3′ NC, 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′ Smad7 siRNA, 5′-GGTGGCCTTGTGATCAATGAAACT-3′ (sense); Anti-miR-367 (an inhibitor of miR-367), 5′-UCAACAUCAGUCUGAUAAGCUA-3′ Anti-miR-C (used as a NC for anti-miR-367 in the antagonism experiment), 5′-GUGGAUAUUGUUGCCAUCA-3′. The oligonucleotide transfection was performed using the Lipofectamine 2000 reagent (Invitrogen). Fifty nanomoles per litre of RNA duplex were used for each transfection.

For alteration expression of circRFWD2, miR-6817-5p, and BMPR2, cell transfection was conducted by Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions after cell reached 70% confluence. The RNA oligoribonucleotides were obtained from GenePharma Co. (Shanghai, China) which were showed in Table S1. The si-circRFWD2, si-BMPR2, miR-6817-5p mimic, miR-6817-5p inhibitor, and negative controls underwent cell transfection with 100 nM concentration. The transfected cells were collected after 2 and 3 days for qRT-PCR and Western blot, respectively.

The human RCC cell lines used in this study included 786–0, ACHN, A498, Caki-2, OS-RC-1, OS-RC-2, and HK-2. They were purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI 1640 (Gibco, Carlsbad, California) supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 maintained at 37°C.
The cells were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacture’s instructions. miR-301a inhibitor (5ʹ-GCUUUGACAAUACUAUUGCACUG-3ʹ), inhibitor NC (5ʹ-CAGUACUUU UGUGUAGUACAAA-3ʹ), miR-301a mimic (5ʹ-CAGUGCAAUAGUAUUGUCAA AGC-3ʹ) and miR-control (5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ) were all transfected at a concentration of 40 nmol/L. The RNA transfection efficiency was at least 60–75%, and the RNA duplex persisted in cells for at least 4 days. All RNA oligoribonucleotides were purchased from GenePharma (Shanghai, P.R. China).