Mir nc

The small interfering RNAs (siRNAs) and plasmid were transfected into ESCC cells using StarFectII High-Efficiency Transfection Reagent (GenStar) in line with the manufacturer’s protocol. Three individual lncRPL34-AS1 siRNAs (si-RPL34-AS1 #1, #2, #3 and si-NC) and plasmid vector (pcDNA 3.1-RPL34-AS1 (pc-RPL34-AS1), pcDNA 3.1-NC (pc-NC)) and pcDNA 3.1-ACAA2 (pc-ACAA2) were purchased from KeyGEN BioTECH. The miR-NC, miR-575 mimics and miR-575 inhibitor were provided by Genomeditech. The all nucleotide sequences were listed in Table S2. After 48 h transfection, cells were acquired for RT-qPCR or western blot analysis.

The partial sequences of TGFBR2 3′UTR which contained the wild or mutant binding sites of miR-20a-5p were amplified and then cloned into the pGL3-Basic luciferase vector (Promega, W.I.) with the aim of constructing pGl3-TGFBR2 (WT) and pGl3-TGFBR2 (Mut). Primers used in plasmid construction were as follows: forward 5′-CAGGCTGGGCCATGTCCAAA-3′ and reverse 5′-GTCAAATGCTAATGCTGRCATG-3′. The two plasmids were, respectively, co-transfected with miR-NC, miR-20a-5p mimic, anti-miR-NC, and anti-miR-20a-5p (Genomeditech). Forty eight hours later, the luciferase activity analysis was conducted on the Dual-Luciferase Reporter assay system (Promega, W.I.), in strict accordance with the instructions of the manufacturer.

Human PTC cell lines (K-1, SW1736, TPC-1, SW579) and human thyroid follicular epithelial cells Nthy-ori 3–1 were available from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Notably, all cells were accordingly cultured in Roswell Park Memorial Institute (RPMI)-1640 medium with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin. The above materials or reagents were from Gibco (Carlsbad, CA, USA).
Small interfering RNA (siRNA) oligonucleotides targeting circ_0062389 (si-circ_0062389#1: 5ʹ-TGCAGACCTCCCGACCTCTTT-3ʹ, si-circ_0062389#2: 5ʹ-ATCTGCTGTCTGCAGACCTCC-3ʹ and si-circ_0062389#3: 5ʹ-GATCTGCTGTCTGCAGACCTC-3ʹ), miR-1179 mimics (5ʹ-AAGCAUUCUUUCAUUGGUUGG-3ʹ) and miR-1179 inhibitors (miR-1179 in, 5ʹ- CCAACCAAUGAAAGAAUGCUU-3ʹ) and their corresponding control (miR-NC, 5ʹ-UCACAACCUCCUAGAAAGAGUAGA-3ʹ) were synthesized by Genomeditech (Shanghai, China). The above vector plasmids or oligonucleotides were respectively transfected into PTC cell lines by lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) when the cells reached 80% confluence.