Anti mir control

MiR-212-5p mimics (sense 5′-ACCUUGGCUCUAGACUGCUUACU-3′, antisense 5′-UAAGCAGUCUAGAGCCAAGGUUU-3′), control miR (sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′), anti-miR-212-5p (sense 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense 5′-ACGUGACACGUUCGGAGAATT-3′), and anti-miR control (sense 5′-UUCUCCGAACGUGUCACGUTT-3′) were purchased from GenePharm (Shanghai, China). MiR-212-5p, anti-miR-212-5p and corresponding control miR were complexed, respectively, with Lipofectamine 3000 (Invitrogen Life Technologies, United States) according to manufacturer’s instructions. SH-SY5Y cells were transfected with MiR-212-5p mimics, miR-212-5p inhibitor, and corresponding control miR for 6 h, followed by followed by stimulation with MPP⁺ (100 μM) for 24 h.
SH-SY5Y cells were transfected with pcDNA3.1(-)-3HA-p53 plasmid (0.5 μg, 1 μg, and 2 μg, respectively) using Lipofectamine 3000 described previously (Shan et al., 2016 (link)). The pcDNA3.1(-)-Flag is used as the mock. In addition, SH-SY5Y cells were transfected with 0.5 μg mTag-Wasabi-LC3 plasmid and manipulated in accordance with the instruction of Lipofectamine 3000. Six hours after transfection, transfection reagents were replaced by nutrient medium containing 10% FBS and cultured overnight.

miRNA overexpression was achieved by transfecting cells with a miRNA mimic (a synthetic RNA oligonucleotide duplex mimicking miRNA precursor), whereas knockdown was achieved by transfecting cells with a miRNA inhibitor (a chemically modified single-stranded antisense oligonucleotide designed to specifically target mature miRNA). Synthetic RNA molecules, including pre-miR-143, pre-miR-145, anti-miR-143, anti-miR-145 and scrambled negative control RNA (pre-miR-control and anti-miR-control), were purchased from GenePharma (Shanghai, China). MCF-7 cells were seeded in 6-well plates and transfected with Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 70% confluent. For overexpression of miRNAs, 100 pmol of pre-miR-143, 100 pmol of pre-miR-145 or 50 pmol of both pre-miR-143 and pre-miR-145 were used. For knockdown of miRNAs, 100 pmol of anti-miR-143, 100 pmol of anti-miR-145 or 50 pmol of both anti-miR-143 and anti-miR-145 were used. After 6 h, the medium was changed to DMEM that was supplemented with 2% fetal bovine serum. The cells were harvested 24 h or 48 h after the transfection for the isolation of total RNA or protein, respectively.

The siRNAs specifically targeting SNHG15 (si-SNHG15–1, si-SNHG15–2 and si-SNHG15–3), scrambled negative control (si-control), pcDNA-SNHG15, empty pcDNA vector (vector), miR-141 mimics (miR-141), mimics negative control (miR-control), anti-miR-141 and anti-miR-control were commercially synthesized by Genepharma (Shanghai, China). For transient transfection, U2OS and MG63 cells were plated into six-well plates (2 × 10⁵/well) and routinely maintained for 24 h at 37 °C. Then the cells were transfected with siRNAs, pcDNA-SNHG15, vector, miR-141, miR-control, anti-miR-141, anti-miR-control, si-SNHG15 + anti-miR-141, si-SNHG15 + anti-miR-control, pcDNA-SNHG15 + miR-141, or pcDNA-SNHG15 + miR-control by Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Subsequent experiments were performed at 48 h post transfection. The sequences of si-RNAs, anti-miR-141 are as follows: si-SNHG15–1 (Sense: 5′-CAG GTA GAC CGT GCA CGT AA-3′, Anti-sence:3′-CCT TGA TGC GTT GCC AGC AGA-5′), si-SNHG15–2 (Sense: 5′-CCG TGC GTA AAC GTT TGC CA-3′, Anti-sence: 3′-TGG CGG TAA CGT AAA TGC G-5′), si-SNHG15–3 (Sense: 5′-ACG GTG GCA ACG TGC GTG GCC A-3′, Anti-sence: 3′-GCC TGC AAC GGT GCA AAT GCG-5′), anti-miR-141 (CCA UCU UUA CCA GAC AGU GU UA).

Overexpression of miR-23a/b was achieved by transfecting gastric cancer cells with miRNA mimics (synthetic RNA oligonucleotides mimicking precursors of miR-23a/b). Knockdown of miR-23a/b was achieved by transfecting with miRNA inhibitors (chemically modified single-stranded antisense oligonucleotides designed to specifically sequester mature miR-23a/b). Synthetic miR-23a/b mimics (pre-miR-23a/b), inhibitors (anti-miR-23a/b) and scrambled negative control RNAs (pre-miR-control and anti-miR-control) were purchased from GenePharma (Shanghai, China). MKN-45 and AGS cells were seeded in 6-well plates using RPMI 1640 medium supplemented with 10% FBS. The cells were transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) using Opti-MEM Reduced Serum Medium (Gibco, Carlsbad, CA, USA) on the following day when the cells were ~60–70% confluent. For each well, equal amounts of pre-miR-23a/b, pre-miR-control, anti-miR-23a/b or anti-miR-control were used. After 6 h, the medium was changed to RPMI 1640 supplemented with 2% FBS. The cells were collected at 24 or 48 h after transfection and subjected to analysis by quantitative RT-PCR or western blotting.

Knockdown or overexpression of miR-221/222 was achieved by transfection with miRNA inhibitors or miRNA mimics. Synthetic miR-221/222 mimics (pre-miR-221/222), inhibitors (antimiR-221/222), and scrambled negative control RNAs (pre-miR-control and anti-miRcontrol) were purchased from GenePharma (Shanghai, China). MCF-7, MDA-MB-231, and SKBR3 cells were seeded in six-well plates using RPMI 1640 medium supplemented with 10% FBS. The cells were transfected with Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.) according to the manufacturer’s protocol. For each well, equal amounts of pre-miR-221/222, pre-miR-control, anti-miR-221/222, or anti-miRcontrol were used. After 6 h, the medium was changed to RPMI 1640 supplemented with 2% FBS. The cells were collected at 24 or 48 h after transfection and used for subsequent analysis.

miRNA overexpression was achieved by transfecting cells with miRNA mimics, whereas knockdown was achieved by transfecting cells with a miRNA inhibitor according to previous publications42 (link). miRNA overexpression was achieved by transfecting cells with miRNA mimics (a synthetic RNA oligonucleotide duplex mimicking miRNA precursor), whereas knockdown was achieved by transfecting cells with a miRNA inhibitor (a chemically modified single-stranded antisense oligonucleotide designed to specifically absorb target miRNA). Synthetic RNA molecules, including pre-miR-221, anti-miR-221 and scrambled negative control RNA (pre-miR-control and anti-miR-control), were purchased from GenePharma (Shanghai, China). MCF-7 or MDA-MB-231 cells were seeded in 6-well plates and transfected with Lipofectamine 2000 (Invitrogen) on the following day when the cells were approximately 70% confluent. Cells were transfected with 50 pmol pre-miR-221 or anti-miR-221 to overexpress or knockdown cellular miR-221. After 6 h, the medium was changed to DMEM or L-15 medium that was supplemented with 2% fetal bovine serum. The cells were harvested 24 or 48 h posttransfection for RNA and protein analysis.