Scrambled mimic control mir nc

NSCLC cell lines (H23, A549, H1299 and SPC-A1) and a normal human bronchial epithelial cell line (16HBE) were obtained from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, Logan City, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1% penicillin/streptomycin at 37°C in a humidified incubator with 5% CO₂.
The small interfering RNAs (siRNAs) targeting circMYLK (si-circMYLK) and GLUT3 (si-GLUT3), the negative control siRNA (si-NC), miR-195-5p mimic and the scrambled mimic control (miR-NC) were designed and synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). The synthesized circMYLK or GLUT3 gene fragment was inserted into pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) to gain the circMYLK-overexpression or GLUT3-overexpression plasmid. An empty vector was used as a negative control. Cells were cultured to 70–80% confluence, and cell transfection was then performed using Lipofectamine 2000 (Invitrogen).

To construct UCA1 overexpression plasmid, the full length of the UCA1 cDNA sequence was amplified, cloned into pcDNA3.1 vector (Invitrogen), and sequenced and named as pcDNA3.1-UCA1 (UCA1). Three specific small interference RNAs (siRNAs) targeting UCA1 (si-UCA1#1, si-UCA1#2, and si-UCA1#3) and scrambled siRNA control (si-NC) were obtained from GenePharma (Shanghai, China). miR-143 mimic (miR-143) and scrambled mimic control (miR-NC) were purchased from RiboBio (Guangzhou, China). All these plasmids and oligonucleotides were transfected into cells by Lipofectamine 2000 reagent (Invitrogen) following the manufacturer’s instructions.

To construct the UCA1 overexpression plasmid, the full length of UCA1 cDNA sequence was amplified, cloned into pcDNA vector (Invitrogen), and sequenced, named as pcDNA-UCA1 (UCA1). Three specific siRNAs targeting UCA1 (si-UCA1#1, si-UCA1#2, and si-UCA1#3) and scrambled siRNA control (si-NC) were obtained from GenePharma (Shanghai, China). The lentivirus vectors containing UCA1 overexpression plasmid (Lv-UCA1) or NC vector (Lv-NC) were amplified and cloned by GeneChem (Shanghai, China). The miR-143 mimic (miR-143) and scrambled mimic control (miR-NC) were purchased from RiboBio (Guangzhou, China). All these plasmids and oligonucleotides were transfected into cells by Lipofectamine 2000 reagent (Invitrogen), following the manufacturer’s instructions.