Pcdna3.1 vector

Two short hairpin RNA (shRNA) sequences targeting PVT1 (sh-PVT1 1 and sh-PVT1 2) or negative control (sh-NC) were designed by Genepharm Co. Ltd. (Shanghai, China). After annealing, double strands of shRNA were inserted into lentiviral pU6-Luc-Puro vector (Genepharm Co. Ltd.). pcDNA3.1 vectors containing full length PVT1 or mutant PVT1 were obtained from RioBio (Guangzhou, China) and transfected into cells using Lipofectamine 2000 (Thermo Fisher Scientific). The miR-16-5p mimics, inhibitor and negative control were synthesized by Genepharma (Shanghai, China) and transfected into cells using Lipofectamine 2000 (Thermo Fisher Scientific).

Four human EC cell lines (KLE, AN3CA, HEC-1-B, and Ishikawa) and human normal endometrial stromal cells (HESCs) were obtained from Cell Culture Center, Chinese Academy of Medical Sciences (Shanghai, China). Cells culture medium was composed of 90% Dulbecco’s modified Eagle’s medium and 10% fetal bovine serum. Cells culture conditions were: 5% CO₂, 37°C, and 95% humidity. The pcDNA3.1 vector expressing HOXB-AS1, HOXB-AS1 siRNA, the miR-149-3p mimics, miR-149-3p inhibitor, and their corresponding negative controls were synthesized by RiboBio (Guangzhou, China). HEC-1-B and Ishikawa cells were harvested at 75-85% confluence. Lipofectamine 3000 (Invitrogen, Thermo Fisher Scientific, Inc.) was used to transfect HOXB-AS1 vector, HOXB-AS1 siRNA, miR-149-3p mimics, and miR-149-3p inhibitor into HEC-1-B and Ishikawa cells. After 24 h, cells were harvested to perform the following studies.

Five human glioblastoma cell lines (U251, LN229, U118, U87, and T98G) were purchased from the Chinese Cell Repository (Shanghai, China). Primary GBM cells (pGBM-1) that derived from a GBM surgical specimen were maintained in DMEM supplemented with 10% FBS. NHAs were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). Oligonucleotide sequences of cMELK were as follows: #shRNA-1: 5′-CATGGTTCTTGAGGTTCTTTT-3′, #shRNA-2: 5′-GGTTCTTGAGGTTCTTTTTCT-3′, and #shRNA-3: 5′-CTTGAGGTTCTTTTTCTAATT-3′. For regulating cMELK the sequence was cloned into pcDNA3.1 vector (RioBio, Guangzhou, China), namely pcDNA-cMELK, with pcDNA-NC as control. miR-593 inhibitors (anti-miR-593), miR-593 mimics (miR-593), and their paired control (anti-NC and miR-NC) were obtained from Genecheom (Shanghai, China). Lentivirus vectors with shRNAs or shCONT and anti-miR-593 or anti-NC were purchased from Genechem (Shanghai, China). Stable U87 and pGBM-1 cell lines were established by lentiviral infection and puromycin selection following the manufacturer’s instructions.

CRC cell lines were acquired from Shanghai Fuxiang Biotechnology Co., Ltd, and cultivated in RPMI‐1640 medium supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin (Hyclone) in a humidified incubator at 37°C with 5% CO₂. miR‐665 mimic/inhibitor and negative controls (miR‐NC or NC inhibitor), and pcDNA.3.1 vector and pcDNA3.1‐E2F3 expression vector were purchased from RiboBio Co. Ltd. Cell transfection was performed using Lipofectamine® 2000 reagent (Thermo Fisher Scientific). In 6‐well plates, 60% confluent cells were transfected with 50 nM of miRNA mimic or inhibitor or 6 μg of plasmid according to manufacturer's instruction. Transfected cells were subjected to subsequent analysis 48 h post‐transfection. To generate cells with stable hsa_circ_0081069 knockdown, pLKO.1‐Puro lentiviral vector was used for shRNA‐mediated gene silencing. Lentivirus with hsa_circ_0081069 shRNA or control shRNA (sh‐NC) were constructed by GenePharma Co. Ltd. 2 × 10⁵ cells were seeded in a 24‐well plates at 50%~60% confluence and the cells were infected with recombinant lentivirus at a MOI (multiplicity of infection) = 5, in the presence of 10 μg polybrene (Sigma, tr‐1003‐g). Infected cells were selected with 1.0 μg/mL puromycin for 2 week. qPCR was performed to confirm the efficiency of shRNA‐mediated knockdown.