Siha htb 35

HPV16-positive cervical cancer cell lines (SiHa (HTB-35) and CaSki (CRL-1550)), HPV-negative cervical cancer cell line (C33A, HTB-31) and human embryonic kidney 293T cell line (HEK, CRL-3216) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). They were maintained in DMEM high glucose medium (cat no. SH30022.02, Hyclone) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA) and 100 U/mL penicillin and streptomycin (Cytiva, Lane Cove West, Australia). All cells were cultured at 37 °C and 5% CO₂. The present study was approved by the Institutional Review Board of the Faculty of Medicine, Chulalongkorn University (COE No. 005/2022, IRB No. 027/65), and the Institutional Biosafety Committee (IBC) of the Faculty of Medicine, Chulalongkorn University (MDCU-IBC026/2021).

Human normal immortalized cervical epithelial cell line (H8) was purchased from Institute of Preclinical Medicine, Peking Union Medical University (Beijing, China), and CC HeLa (CCL-2) and SIHA (HTB-35) cells were acquired from the American Type Culture Collection (ATCC, Rockville, MD, USA). The aforementioned cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco BRL, Gran Island, NY, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C with 5% CO₂ and 95% air.
Small interference RNA (siRNA) targeting PVT1 (si-PVT1) and its negative control (si-NC), miR-503 inhibitor (anti-miR-503) and its negative control miR-NC inhibitor (anti-miR-NC), and miR-503 mimic (miR-503) and its negative control miR-NC mimic (miR-NC) were designed and synthesized by GenePharma Co., Ltd (Shanghai, China). For upregulation of PVT1 and ARL2, corresponding overexpression plasmids pcDNA-PVT1 (PVT1) and pcDNA-ARL2 (ARL2) were constructed by Hanbio Biotechnology Co., Ltd (Shanghai, China), with non-targeting plasmid (pcDNA) as negative control. The aforementioned oligonucleotides or plasmids were transfected into CC HeLa and SIHA cells using Lipofectamine 3000 (Life Technologies Corporation, Carlsbad, CA, USA) based on the user’s manual.