Sirna2

Human PTC cell lines (SW579 and TT) were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cell lines were cultured in RPMI-1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen) at 37°C in a humid atmosphere containing 5% CO₂. Three siRNAs were initially designed and used in our study. The siRNA-1, siRNA-2, and siRNA-3 sequences were purchased from RiboBio (Guangzhou, Guangdong, China) and are as follows: siRNA-1 (Si-1, 5′-AGAAAGUGUUGUUGAAUAACU-3′), siRNA-2 (Si-2, ′-GGAUGGUCAAGUUCUAGAACA-3′), siRNA-3 (Si-3, 5′-UUCUAGAACUUGACCAUCCUU-3′), and negative control siRNA (si-NC; 5′- UUCUCCGAACGUGUCACGUTT-3′). The TT and SW579 cells were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 48 h of transfection, lncRNA NONHSAT129183 expression was determined using qPCR. Preliminary qPCR results indicated that siRNA-2 had a significantly higher silencing efficiency than siRNA-1 and siRNA-3. Thus, siRNA-2 was chosen for the cellular physiological function assays.

The siRNAs (siRNA1 and siRNA2) for knockdown of H19 and the scramble siRNA for negative control were purchased from RiboBio (Guangzhou, People’s Republic of China). The constructed H19-overexpressing vectors (pcDNA3.1-H19) and pcDNA3.1 empty vectors were obtained from GenePharma (Shanghai, People’s Republic of China). For siRNA and vector transfection, cells were seeded into six-well plates at a density of 5 × 10⁴ cells/well to reach about 50%–80% confluence for transfection. The transient transfection was performed by using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. At 24 h after transfection, cells were processed for further experimentation.

The RT4 and T24 human BC cell lines (The Second Affiliated Hospital of Kunming Medical University, Yunnan Institute of Urology, Kunming, China) were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Invitrogen RPMI-1640 medium, PBS, Opti-MEM I, Lipofectamine 2000 and glutamine were purchased from Thermo Fisher Scientific, Inc.
Two siRNA sequences targeting LASS2 [National Center for Biotechnology Information (NCBI) accession nos. NM013384, NM181747 and NM022075] were synthesized by Guangzhou RiboBio Co., Ltd. (Guangzhou, China): siRNA-1, 5′-GGCUAUUACUUCUUCAAUUTT-3′ and siRNA-2, 5′-CAGUAUUGGUACUACAUGATT-3′. Unspecific control siRNA (si-NC) was also obtained from Guangzhou RiboBio Co., Ltd. The siRNAs were transfected into the RT4 cell line using Lipofectamine 2000, according to the manufacturer's protocol. siRNA was used for the in vitro experiments, and shRNA was used for cell selection and then for the Xenograft model.