Lipofectamine 2000

The mouse microglial cell line BV2 and mouse hippocampal neuronal cell line HT22 were obtained from Procell Life Science & Technology Co.,Ltd. (Wuhan, China) and cultivated in their specific medium (Procell) comprising 10% fetal bovine serum in an moist incubator containing 5% CO₂ at 37°C. The siRNA targeting XIST (si-XIST) and the siRNA control (si-NC); miR-107 inhibitor (anti-miR-107), control (anti-NC), and LY294002 (a PI3K/Akt signaling inhibitor) were obtained from Genechem. Lipofectamine 2000 (Solarbio) was used to implement cell transfection according to the manufacturer’s instructions.
For preparation of Aβ_1–42 oligomers, Aβ_1–42 was dissolved in 100% hexafluoroisopropanol into 1 mM solution, and then allowed to evaporate at 25°C for 2 h to remove residual hexafluoroisopropanol. The peptide film was dissolved into 1 mM solution with DMSO. This solution was diluted to 100 μM solution in PBS and incubated for 24 h at 4°C. To mimic the Aβ_1–42-induced microglia-mediated neurotoxicity in AD, the BV2 cells were transfected with si-NC or si-XIST and then stimulated with Aβ_1–42 (10 μM) for 24 h, and afterward the mediums were collected as conditioned mediums. The conditioned mediums were used to incubate HT22 cells for 48 h. To inhibit the PI3K/Akt signaling pathway, BV2 cells were coped with LY294002 (10 μM; Genechem) for 1 h before transfection.

Human gastric adenocarcinoma cell lines AGS and MGC-803 were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, CAS (China). The cells were cultured in 89% DMEM (Gibco) (supplemented with 10% FBS (Gibco) and 1% streptomycin-penicillin (Gibco)) and incubated at 37°C with 5% CO₂. Linc-ROR silencing vector possessing puromycin resistance was constructed by Genechem (Shanghai, China). Lentiviral particles were synthesized by co-transfection with lentiviral packaging plasmids and Linc-ROR silencing vector into 293T cells. 40 nM of miR-212-3p mimics or antagomir (Sangon Biotechnology Company, Shanghai, China) were transfected into the human gastric adenocarcinoma cells (5×10⁵ cells/well in six-well plate) using Lipofectamine 2000 (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China). The cells transfected with nonsense miRNA served as the control group.

The miR‐873 inhibitor and empty vector (mock) were obtained from Shanghai Tuoran Biology Co., Ltd (Shanghai, China). The negative control siRNA (MBS 8241404) and GLI1 siRNA (MBS8208749) were purchased from MyBio Source (San Diego, CA, USA). PC9 cells were transfected with mimics or siRNA (50 pmol) using Lipofectamine 2000 (Solarbio) for 48 hours according to a standardized method.
Using data from the miRanda and Targetscan websites, we predicted the binding site of the GLI1 gene to miR‐873. The 3'UTR of GLI1 with affinity for miR‐873 and a mutant reporter were cloned to the downstream of firefly luciferase of psiCHECK‐2 vector (Hibio, Hangzhou, China). MiR‐873 was then co‐transfected into HEK293T cells using Lipofectamine 2000. After transfection, luciferase activity analysis was performed.

The potential complementary sequences of circ-ACACA and miR -1183 were forecasted by circular RNA Interactome (24 (link)). The wild type (WT) sequence of circ-ACACA harboring the binding sites of miR-1183 was inserted into the pGL3 vector (Promega Corporation) to establish the luciferase reporter vector WT-circ-ACACA. Similarly, the mutant (MUT)-circ-ACACA reporter vector was established by mutating the potential target sites of miR-1183. Then, the luciferase reporter vectors (100 ng) were cotransfected with 50 ng miR-1183 or miR-NC into A549 and H1299 cells for 24 h using Lipofectamine 2000 (Beijing Solarbio Science & Technology, Co., Ltd.). Firefly luciferase activities were normalized by comparison with Renilla luciferase. The Dual-Glo Luciferase Assay System kit (Promega Corporation) was utilized to measure luciferase activity.