Pmirglo vector

To construct vectors for ceRNA study, public online tools including ENCORI (http://starbase.sysu.edu.cn/index.php)⁴³ and TargetScan (http://www.targetscan.org/vert_72/)⁴⁴ were applied to analyze the recognizing sequences of miR‐223‐3p in GAS5 and P2Y12. The sequences of the GAS5 and P2Y12 3'‐untranslated region (3'‐UTR) containing the predicted miR‐223‐3p binding sequences were amplified by PCR with a DNA template from a healthy volunteer. The PCR products were purified from agarose gel. They were then subcloned into the NheI (Thermo Fisher Scientific, MA, USA) and XbaI (Thermo Fisher Scientific, MA, USA) restriction sites downstream the firefly luciferase (Luc) gene of the pmirGLO vector (YouBio, Changsha, China). These wild‐type plasmids were designed as pmirGLO‐GAS5‐wt and pmirGLO‐P2Y12‐wt. Five base pair of the predicted recognizing sequences of miR‐223‐3p in GAS5 and P2Y12 3'‐UTR were mutated to generate the corresponding mutant plasmids, which were named as pmirGLO‐GAS5‐mut and pmirGLO‐P2Y12‐mut, respectively. The PCR primers were synthesized by Sangon Biotech (Shanghai, China), and the detailed sequences information is listed in Table S1.

Endothelial Cell Growth Supplement (ECGS) and Matrigel were procured from BD technologies (New Jersey, USA). Fetal bovine serum (FBS), DMEM, penicillin, and streptomycin were obtained from HyClone Laboratories Inc. (Logan, USA). Lipofectamine®3000, pmirGLO vector, pcDNA3.1 vector, and Trizol reagent were purchased from Invitrogen (Waltham, USA). The cell counting kit (CCK)-8 test kit was acquired from Dojindo Corp. Ltd. (Kyushu, Japan). Dual-Luciferase Reporter assay system was obtained from Promega Corp. Ltd. (Madison, USA). The protein extraction kit was acquired from Beyotime Technologies (Shanghai, China). PVDF membranes from Millipore (Billerica, US), electrochemical luminescence (ECL) kit from Tanon (Shanghai, China), antibodies against SUSD2, ACTIN, VEGF and CD31 from Cell Signaling Technology (Danvers, US), and Annexin V‑propidium iodide (PI) dual-staining apoptosis test kit from Nanjing Keygen Biotech Co., Ltd., (Nanjing, China) were obtained.

Mouse SOCS1 3′ UTR fragment wild type (SOCS1-Wt) containing the potential binding sites of mmu-miR-222-3p and its corresponding mutant (SOCS1-Mut) were cloned by PCR methods into pmirGLO vector (Invitrogen). SOCS1-Mut was replaced UGUAGC with ACAUCG in SOCS1 sequence. Splenocytes were transfected according to the following groups: SOCS1-Wt+miR-NC mimic (miR-NC), SOCS1-Wt+miR-222-3p mimic (miR-222-3p), SOCS1-Mut+miR-NC, and SOCS1-Mut+miR-222-3p; or SOCS1-Wt+miR-NC-in, SOCS1-Wt+miR-222-3p-in, SOCS1-Mut+miR-NC-in, and SOCS1-Mut+miR-222-3p-in. The pmirGLO vector itself provided a strong Renilla luciferase signal acting as a control reporter for normalization. After 24 h incubation, cells were collected to measure Firefly and Renilla luciferase activity using the dual-luciferase reporter assay system (Promega, Madison, WI, USA). All data were the average of at least three independent transfections, and were normalized to SOCS1-Wt+miR-NC/miR-NC-in.

First, we predicted the downstream targets of NEAT1 and miR-181a using a bioinformatic algorithm. Next, we amplified and inserted 3′UTR of wild-type NEAT1 (NEAT1-WT), mutant-type NEAT1 (NEAT1-MUT), wild-type GPD1L (GPD1L-WT), and mutant-type GPD1L (GPD1L-MUT) into the pmirGLO vector (Invitrogen, USA). For the luciferase reporter assay, these plasmids were co-transfected with miR-181a mimic or mimic-NC into cells using Lipofectamine 3000 (Invitrogen, USA). At 48 h after transfection, the Dual-Luciferase Reporter system (Promega, USA) was used to measure luciferase activity.