Sirna

BV2 cells or primary cultured astrocytes were transiently transfected with siRNA targeting GAS6 (100 nM), siRNA targeting AXL (100 nM), or control siRNA (Genomeditech, Shanghai, China) using 7.5 μL of Lipofectamine™ 3000 (Invitrogen, California, USA) according to the manufacturer’s protocol. The cells were incubated in Opti-MEM for 48 h, then changed to astrocyte culture and continually incubated for 24 h, and the GAS6 knockdown BV2 supernatant fluid was collected for the next step. The sequences used for GAS6 knockdown were sense 5-CGGAGUAUUUCUAUCCACGAU-3 and antisense 5-AUCGUGGAUAGAAAUACUCCG-3; the sequences used for AXL knockdown was sense 5-GGAGACCCGUUAUGGAGAATT-3 and antisense 5-UUCUCCAUAACGGGUCUCCTT-3.

PAX6 was silenced in A549 cells with siRNA (RiboBio Co., Ltd., Guangzhou, China), according to the manufacturer’s instructions; the target sequences were as follows: si-h-PAX6_001, GCGACTCCAGAAGTTGTAA; si-h-PAX6_002, GCAGACGGCATGTATGATA; si-h-PAX6_003, GCTTCACCATGGCAAATAA. The corresponding negative control was purchased from RiboBio Co., Ltd. To stably knock down PAX6 in cells, siRNA targeting the si-h-PAX6_002 coding sequence 5′-GCAGACGGCATGTATGATA-3′ was designed and inserted into a pGMLV-SC5RNAi lentiviral vector (Genomeditech Co., Ltd, Shanghai, China); a scramble siRNA was used as a negative control.
To overexpress PAX6 in cells, an expression construct was generated by subcloning PCR-amplified, full-length human PAX6 cDNA into a pGMLV-CMV-PAX6 lentiviral vector (Genomeditech); an empty vector was used as the negative control. These procedures were performed, as described previously.^{24 (link)} The knockdown and overexpression efficiencies were evaluated by quantitative reverse transcription PCR (RT-qPCR) and western blotting.

The NSCLC cell line A549 was obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Non-fetal-derived human brain microvascular endothelial cells (HBMECs) were purchased from Bioleaf Biotechnology (Shanghai, China). A549 and HBMECs were grown in Roswell Park Memorial Institute-1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum at 37 °C with 5% CO₂. The lnc-MMP2-2 overexpression and silencing lentivirus and their control lentivirus were packaged by Genomeditech (Shanghai, China). EPB41L5-targeting siRNA and scramble control siRNA were obtained from Ribobio (Guangzhou, China). The EPB41L5-targeting sequences were as follows: siRNA#1, 5′-GGATCACGATTTA GATATA-3′; siRNA#2, 5′-GTCCTGAACTTGTCTCAGA-3′; siRNA#3: 5′-CGACTATTTTGGTCTGAG A-3′. The overexpression plasmid (pcDNA3.1-EPB41L5) and the empty vector (pcDNA3.1) were obtained from Genomeditech (Shanghai, China). miR-1207-5p antagomir, antagomir control, and miR-1207-5p agomir and agomir control were purchased from Ribobio (Guangzhou, China). Cell transfection was performed using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. For exosome transfection, lnc-MMP2-2 Smart Silencer (RiboBio Co, Ltd., Guangzhou, China) were loaded in exosomes using the Exo-Fect Exosome Transfection Kit.