293t cells

The target circLphn3 sequences or target mRNA (ZO-1) 3′ untranslated region (UTR) sequences were cloned downstream of hRluc in psiCHECK2 plasmid to construct vectors containing potential miR-185-5p-binding sites. The reporter vectors (HanBio) and miR-185-5p mimics (HanBio) were co-transfected into 293T cells (HanBio) and the renilla luciferase activity (reporter gene) and firefly luciferase activity (internal reference gene) were determined by the Dual-Lumi™ II Luciferase Reporter Gene Assay Kit (BeyoTime). Relative luciferase activity = renilla luciferase activity/firefly luciferase activity.

For ectopic expression, plasmid pHBAd-MCMV-GFP-NONMMUG014387 and pHBAd-MCMV-GFP were transfected into Schwann cells. Full-length NONMMUG014387 (Hanbio Biotechnology Co., Shanghai, China) was subcloned into pHBAd/MCMV/GFP vector (Hanbio Biotechnology Co.) and regulated by the murine cytomegalovirus (MCMV) promoter. Green fluorescent protein (GFP) was regulated by the CMV promoter. After sequencing, adenoviral vector DNAs and packaging vectors were transfected into 293T cells (Hanbio Biotechnology Co.). Lipofiter™ (Hanbio Biotechnology Co.) was used for transfection. Forty-eight hours after cotransfection, adenovirus in supernatants was collected and filtered through 0.45 μm filters. Finally, adenovirus was purified using ultracentrifugation and titer determination. The final concentration of recombinant Ad-GFP was 2 × 10¹⁰ PFU/mL, and recombinant Ad-NONMMUGO148387 was 1 × 10¹⁰ PFU/mL. Schwann cells were then transfected with Ad-GFP and Ad-NONMMUGO148387 for 36 hours, and subsequently reseeded for total RNA extraction. Polymerase chain reaction (PCR) was used to detect expression of NONMMUGO148387.