Pgl3 vector

To construct the Dmd minigene, exons 50, 51, and 52 of the mouse Dmd gene were amplified by PCR. The PCR products were purified using the EasyPure Quick Gel Extraction Kit (Transgen #EG101-01) and constructed into the pcDNA 3.1 vector through HindIII (New England Biolabs (NEB) #R3104) and EcoRI (NEB #R3101) restriction site using the ClonExpress II One Step Cloning Kit (Vazyme #C112). To construct a vector expressing linear AS-RNA, the PGL3 vector (Addgene #107721) was linearized by BsaI (NEB #R3733) and EcoRI (NEB #R3101). Antisense sequences of less than 50 base pairs (bp) were chemically synthesized, annealed, and ligated to the linearized PGL3 vector by T4 DNA ligase (Vazyme #C301). Antisense sequences greater than 100 bp were amplified by PCR and ligated to the linearized PGL3 vector using the ClonExpress II One Step Cloning Kit (Vazyme #C112). The transcription of AS-RNA was driven by the U6 promoter. DNA, including a Twister P3 U2A, a 5′ ligation sequence, a 3′ ligation sequence, and a Twister P1, was synthesized and cloned into a linearized PGL3 vector to construct the AS-circRNA expression vector. Antisense sequences of different lengths were ligated between the 2 ligation sequences by the method described above. The sequence of U7 has been published previously [10 (link)]. Primers and antisense sequences are listed in Table S2.

For the miR‐181a‐5p sponge vector, 9 copies of the miR‐181a‐5p sponge sequence and WPRE were cloned into the Fuw vector (Addgene). For the shPTEN vector, short hairpin RNAs (shRNAs) targeting PTEN were cloned into the pLKO.1 vector (Addgene). For the luciferase reporter vector, full‐length Pten 3'UTR or 3'UTR with 1–3 mutant miR‐181a‐5p binding sites were cloned into the pGL3 vector (Addgene). Mutant vectors changed the miR‐181a‐5p target site in the PTEN 3'UTR from 5′‐TGAATGT‐ 3′ to 5'‐ACATTCA‐3′. For the AAV miR‐181a‐5p OE vector, the miR‐181a‐5p sequence, WPRE, ubiquitin promoter sequence, and a GFP fluorescent reporter were cloned into the AAV vector (VPK‐410, Cell Biolabs). All vectors were verified by DNA sequencing. Detailed primer sequences are listed in Table S1.

Two short hairpin RNAs (shRNAs) aimed to down-regulate the expression of FOXD3-AS1 were designed by BLOCK-iT™ RNAi Designer (ThermoFisher Scientific, USA). The nucleotide sequences of shRNA#1 and shRNA#2 were shown in Supplemental Table 1. The full-length of lncRNA FOXD3-AS1 was cloned from SUNE1 cell cDNA and cloned into the pcDNA3.1(+) or pcDNA3.1(-) to construct over expression plasmid. The wild-type and mutant-type promoters of YBX1 were cloned into the pGL3 vector (Addgene, USA). Knockdown or overexpression of FOXD3-AS1 was performed through transiently transfection using lipotamine 3000. The shFOXD3-AS1 or its scramble control (shCtrl) plasmid was co-transfected into HEK293T cells with the lentivirus packaging plasmids psPAX2 and pMD2G (Addgene, USA). The lentivirus supernatant was collected after 48h incubated and used to infect SUNE1 cells, and positive stably transfected cells were selected and maintained using puromycin.