Fig. 2

Nuclear lncRNAs direct interact with miRNAs in the nucleus of sow GCs. a miRNA response elements (MREs) of four miRNAs in the corresponding nuclear lncRNAs were predicted by RNAhybrid. For LOC102164325, three MRE motifs (MRE1, MRE2 and MRE3) of miR-339 were detected, and the minimum free energy (mfe) of MRE2 and MRE3 motifs binding to LOC102164325 are shown in Additional file 2: Fig. S3a, b. b Subcellular localization of miRNAs in sow GCs. Levels of miRNAs in nuclear and cytoplasm fractions isolated from sow GCs were detected by qPCR. GAPDH and U6 were used as marker genes in the cytoplasm and nucleus, respectively. n = 3. c Schematic showing the construction of reporter vector. LncRNAs containing MRE motif was amplified and cloned into a pGL3-basic vector. Diagram of reporter vectors of LOC102164325 with the other two MRE motifs are shown in Additional file 2: Fig. S3c. WT, wild-type. MT, mutant-type. WT1, wild-typed MRE1 of miR-339. MT1, mutant-typed MRE1 of miR-339. d Luciferase assay. GC line KGN were co-transfected with reporter vectors and the mimics for the corresponding miRNAs, and luciferase activity was detected. Luciferase activity of reporter vectors of LOC102164325 with MRE2 and MRE3 motifs are shown in Additional file 2: Fig. S3c. n = 3. e RNA pull-down assay. The physical interaction between lncRNAs and the corresponding miRNAs was detected. lncRNA-RNA complexes were pulled down from nuclear RNAs in sow GCs by using biotinylated probes for lncRNAs, levels of lncRNAs and miRNAs were determined by qPCR. PD, pull-down. Data for miR-378 experiments were analyzed using Moses Extreme Reactions in SPSS v26.0 software. n = 3 for LOC100626841 and LOC102160522, n = 2 for LOC100512907 and LOC102164325. Values are means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant