Fig. 5

CircItgb5 serves as a sponge for miR-96-5p. A Total RNA was extracted from PASMCs and immunoprecipitated by AGO2 or IgG antibody. CircItgb5 in the immunoprecipitate was determined by RT-PCR using agarose gel electrophoresis. B Schematic illustration showed the binding of miRNAs on circItgb5 using CirMIR1.0. C MiRNAs interacted with circItgb5 were predicted by RNAhybrid and miRanda. D A cytoscape drawing circItgb5-miRNAs interaction network. Eight miRNAs were previously reported to be related to PH (shown in orange). E The selected eight miRNAs (from D) were validated by RT-qPCR. *0.01 ≤ P ≤ 0.05 versus Lsh-NC. F Schematic drawing showed the putative binding sites for miR-96-5p on circItgb5. G RT-qPCR was used to detect the relative level of miR-96-5p. *0.01 ≤ P ≤ 0.05 versus mimic control. H, I The luciferase activities of wild-type or mutant pGL3-circItgb5 in 293 T cells transfected with miR-96-5p mimic or mimic control. The red letter represents mutated sequences. The mutation principle: G to U, U to G, C to A, A to C. *0.01 ≤ P ≤ 0.05 versus mimic control. J Relative miR-96-5p level was detected in lungs of rats and PASMCs after the corresponding treatment. **0.001 ≤ P ≤ 0.009 versus control, ^#0.01 ≤ P ≤ 0.05 versus Lsh-NC. All data are presented as means ± SD (n = 3 biological replicates)