Figure 3 Xist competitively binds to miR-101-3p to regulate HMGB3. (A) RT-qPCR showed that mRNA expression of HMGB3 in the BPD group was evidently higher than that in the control group. (B) Starbase verified the sequence binding sites between Xist and miR-101-3p and between miR-101-3p and HMGB3. (C) Dual-luciferase reporter gene assay verified the targeting relations between Xist and miR-101-3p and between miR-101-3p and HMGB3. (D) RNA pull-down assay further affirmed the targeting relation between Xist and miR-101-3p. (E) RT-qPCR assessed miR-101-3p expression in mice with different treatment. (F, G) RT-qPCR and Western blot analysis showed that mRNA expression and protein level of HMGB3 in BPD mice were decreased with overexpressed miR-101-3p. Two-way ANOVA was used to assess data in panels A and C, one-way ANOVA was used to assess data in panels D and E. Tukey’s multiple comparisons test was applied for post hoc test. The t test was used for analyzing data in remaining panels. ** p<0.01, ## p<0.01, n=3. Xist – X inactive specific transcript; miR – microRNA; HMGB3 – high-mobility group protein B3; RT-qPCR – reverse transcription-quantitative polymerase chain reaction; BPD – bronchopulmonary dysplasia; ANOVA – analysis of variance.