Figure 5. Circ‐myh8 promotes the pulmonary artery smooth muscle cells (PASMCs) proliferation and cell‐cycle progression by upregulating the transcription activity of HIF1α.

A, Circular RNA (circRNA) sequencing and pathway analysis of circ‐myh8 mediated mRNA expression profiles. B, Representative images and summarized data of HIF1α protein expression after overexpression or knockdown of circ‐myh8. For the circ‐myh8 overexpression, PASMCs were transfected with circ‐myh8 plasmid or pcDNA following culture in normoxia condition for 24 hours. For the circ‐myh8 knockdown, PASMCs were transfected with circ‐myh8 siRNA, gapmers, or NC, then exposed to hypoxia (0.3% FiO₂) for 24 hours. C through F, Reverse transcription‐quantitative polymerase chain reaction (RT‐qPCR) assay for HIF1α (C) HIF1β (D), HIF2α (E), and HIF2β (F) mRNA expression in the above cells in (B). The normal overexpression of circ‐myh8 can upregulate mRNA and protein levels, while the hypoxic knockdown of circ‐myh8 inhibits HIF1α mRNA and protein expression. G, Representative images and summarized data of EdU staining in cells. PASMCs were transfected with circ‐myh8 overexpression plasmid and control plasmid, with siRNA targeting HIF1α and siNC, then exposed to hypoxia (0.3% FiO₂) for 24 hours. H through J, Representative images and summarized data of PCNA expression (H), Ki67 expression (I), and cyclins expression (J) of the above cells in (G). Scale bar, 25 μm. K, The luciferase activity of HIF1α promoters after circ‐myh8 overexpression or knockdown. PASMCs in 24‐well plates were transfected with circ‐myh8 plasmids or control, together with pGL3‐basic‐HIF1α (pGL3‐HIF1α) plasmid. The luciferase activity was measured 24 hours later. For the circ‐myh8 knockdown, PASMCs were transfected with circ‐myh8 small interfering RNA, gapmers, or NC, together with pGL3‐basic‐HIF1α (pGL3‐HIF1α) plasmid, then exposed to hypoxia (0.3% FiO₂) for 24 hours. Data represent means±SEM from indicated independent experiments. Student's t test (for 2 means) or 1‐way ANOVA followed by Dunnett's test (for >2 means). HIF1α indicates hypoxia‐inducible factor alpha; Hyp, hypoxia; NC, negative control; Nor, normoxia; PCNA, proliferating cell nuclear antigen; and veh, vehicle. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.