CircHIPK3 regulates cardiac fibroblast proliferation, migration and phenotypic switching through the miR-152-3p/TGF-β2 axis under hypoxia

Figure 1. circHIPK3 is upregulated in cardiac fibroblasts and accelerates the process of fibrosis. (A) The OD values from the CCK-8 analysis revealed cell viability of cardiac fibroblasts treated with hypoxia at different times. n = 4. *P < 0.05. (B–D) The cell cycle was assayed by a cell cycle assay in cardiac fibroblasts. The results represent the percentage of cells in G1 and S + G2/M phases for the indicated condition. n = 3. *P < 0.05. (E–G) Wound scratch assay for the normal (Nor) and hypoxia groups. The average sizes of the gaps were measured at 48 h. Scale bar = 200 μm. n = 5. *P < 0.05. (H and I) The protein expression levels of α-SMA, Col I and Col III were measured by western blot analysis. n = 3. *P < 0.05. (J–O) Representative immunofluorescence images of Col III (green), DAPI (blue) and merged images. Scale bar = 20 μm. n = 3. (P) RT-qPCR analysis of circHIPK3 expression in normal or hypoxia-treated CFs. n = 3. *P < 0.05. *P < 0.05, vs. normal group.

Figure 2. circHIPK3 promoted proliferation, migration and phenotypic modulation in cardiac fibroblasts exposed to hypoxia. CFs were transfected with negative control (LV), circHIPK3, or circHIPK3 siRNA (si-circHIPK3) for 48 h. (A) The relative expression of circHIPK3 in CFs after different treatments was examined by RT-qPCR analysis. n = 3. *P < 0.05, #P < 0.05. (B–G) The cell cycle was investigated by a cell cycle assay in cardiac fibroblasts. The results represent the percentage of cells in G1 and S + G2/M phases for the indicated conditions. n = 3. *P < 0.05, #P < 0.05. (H–M) Migration was determined by the wound scratch assay. The average sizes of the gaps were measured at 48 h. Scale bar = 200 μm. n = 5. *P < 0.05, #P < 0.05. (N and O) The protein expression levels of α-SMA, Col I and Col III were measured by western blot analysis. n = 3. *P < 0.05, #P < 0.05. *P < 0.05, vs. normal group. #P < 0.05, vs. hypoxia group.

Figure 3. circHIPK3 acts as a miR-152-3p sponge. (A–C and E–H) RNA FISH assays were conducted to detect circHIPK3 and miR-152 localization in CFs using a Cy3-labeled circHIPK3 probe and FITC-labeled miR-152 probe. Nuclei were stained with DAPI. Scale bar = 20 μm. n = 3. (D) A luciferase reporter assay was conducted to detect the luciferase activity of RLuc-circHIPK3-WT and RLuc-circHIPK3-Mut in HEK-293T cells transfected with miR-152-3p mimics or NC mimic. Luciferase activity was detected 48 h after transfection. n = 3. *P < 0.05, vs. circHIPK3-WT + NC group. #P < 0.05, vs. circHIPK3-WT + miR-152-3p group. (I and J) Relative expression of circHIPK3 and miR-152-3p in CFs after different treatments was examined by RT-qPCR analysis. n = 3. *P < 0.05, vs. hypoxia group.

Figure 4. MiR-152-3p inhibited the phenotypic transformation by targeting TGF-β2. To determine the role of miR-152-3p in CFs, CFs were transfected with the miR-152-3p mimic or inhibitor. (A–G and J–P) Representative images of EdU-stained CFs from different groups. Quantification of EdU+ cells presented as the % EdU-positive cells and Hoechst-stained nuclei. The orange color is EdU-positive cells, and the blue color is nuclei stained by Hoechst 33342. Scale bar = 20 μm. n = 10. *P < 0.05, vs. MNC group or INC group. (H, I and Q, R) The protein expression levels of Col I and Col III were measured by western blot analysis. n = 3. *P < 0.05, vs. MNC group or INC group. (S) Relative expression of miR-152-3p in CFs after transfection with miR-152-3p mimic or inhibitor was examined by RT-qPCR analysis. n = 3. *P < 0.05, vs. hypoxia group. (T) TGF-β2 was predicted as a target gene of miR-152-3p using the TargetScan database. HEK-293T cells were cotransfected with RLuc-TGF-β2-WT or RLuc-TGF-β2-Mut and miR-152-3p mimics or NC mimic. Luciferase activity was detected using the dual luciferase reporter assay at 48 h post-transfection. n = 3. *P < 0.05, vs. TGF-β2-WT + NC group. #P < 0.05, vs. TGF-β2-WT…

Figure 5. circHIPK3 sponges miR-152-3p and inhibits its activity. (A–E) The cell cycle was investigated by a cell cycle assay in cardiac fibroblasts. The results represent the percentage of cells in G1 and S + G2/M phases for the indicated conditions. n = 3. *P < 0.05, #P < 0.05. (F–J) Wound scratch assay for the different groups. The average sizes of the gaps were measured at 48 h. Scale bar = 200 μm. n = 5. *P < 0.05, #P < 0.05. (K and L) The protein expression levels of Col I, Col III, TGF-β2, p-Smad2 and p-Smad3 were measured by western blot analysis. n = 3. *P < 0.05, #P < 0.05. *P < 0.05, vs. hypoxia group, #P < 0.05, vs. si-circHIPK3 group.