Figures & data
Table 1. Primer sequences for RT-qPCR.
Figure 1. TGF-β1 induced upregulation of Circ_0007535 in HFL1 cells. (A) RT-qPCR analysis of circ_0007535 expression in lung tissues from 27 pulmonary fibrosis patients and 27 normal controls. (B) Circ_0007535 level was determined by RT-qPCR after HFL1 cells were treated with 10 ng/mL TGF-β1. (C,D) Circ_0007535 and GAPDH levels were analysed using RT-qPCR after total RNA was digested with RNase R (C) or reverse transcription by Oligo (dT)18 primers/random primers (D). (E) RT-qPCR was used for expression analysis of circ_0007535, U6, and GAPDH after RNA isolation from cytoplasm and nucleus. ***p < 0.001, ****p < 0.0001, n = 3.
Figure 2. Circ_0007535 inhibition reversed TGF-β1-induced proliferation, motility, ECM accumulation, and inflammation in HFL1 cells. HFL1 cells were performed with the treatment of control, TGF-β1, TGF-β1 + si-NC, TGF-β1 + si-circ_0007535. (A) Expression detection for circ_0007535 was carried out by RT-qPCR. (B) Cell viability examination was conducted using CCK-8 assay. (C,D) The proliferation analysis was performed via EdU assay. (E-F) The assessment of invasion (E) and migration (F) was implemented through transwell assay and scratch assay. (G-H) EMT-associated markers (G) and ECM proteins (H) were measured by western blot. (I) Inflammation evaluation was administrated using ELISA. **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3.
Figure 3. Circ_0007535 was affirmed to interact with miR-18a-5p. (A) The binding regions between circ_0007535 and miR-18a-5p in starbase. (B) The transfection efficiency of miR-18a-5p was assessed via RT-qPCR. (C–E) Target binding between circ_0007535 and miR-18a-5p was analysed via dual-luciferase reporter assay (C), RIP assay (D) and pull-down assay (E). (F) miR-18a-5p level was detected using RT-qPCR in lung tissues from 27 pulmonary fibrosis patients and 27 normal controls. (G) Pearson correlation analysis was applied to evaluate the expression association between circ_0007535 and miR-18a-5p in pulmonary fibrosis patients. (H) RT-qPCR was performed for miR-18a-5p quantification after TGF-β1 treatment in HFL1 cells. **p < 0.01, ****p < 0.0001, n = 3.
Figure 4. Circ_0007535/miR-18a-5p network affected biological behaviours in TGF-β1-treated HFL1 cells. Control, TGF-β1, TGF-β1 + si-NC, TGF-β1 + si-circ_0007535, TGF-β1 + si-circ_0007535 + anti-miR-NC, and TGF-β1 + si-circ_0007535 + anti-miR-18a-5p groups were designed in HFL1 cells. (A) The miR-18a-5p expression was examined using RT-qPCR. (B) Cell viability was tested via CCK-8 assay. (C,D) Cell proliferation was evaluated through EdU assay. (E-F) Cell invasion (E) and migration (F) were determined by transwell assay and scratch assay. (G,H) Western blot was used for EMT (G) and ECM (H) associated protein detection. (I) Inflammatory cytokines were detected by ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3.
Figure 5. Circ_0007535 induced TGFBR1 upregulation by targeting miR-18a-5p. (A) Starbase was used for binding prediction between miR-18a-5p and TGFBR1. (B-D) Dual-luciferase reporter assay (B), RIP assay (C) and pull-down assay (D) were applied to confirm miR-18a-5p interaction with TGFBR1. (E) TGFBR1 level was measured in lung tissues from 27 pulmonary fibrosis patients and 27 normal controls via using RT-qPCR. (F) The expression correlation between miR-18a-5p and TGFBR1 in pulmonary fibrosis patients was analysed by Pearson correlation analysis. (G) Western blot was performed for protein analysis of TGFBR1 in TGF-β1-treated HFL1 cells. (H) RT-qPCR was used to determine miR-18a-5p level after anti-miR-NC or anti-miR-18a-5p transfection. (I) TGFBR1 protein level was examined by western blot after miR-18a-5p, anti-miR-18a-5p or control transfection. (J) TGFBR1 protein determination was performed via western blot after TGF-β1-treated HFL1 cells were transfected with si-circ_0007535, si-circ_0007535 + anti-miR-18a-5p or relative control groups. ***p < 0.001, ****p < 0.0001, n = 3.
Figure 6. Overexpression of miR-18a-5p relieved TGF-β1-evoked regulation in HFL1 cells via downregulating TGFBR1. HFL1 cells were treated with control, TGF-β1, TGF-β1 + miR-NC, TGF-β1 + miR-18a-5p, TGF-β1 + miR-18a-5p + pcDNA, and TGF-β1+ miR-18a-5p + TGFBR1. (A) Western blot was used to measure TGBR1 protein expression. (B) CCK-8 assay was performed to determine cell viability. (C,D) EdU assay was applied to analyse cell proliferation. (E,F) Transwell assay and scratch assay were performed for the assessment of cell invasion (E) and migration (F). (G,H) the protein detection related to EMT (G) and ECM (H) was carried out using a western blot. (I) ELISA was employed to assess inflammatory response. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3.
