Fig 2: The hsa_circ_0004018 suppressed the proliferation and activation of HSCs through upregulating the expression of TEP1. (A) After transfection with TEP1 containing plasmid and the vector control, the RNA levels of TEP1 were detected by real-time PCR. The data are from three independent experiments. (B, C) After overexpression with TEP1, the growth curves of the HSCs were examined by CCK8 assay (B), while the expression of α-SMA and COL1A1 in HSCs was detected by western blotting (C). (D, E) The RNA levels (D) and the protein levels (E) were respectively detected after overexpression with hsa_circ_0004018. The vector was transfected as control. (F, G) After transfection with the indicated mimic hsa-miR-660-3p or negative control (NC) plus hsa_circ_0004018 or the vector control, the RNA levels (F) and the protein levels (G) of TEP1 in HSCs were respectively detected by real-time PCR and western blotting.

Fig 3: Overexpression of circSKIL promotes the osteogenic differentiation and activates the JNK/STAT3 pathway in control cells. Control cells were transfected with either a circSKIL-overexpressing plasmid or an empty vector (NC). (A) mRNA expression levels of RUNX2, ALP, COL I, OCN, and OPN (n=3). (B) Western blot analysis of the protein expression levels of RUNX2, ALP, COL1A1, OCN, and OPN (n=3). (C) Western blot analysis of the protein levels of p-JNK, JNK, p-STAT3, and STAT3 (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. NC. circ, circular RNA; NC, negative control; RUNX2, runt-related transcription factor 2; ALP, alkaline phosphatase; COL I, collagen I; OCN, osteocalcin; OPN, osteopontin; COL1A1, collagen type I α 1 chain; p-, phosphorylated.

Fig 4: Downregulation of circSKIL suppresses osteogenic differentiation and inhibits the JNK/STAT3 pathway in OPLL cells. OPLL cells were transfected with either a circSKIL-targeting siRNA or a negative control siRNA (si-NC). (A) mRNA expression levels of RUNX2, ALP, COL I, OCN, and OPN (n=3). (B) Western blot analysis of the protein expression levels of RUNX2, ALP, COL1A1, OCN, and OPN (n=3). (C) Western blot analysis of the protein levels of p-JNK, JNK, p-STAT3, and STAT3 (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. si-NC. circ, circular RNA; OPLL, ossification of the posterior longitudinal ligament; si, small interfering; NC, negative control; RUNX2, runt-related transcription factor 2; ALP, alkaline phosphatase; COL I, collagen I; OCN, osteocalcin; OPN, osteopontin; COL1A1, collagen type I α 1 chain; p-, phosphorylated.

Fig 5: Overexpression of hsa_circ_0004018 alleviated CCl4-induced mouse liver fibrosis through suppressing the expression of hsa-miR-660-3p and upregulating TEP1 in HSCs. (A) The representative section staining of the liver samples isolated from the CCl4-induced liver fibrosis mice respectively injected with lentivirus expressing hsa_circ_0004018 (cirRNA group) or the negative control (NC group). (B, C) They were detected by real-time PCR the RNA levels of hsa_circ_0004018 (B) and hsa-miR-660-3p (C) in the liver samples of the cirRNA group and the NC group mice. (D, E) The RNA levels (D) and the protein levels of TEP1 (E) were respectively detected in the liver samples of the cirRNA group and the NC group mice. (F–H) It was detected the expression of α-SMA and COL1A1 in liver samples of the cirRNA group and the NC group by western blotting (F) and immunohistochemistry (G, H), respectively.