Downregulating ANGPTL3 by miR-144-3p promoted TGF-β1-induced renal interstitial fibrosis via activating PI3K/AKT signaling pathway

Figures

• 

  Fig. 1. Expression levels of miR-144-3p, ANGPTL3 and PI3K/AKT pathway markers in UUO mouse model. Representative micrographs of hematoxylin–eosin (H&E) staining (A) and Masson trichrome staining (B) in kidney tissues derived from UUO and sham group with five mice in each group. Scale bars, 100 μm; (C–D) Quantitative real-time PCR-based expressions of miR-144-3p and ANGPTL3 in the sham and UUO groups, and the internal reference miRNA was U6. Data were expressed as means ± SD. ***p < 0.001, compared with sham; (E) Western blot results including ANGPTL3, α-SMA, PI3K, p-PI3K, AKT, p-AKT, Collagen I and Fibronectin in the sham and UUO groups. GAPDH was used as an internal reference for the other proteins.

• 

  Fig. 2. Elevating ANGPTL3 levels mitigated the inflammation, fibrosis, and PI3K/AKT pathway activation induced by TGF-β1 in HK-2 cells. (A) The protein level of ANGPTL3 was determined in HK-2 cells after transfection with pcDNA3.1-ANGPTL3 or pcDNA3.1. HK-2 cells were transfected with pcDNA3.1-ANGPTL3 or pcDNA3.1, followed by treatment with 10 ng/mL TGF-β1. ***p < 0.001, compared with pcDNA3.1; (B) ELISA assay was used to determine the concentration levels of TNF-α and IL-6 in the above HK-2 cells. (C) Cell apoptotic rate was measured in the above HK-2 cells. (D) The protein levels of ANGPTL3, α-SMA, PI3K, p-PI3K, AKT, p-AKT, Collagen I and Fibronectin were detected in the above HK-2 cells via Western blot analysis. Data were expressed as means ± SD. **p < 0.01, ***p < 0.001, compared with control; #p < 0.05, ##p < 0.01, ###p < 0.001, compared with TGF-β1 + pcDNA3.1.

• 

  Fig. 3. ANGPTL3 identified as a direct target of miR-144-3p. (A) Binding of miR-144-3p with ANGPTL3 3′-UTR was predicted by TargetScan. (B) Dual luciferase reporter gene assay revealed that miR-144-3p inhibitor significantly increased the luciferase activity of ANGPTL3 wild-type (WT) 3′-UTR. (C) The expression level of miR-144-3p was determined in TGF-β1-stimulated HK-2 cells. The expression level of miR-144-3p (D) and the protein level of ANGPTL3 (E) were measured in HK-2 cells transfected with miR-144-3p inhibitor, followed by TGF-β1 treatment. Data were expressed as means ± SD. ***p < 0.001, compared with miR-NC or control.

• 

  Fig. 4. Knockdown of ANGPTL3 reversed the effects of miR-144-3p inhibitor on TGF-β1-induced HK-2 cells. (A) Knockdown of ANGPTL3 was confirmed via Western blot analysis in TGF-β1-stimulated HK-2 cells transfected with si-ANGPTL3. **p < 0.01, compared with si-NC; HK-2 cells were co-transfected with miR-144-3p inhibitor and si-ANGPTL3, followed by treatment with 10 ng/mL TGF-β1. (B) ELISA assay was used to determine the concentration levels of TNF-α and IL-6 in the above HK-2 cells. (C) Cell apoptotic rate was measured in the above HK-2 cells. (D) The protein levels of α-SMA, PI3K, p-PI3K, AKT, p-AKT, Collagen I and Fibronectin were detected in the above HK-2 cells via Western blot analysis. Data were expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with miR-NC; #p < 0.05, ##p < 0.01, compared with miR-144-3p inhibitor.

• 

  Fig. 5. LY294002 rescued the effects of ANGPTL3 knockdown on TGF-β1-induced HK-2 cells. TGF-β1-stimulated HK-2 cells were transfected with si-ANGPTL3, followed by treatment with PI3K/AKT signaling pathway inhibitor LY294002. (A) ELISA assay was used to determine the concentration levels of TNF-α and IL-6 in the above HK-2 cells. (B) Cell apoptotic rate was measured in the above HK-2 cells. (C) The protein levels of α-SMA, PI3K, p-PI3K, AKT, p-AKT, Collagen I and Fibronectin were detected in the above HK-2 cells via Western blot analysis. Data were expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, compared with si-NC; #p < 0.05, ##p < 0.01, compared with si-ANGPTL3.

• 

  Figure S1. All the uncropped protein bands included in all figures.