LincRNA 1700020I14Rik alleviates cell proliferation and fibrosis in diabetic nephropathy via miR-34a-5p/Sirt1/HIF-1α signaling

Fig. 1. Differentially expressed lincRNAs in renal tissues or MCs and characterization of 1700020I14Rik in MCs. a Seven lncRNAs were validated in renal tissues by qRT-PCR and shown in consistent expression with RNA-Seq results. b Seven lncRNAs were further validated in MCs cultured with high or low glucose by qRT-PCR and 5 lincRNAs were in consistent with the previous results. c Expression of 1700020I14Rik was showed with confocal FISH images and its mainly cytoplasmic localization in MCs and highly expressed in MCs culture with high glucose medium. White scale bar, 50 µm. Green scale bar, 25 µm. Data were represented as mean ± SD. Significance was calculated using the Student’s t-test: *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 2. 1700020I14Rik suppressed the proliferation of MCs. a The expression of 1700020I14Rik was measured by qRT-PCR in cells transfected with 1700020I14Rik (+) or siRNA-832 or their matched controls, which showed obviously upregulating or downregulating the expression of 1700020I14Rik compared with their matched controls, respectively. b CCK8 assay was performed to evaluate the effect of 1700020I14Rik on the proliferation in MCs after 48 h transfection, which suggested 1700020I14Rik could suppressed the proliferation of MCs cultured with high glucose medium. c Flow cytometric assay was performed to analyze the effect of 1700020I14Rik on the cell cycle in MCs transfected with 1700020I14Rik (+), siRNA-832, and their matched controls, respectively. Results showed that 1700020I14Rik regulated the S phase. d Proliferating mesangial cells were labeled with EdU. Scale bar, 200 µm. e The expressions of cell-cycle-related proteins cyclin D1 and p21 regulated by 1700020I14Rik were analyzed by western blot. Data were represented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, n.s: no statistical significance

Fig. 3. Effects of 1700020I14Rik on the regulation of fibrosis-related proteins. a–c The mRNA expression level of Col-4 (a), FN (b), and TGF-β1 (c) in MCs transfected with plasmids or siRNA of 1700020I14Rik was measured by qRT-PCR. d–e The protein expression level of Col-4, FN, and TGF-β1 in MCs transfected with plasmids (d) or in MCs transfected with siRNA-832 (e) was analyzed by western blot. f, g Abnormal expressions of Col-4, FN, and TGF-β1 in MCs transfected with plasmids (f) or in MCs transfected with siRNA (g) were assessed by immunofluorescence analysis. Scale bar, 200 µm. Overall, these results suggested that 1700020I14Rik alleviated renal fibrosis in DN. Data were represented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, n.s: no statistical significance

Fig. 4. 1700020I14Rik interacted with miR-34a-5p by both directly targeting and Ago2-dependent manner. a Predicted binding sites of miR-34a-5p of four species on 1700020I14Rik transcript. The red part of the column indicates the potential binding sites. b The inverse expression of 1700020I14Rik and miR-34a-5p in MCs cultured in high glucose or low glucose medium was assessed by qRT-PCR. c The expression of miR-34a-5p regulated by 1700020I14Rik plasmid or its siRNA was measured by qRT-PCR. d The expression of miR-34a-5p regulated by miR-34a-5p mimics and inhibitor was measured by qRT-PCR. e The expression of 1700020I14Rik regulated by miR-34a-5p mimics or inhibitor was detected by qRT-PCR. Data suggested that 1700020I14Rik had an opposite expression comparing with miR-34a-5p in MCs. f Dual-luciferase reporter assay was performed to measure the luciferase activity in cells co-transfected with miR-34a-5p and luciferase reporters containing 1700020I14Rik (1700020I14Rik wt), mutant transcript (1700020I14Rik mut), or miR-34a-5p inhibitor (as positive control, PC). These results indicated that there was direct interaction between 1700020I14Rik and miR-34a-5p. Data were presented as the relative…

Fig. 5. Effects of miR-34a-5p on the proliferation of MCs. a–c The proliferation of MCs transfected with miR-34a-5p mimics or inhibitor was measured by EdU assay (a) (Scale bar, 200 µm), by flow cytometric analysis (b) and by CCK-8 assay (c). d–e Western blot was used to analyze the expressions of cell-cycle-related proteins cyclin D1 and p21 (d), and the expressions of Col-4, FN and TGF-β1 regulated by miR-34a-5p (e). These results indicated that miR-34a-5p promotes proliferation and fibrosis in MCs. Data were represented as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001, n.s: no statistical significance

Fig. 6. MiR-34a-5p affects the fibrosis of MCs through Sirt1/HIF-1α signaling. a Predicted miR-34a-5p target sequence of the Sirt1 3′-UTR showed the highly conserved binding sites in different species. The red nucleotides are the seed sequences of miR-34a-5p. b Dual-luciferase reporter assay was performed to measure the luciferase activity in HEK 293 cells co-transfected with miR-34a-5p and luciferase reporters containing Sirt1 (Sirt1 wt), mutant transcript (Sirt1 mut), or miR-34a-5p inhibitor (as positive control, PC). These results indicated that there was direct interaction between Sirt1 and miR-34a-5p. Data were presented as the relative ratio of firefly luciferase activity to renilla luciferase activity. c, d The mRNA expression level of Sirt1 (c) and HIF-1α (d) regulated by miR-34a-5p mimics or inhibitor was analyzed by qRT-PCR. e–f Protein expression levels of Sirt1 and HIF-1α regulated by miR-34a-5p inhibitor (e) or miR-34a-5p mimics (f) were measured by western blot. g Protein expression levels of HIF-1α, as well as its downstream proteins expression level regulated by combined effects of siRNA-Sirt1 and miR-34a-5p inhibitor were measured by western blot. These results suggested…

Fig. 7. 1700020I14Rik reduced the expressions of fibrosis factors in MCs by regulating miR-34a-5p/Sirt1/HIF-1α signaling. a–b Protein expression levels of Sirt1 and HIF-1α in MCs influenced by 1700020I14Rik plasmids (a) and by siRNA (b) were tested by western blot. c, d Protein expressions of Sirt1 and HIF-1α regulated by the effect of 1700020I14Rik combined with miR-34a-5p mimics (c) or inhibitor (d) were tested by western blot. These results indicated that 1700020I14Rik regulates the expressions of Sirt1 and HIF-1α by regulating miR-34a-5p. e, f Protein expression levels of Col-4, FN, and TGF-β1 regulated by the effect of 1700020I14Rik combined with miR-34a-5p mimics (e) or inhibitor (f) were measured by western blot. Results showed that the expressions of renal fibrosis genes TGF-β1, FN, and Col-4 were reduced by overexpression of 1700020I14Rik, the expressions of these proteins were abolished by the participation of miR-34a-5p mimics. The expressions of TGF-β1, FN, and Col-4 were upregulated by silence of 1700020I14Rik, these changes were blocked by miR-34a-5p inhibitor. Data were represented as the mean ± SD of three independent experiments. *p < 0.05; **p < 0.01, and ***p < 0.001,…