MiR-29a-3p inhibits fibrosis of diabetic kidney disease in diabetic mice via downregulation of DNA methyl transferase 3A and 3B

Figure 1 Expression of DNA methyl transferase 3A/3B and fibrosis-related molecules. A: Quantitative reverse transcription PCR to detect the expression of miR-29a-3p; B: Western blotting to detect the expression of DNA methyl transferase 3A/3B (DNMT3A/3B), and the right picture shows the statistical analysis map; C: Western blotting; D: Immunofluorescence (× 400) to detect alpha smooth muscle actin (α-SMA), collagen I, and fibronectin expression, and the right picture shows the statistical analysis chart (n = 3). CON: Control group; HG: High glucose group.

Figure 2 Expression of alpha smooth muscle actin, collagen I, and fibronectin. Alpha smooth muscle actin (α-SMA), collagen I, and fibronectin were significantly decreased after transfection of cells in the high glucose (HG) group with miR-29a-3p mimics. A: Western blot including the statistical analysis chart; B: Immunofluorescence (× 400) (n = 3). HG: High glucose group; NC: Negative control.

Figure 3 Expression of alpha smooth muscle actin, collagen I, and fibronectin. Alpha smooth muscle actin (α-SMA), collagen I, and fibronectin were significantly increased in cells transfected with inhibitor-miR-29a-3p in the normal glucose group. A: Western blot including the statistical analysis chart; B: Immunofluorescence (× 400) (n = 3). ^bP < 0.01. ^cP < 0.001. CON: Control group; NC: Negative control.

Figure 4 Effect of adding inhibitor-mir-29a-3p to cells in the normal glucose group or adding miR-29a-3p mimics to cells in the high glucose group on the expression of DNA methyl transferase 3A/3B. A and C: Quantitative reverse transcription PCR for detecting the transfection efficiency; B and D: Western blotting to detect the expression of DNA methyl transferase 3A/3B (DNMT3A/3B) (n = 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. CON: Control group; HG: High glucose group; NC: Negative control.

Figure 5 Inhibition of DNA methyltransferase 3A/3B can reverse the effect of downregulation of miR-29a-3p on diabetic kidney disease-related fibrosis in SV40MES13 cells cultured with high glucose. Quantitative reverse transcription PCR detected the expression levels of alpha smooth muscle actin (α-SMA), collagen I, and fibronectin in the diabetic kidney disease model. Results are represented as mean ± SD (n = 3). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NC: Negative control.

Figure 6 After antagomiR-29a-3p therapy, the renal index of diabetic mice increased, serum creatinine and blood urea nitrogen levels were within the normal range, and the renal pathological changes were relieved. A: Renal pathological changes in mice at 10, 12, and 16 weeks hematoxylin-eosin staining (HE), periodic acid-Schiff staining (PAS), × 400]; B and C: Results of serum creatinine (CR) and blood urea nitrogen (BUN) levels in mice at different weeks of age; D: Quantitative reverse transcription PCR to detect the expression of miR-29a-3p; E: Renal index of diabetic (db/db) mice in each group (bilateral renal body weight/mouse body weight), (n = 5); F and G: Serum biochemical test results for mice in each group; H: Random caudal vein blood glucose levels; I and J: Renal pathological changes in the mice: The pathological changes in db/db (agomiR-29a-3p) mice were more severe than that in the db/m mice, but were less severe than that in the db/db mice without agomiR-29a-3p treatment. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: Not significant; NC: Negative control; CON: Control group.

Figure 7 Change in the expression of DNA methyl transferase 3A/3B and fibrosis-related molecules in the mice. The expression levels of DNA methyl transferase 3A/3B (DNMT3A/3B), alpha smooth muscle actin (α-SMA), collagen I, and fibronectin in diabetic (db/db) mice treated with agomiR-29a-3p were significantly lower than those in the db/db mice without agomiR-29a-3p treatment (n = 3). CON: Control group; NC: Negative control.

Figure 8 Change in the expression of Wnt3a/β-catenin and Janus kinase/signal transducer and activator of transcription pathways in the diabetic mice with agomiR-29a-3p treatment. The expression of Wnt3a, β-catenin, Janus kinase (JAK) 2, and signal transducer and activator of transcription (Stat) 3 in the diabetic (agomiR-29a-3p) mice were significantly lower than that in the diabetic mice without agomiR-29a-3p treatment (n = 3). CON: Control group; NC: Negative control.