1. MicroRNA-29b Inhibits Diabetic Nephropathy in db/db Mice
Hai-Yong Chen, Xiang Zhong, Xiao R Huang, Xiao-Ming Meng, Yongke You, Arthur CK Chung, Hui Y Lan 
Molecular Therapy 
Volume 22, Issue 4, Pages (April 2014)
DOI: /mt
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

2. Figure 1
Overexpression of miR-29b attenuates advanced glycation end (AGE)-induced TGF-β/Smad3-mediated fibrosis in mesangial cells in vitro. (a) Real-time PCR detects miR-29b expression in response to AGE (50 µg/ml) stimulation by mesangial cells in the presence or absence of Dox (2 µg/ml)-induced miR-29b overexpression. (b–d) Real-time PCR shows that Dox-induced miR-29b overexpression inhibits AGE-induced collagen I, collagen IV, and fibronectin mRNA expression in mesangial cells. (e) Western blot analysis shows that Dox-induced miR-29b overexpression inhibits AGE-induced upregulation of fibronectin, collagen I, collagen IV, and phosphorylation of Smad3 in mesangial cells. (f) Real-time PCR shows that Dox-induced overexpression of miR-29b inhibits AGE-induced TGF-β mRNA expression by mesangial cells. Data represent the mean ± SE for at least four independent experiments. *P < 0.05, **P < 0.01, ***P < compared to normal medium (0 hour). #P < 0.05, ##P < 0.01, ###P < compared to Dox(−). Dox(+), Doxycycline induced-miR-29b overexpression.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

3. Figure 2
Knockdown of miR-29b enhances advanced glycation end (AGE)-induced TGF-β/Smad3-mediated fibrosis in mesangial cells. (a) Real-time PCR detects miR-29b expression in mesangial cells with/without miR-29b knockdown. (b–d) Real-time PCR detects that knockdown of miR-29b enhances AGE (50 µg/ml) induced collagen I, collagen IV, and fibronectin mRNA expression by mesangial cells. (e) Western blot analysis reveals that knockdown of miR-29b enhances AGE (50 µg/ml) induced collagen I, collagen IV, and fibronectin protein expression, and phosphorylation of Smad3. (f) knockdown of miR-29b enhances AGE (50 µg/ml) induced TGF-β expression in mesangial cells. Data represent the mean ± SE for at least four independent experiments. *P < 0.05, **P < 0.01, ***P < compared to normal medium control (0 hour). #P < 0.05, ##P < 0.01, ###P < compared to VC. VC, Vector control, KD, miR-29 knockdown.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

4. Figure 3
miR-29b therapy attenuates microalbuminuria and histological damage in db/db mice. (a,b) Real-time PCR and in situ hybridization show that miR-29b expression is significantly reduced in the diabetic kidney of db/db mice, which is restored by miR-29b gene therapy using the ultrasound-microbubble gene transfer technique. (c) miR-29b treatment attenuates microalbuminuria in db/db mice. (d,e) miR-29 therapy inhibits histological injury including mesangial matrix index (PAS staining). (i) week 10 db/m mice, (ii) week 10 db/db mice, (iii) week 20 db/m mice, (iv) week 20 db/db mice, (v) week 20 db/db mice treated with empty vector (VC), and (vi) week 20 db/db mice treated with miR-29b. Scr, scramble probe as negative control for in situ hybridization (ISH). Data represent the mean ± SE for at groups of eight mice. **P < 0.01, ***P < compared to week 20 db/m mice. #P < 0.05, ###P < compared to week 20 db/db mice. †††P < compared to week 10 db/m. g, glomerulus; m+, db/m; db, db/db, VC, db/db mice received empty vector control treatment; miR-29, db/db mice received miR-29b treatment. Original magnification: (b) ×200, (d) ×400.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

5. Figure 4
Immunohistochemistry and renal-time PCR detect that miR-29b therapy inhibits collagen I and collagen IV expression in the diabetic kidney of db/db mice. (a) Collagen I expression by immunohistochemistry and quantitative data in glomerular and tubulointerstitial areas. (b) Collagen I mRNA expression by real-time PCR. (c) Collagen IV expression by immunohistochemistry. (d) Collagen IV mRNA expression by real-time PCR. (i) week 10 db/m mice, (ii) week 10 db/db mice, (iii) week 20 db/m mice, (iv) week 20 db/db mice, (v) week 20 db/db mice treated with empty vector (VC), and (vi) week 20 db/db mice treated with miR-29b. Data represent the mean ± SE for at groups of eight mice. *P < 0.05, **P < 0.01, ***P < compared to week 20 db/m mice. ##P < 0.01, ###P < compared to week 20 db/db mice. ††P < 0.01 compared to week 10 db/m. m+, db/m; db, db/db, VC, db/db mice received empty vector control treatment; miR-29, db/db mice received miR-29b treatment. Original magnification: (a) ×200, (b) ×400.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

6. Figure 5
Western blot, immunohistochemistry, and real-time PCR reveal that miR-29b therapy inhibits renal fibrosis in db/db mice. (a) Western blot analysis shows that miR-29b therapy significantly reduces protein levels of collagen I, collagen IV and fibronectin. (b) Fibronectin immunostaining and quantitative data in the glomerular and tubulointerstitial areas. (c) Fibronectin mRNA expression by real-time PCR. (i) week 10 db/m mice, (ii) week 10 db/db mice, (iii) week 20 db/m mice, (iv) week 20 db/db mice, (v) week 20 db/db mice treated with empty vector (VC), and (vi) week 20 db/db mice treated with miR-29b. Data represent the mean ± SE for at groups of 8 mice. *P < 0.05, **P < 0.01, ***P < compared to week 20 db/m mice; #P < 0.05, ##P < 0.01, ###P < compared to week 20 db/db mice. †P < 0.05, †††P < compared to week 10 db/m. m+, db/m; db, db/db, VC, db/db mice received empty vector control treatment; miR-29, db/db mice received miR-29b treatment. Original magnification: (b) ×400.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

7. Figure 6
miR-29b therapy blocks activation of TGF-β/Smad3 signaling in the diabetic kidney of db/db mice. (a) Immunohistochemistry for TGF-β1 expression. (b) Western blot analysis for phosphorylation of Smad3. (c) Real-time PCR for TGF-β1 mRNA expression. (i) week 10 db/m mice, (ii) week 10 db/db mice, (iii) week 20 db/m mice, (iv) week 20 db/db mice, (v) week 20 db/db mice treated with empty vector (VC), and (vi) week 20 db/db mice treated with miR-29b. Data represent the mean ± SE for at groups of 8 mice. *P < 0.05, **P < 0.01, ***P < compared to week 20 db/m mice. #P < 0.05, ##P < 0.01 compared to week 20 db/db mice. ††P < 0.01 compared to week 10 db/m. m+, db/m; db, db/db, VC, db/db mice received empty vector control treatment; miR-29, db/db mice received miR-29b treatment. Magnification ×100.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

8. Figure 7
miR-29b therapy inhibits Sp1/NF-κB–driven renal inflammation in the diabetic kidney of db/db mice. (a) Western blot analysis of Sp1 expression and NF-κB/p65 phosphorylation. (b) Real-time PCR analysis of mRNA expression of Sp1, NF-κB/p65, TNF-α, and MCP-1. (c) F4/80+ macrophages infiltrating the glomerulus and tubulointerstitium. Data represent the mean ± SE for at groups of eight mice. *P < 0.05, **P < 0.01, ***P < compared to week 20 db/m mice. #P < 0.05, ##P < 0.01, ###P < compared to week 20 db/db mice. †††P < 0.01 compared to week 10 db/m. m+, db/m; db, db/db, VC, db/db mice received empty vector control treatment; miR-29, db/db mice received miR-29b treatment.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions

9. Figure 8
miR-29b treatment inhibits T-bet–dependent Th1-mediated immune response in the diabetic kidney of db/db mice. (a)Western blot analysis of T-bet expression. (b,c) Real-time PCR analysis of T-bet and IFN-γ mRNA expression. (d) ELISA analysis of plasma IFN-γ levels. (e) CD4+IFN-γ+ T cells by two-color immunofluorescence. CD4 (green), IFN-γ (red), nuclei were counterstained with DAPI (blue). Results reveal that miR-29 treatment blocks Th1-mediated immune response as evidenced by inhibition of T-bet and IFN-γ expression and CD4+IFN-γ+ T helper cell infiltration. Data represent the mean ± SE for at groups of 8 mice. *P < 0.05, **P < 0.01 compared to week 20 db/m mice. #P < 0.05, ##P < 0.01 compared to week 20 db/db mice. ††P < 0.01 compared to week 10 db/m. m+, db/m; db, db/db, VC, db/db mice received empty vector control treatment; miR-29, db/db mice received miR-29b treatment. Original magnification ×800.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2014 The American Society of Gene & Cell Therapy Terms and Conditions