1. Volume 80, Issue 4, Pages 358-368 (August 2011)
A microRNA circuit mediates transforming growth factor-β1 autoregulation in renal glomerular mesangial cells 
Mitsuo Kato, Laura Arce, Mei Wang, Sumanth Putta, Linda Lanting, Rama Natarajan 
Kidney International 
Volume 80, Issue 4, Pages (August 2011)
DOI: /ki
Copyright © 2011 International Society of Nephrology Terms and Conditions

2. Figure 1
Auto-upregulation of transforming growth factor-β1 (TGF-β1) in mouse mesangial cells (MMCs). (a) Dose-response curve of TGF-β1 mRNA levels (assessed by quantitative real-time PCR) in MMCs treated with exogenous recombinant TGF-β1 for 24h (mean and s.e., n=4). Significant increase was detected at 5–10ng/ml of TGF-β1. (b) Time course study of TGF-β1 mRNA levels in MMCs treated with 10ng/ml of TGF-β1 (mean and s.e., n=4). Significant increase was detected only at 24h. (c) Mouse TGF-β1 promoter (including upstream glucose response element, CACGTG, also known as E-box) Luc reporter (pA835luc) activity was significantly increased by TGF-β1(10ng/ml for 24h) (mean and s.e., n=4). SD, serum depletion. (d) miR-192 mimic oligonucleotides (oligos) increased TGF-β1 promoter Luc reporter (pA835luc) activity, whereas miR-192 inhibitor oligos inhibited the TGF-β1-induced reporter activity (mean and s.e., n=4). NC, negative control oligo. ***P<0.001, **P<0.01, and *P<0.05, respectively.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

3. Figure 2
The expression of miR-200 family members in mouse mesangial cells (MMCs) treated with transforming growth factor-β1 (TGF-β1) and in the glomeruli of diabetic mice. (a) Mature sequences of miR-200 family members. miR-200 family members are divided into two groups, group 1 (miR-141 and miR-200a) and group 2 (miR-200b, miR-200c, and miR-429), based on their seed sequences underlined. (b) Expression levels of miR-200 family member and miR-192 and miR-215 in MMCs treated with or without TGF-β1 (10ng/ml for 24h). miR-200b and miR-200c levels were significantly increased by TGF-β1, with no significant change in miR-141 or miR-429 (mean and s.e., n=3). miR-192 levels were increased and miR-215 levels decreased, as reported previously.4 (c) miR-200b and miR-200c levels were increased in diabetic mice glomeruli relative to respective control non-diabetic mice (mean and s.e., n=4). CTR, control; STZ, streptozotocin. ***P<0.001, **P<0.01, and *P<0.05, respectively.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

4. Figure 3
miR-200 family members and transforming growth factor-β1 (TGF-β1) are upregulated by miR-192 in mouse mesangial cells (MMCs). (a, b) miR-192 mimic or inhibitor oligonucleotides (oligos) were transfected into MMCs and miR-200b/c levels examined by real-time quantitative PCR (qPCR). Effects of miR-192 mimic and inhibitor on the expression of (a) miR-200b and (b) miR-200c. SD, serum depletion. miR-192 significantly increased miR-200b and miR-200c levels in MMCs compared with negative control (NC). miR-192 inhibitor ameliorated the TGF-β1(10ng/ml for 24h)-induced increases in miR-200b and miR-200c levels (mean and s.e., n=3). (c) Effects of miR-192 and miR-200b mimics and inhibitors on the expression of TGF-β1. miR-192 or miR-200b significantly increased TGF-β1 levels in MMCs. Conversely, miR-192 or miR-200b inhibitor significantly attenuated the TGF-β1-induced increase in TGF-β1 levels (mean and s.e., n=3). ***P<0.001, **P<0.01, and *P<0.05, respectively.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

5. Figure 4
Zeb1 is inhibited by miR-200b and miR-200c. (a) Zeb1 protein levels are significantly decreased by miR-200b or miR-200c (or miR-200a) in mouse mesangial cells (MMCs). NC, negative control of mimic oligonucleotide (oligo). (b) Quantification of Zeb1 protein levels (mean and s.e., n=3). (c) Decreased levels of Zeb1 in transforming growth factor-β1 (TGF-β1) (10ng/ml for 24h)-treated MMCs. SD, serum depletion. (d) Quantification of Zeb1 protein levels (mean and s.e., n=3). (e) Increase of Zeb1 protein levels by miR-200b inhibitor. All samples were treated with TGF-β1 (10ng/ml for 24h). (f) Quantification of Zeb1 protein levels (mean and s.e., n=3). ***P<0.001, **P<0.01, and *P<0.05, respectively.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

6. Figure 5
Zeb1 3′ untranslated region (UTR) is a bona fide target of miR-200b. (a) Luciferase reporter constructs of Zeb1 3′UTR. Two potential miR-200b/c target sites (1 and 2) were found in this 3′UTR sequence (wild type (WT)). Mutations were introduced into these sites by site direct mutagenesis. Zeb1 3′UTR MT1 and MT2 are single mutants of site 1 (M1) and site 2 (M2), respectively. Zeb1 3′UTR MT1&2 is a double mutant of both sites (M1M2). (b) miR-200b and miR-200c significantly inhibited the reporter activity of WT Zeb1 3′UTR (mean and s.e., n=4). (c) The constructs with single mutation in either site1 or site2 in the Zeb1 3′UTR showed less inhibitory response to miR-200b mimic, whereas the double mutant of both sites completely lost the response to miR-200b, indicating that these two sites are real targets of miR-200b (mean and s.e., n=4). ***P<0.001.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

7. Figure 6
miR-200b regulates collagen type I α2 (Col1a2) expression through the upstream E-boxes. (a) Col1a2 mRNA levels were significantly increased by a combination of miR-192 and miR-200b mimics (mean and s.e., n=3). NC, negative control. (b) Mouse Col1a2 promoter (including upstream E-boxes) Luc reporter activity was increased by miR-192+miR-200b mimics (mean and s.e., n=4). (c) miR-200b inhibitor inhibited the transforming growth factor-β1 (TGF-β1)-induced expression of Col1a2 (mean and s.e., n=3). (d) miR-192 or miR-200b/c inhibitors inhibited TGF-β1 (10ng/ml for 24h)-induced Col1a2 promoter reporter activity (mean and s.e., n=4). (e) Col4a1 mRNA levels were significantly increased by miR-192 or miR-200b mimics as well as TGF-β1 in mouse mesangial cells (MMCs), whereas inhibitors of miR-192 or miR-200b attenuated Col4a1 mRNA levels in TGF-β1-treated MMCs (mean and s.e. n=3). SD, serum depletion. ***P<0.001 and **P<0.01, respectively.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

8. Figure 7
Chromatin immunoprecipitation (ChIP) analysis of E-box regulators (upstream stimulatory factor 1 (USF1), Tfe3, and Zeb1) on the transforming growth factor-β1 (TGF-β1) promoter and collagen type I α2 (Col1a2) enhancer E-box regions. (a) Upstream promoter region of the TGF-β1 gene. Arrows indicate positions of primers used for ChIP analysis. (b) ChIP analysis of USF1 on the TGF-β1 promoter E-box region. Significant increase in USF1 occupancy at 24h after HG treatment. HG, high glucose (25mmol/l); Man, Mannitol (19.5mmol/l); NG, normal glucose (5.5mmol/l). Mean±s.e.m. (n=3). (c) ChIP analysis of USF1, Tfe3, and Zeb1 on the TGF-β1 promoter E-box region. Results show significant increases in the occupancies of USF1 and Tfe3, but significant decrease in that of Zeb1 at 24h of TGF-β1 (10ng/ml) treatment relative to control (serum depletion (SD)). Mean±s.e.m. (n=3). (d) Upstream enhancer region of the Col1a2 gene. Arrows indicate positions of primers used for ChIP analysis. (e) ChIP analysis of USF1, Tfe3, and Zeb1 on Col1a2 far-upstream E-box region. Significant increase in the occupancy of Tfe3 and reciprocal decrease in that of Zeb1 were detected at 24h of TGF-β1 treatment. No significant change was detected in USF1 occupancy. Mean±s.e.m. (n=3). ***P<0.001, **P<0.01, and *P<0.05, respectively.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

9. Figure 8
miR-192 inhibitors attenuate the expression of miR-200 family members, transforming growth factor-β1 (TGF-β1), and collagen type I α2 (Col1a2) genes in mouse mesangial cells MMCs, in vitro) and mouse kidney cortex (in vivo). (a) miR-192 inhibitor significantly inhibited the expression of miR-141, miR-200b, miR-200c, Col1a2, Col4a1, and TGF-β1 in MMCs in vitro (mean and s.e., n=3). (b) In vivo effects of injecting normal mice with locked nucleic acid (LNA)-antimiR-192: 21-mer, 5′-gGctGtcAatTcaTagGtcAg-3′ (uppercase: LNA; lowercase: DNA).22 LNA-antimiR-192 significantly inhibited the expression of miR-141, miR-200b, miR-200c, Col1a2, Col4a1, and TGF-β1 in mouse kidney cortex relative to those in saline-injected control mice (mean and s.e., n=3). ***P<0.001, **P<0.01, and *P<0.05, respectively.
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions

10. Figure 9
Auto-upregulation of transforming growth factor-β1 (TGF-β1) mediated by a microRNA (miRNA) cascade in mouse mesangial cells (MMCs). Diabetic conditions increase the expression of TGF-β1 that induces miR-192 (inhibitor of Zeb1/2) probably via mechanisms involving the actions of Smad3 and p53. miR-200b/c are also upregulated because of the downregulation of Zeb1/2 by miR-192. Because miR-200b/c target Zeb1/2, this can lead to a further decrease of Zeb1/2 and further augment the expression of miR-200b/c, TGF-β1, collagen type I α2 (Col1a2), and Col4a1 through loss of repression, along with gain of activation (via Tfe3 and/or upstream stimulatory factor 1 (USF1)) at their E-boxes. These signaling loops accelerate TGF-β1 signaling and downstream gene expression involved in the progression of chronic kidney diseases like diabetic nephropathy (DN).
Kidney International  , DOI: ( /ki )
Copyright © 2011 International Society of Nephrology Terms and Conditions