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Differentially expressed microRNAs in bone marrow mesenchymal stem cell-derived microvesicles in young and older rats and their effect on tumor growth factor-β1-mediated epithelial-mesenchymal transition in HK2 cells.

Wang Y, Fu B, Sun X, Li D, Huang Q, Zhao W, Chen X - Stem Cell Res Ther (2015)

Bottom Line: A TGF-β1-mediated EMT model was used to study the effects of BM-MSC-MVs and differentially expressed miRNAs on EMT.MiR-344a, miR-133b-3p, and miR-294 affected TGF-β1-mediated EMT in HK2 cells.Among these, miR-133b-3p and miR-294 significantly inhibited TGF-β1-mediated EMT in HK2 cells (P < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu, 210029, China. wangwang_890125@sina.com.

ABSTRACT

Introduction: The prevalence of renal fibrosis is higher in older than in younger individuals. Through paracrine activity, bone marrow mesenchymal stem cell-derived microvesicles (BM-MSC-MVs) influence the process of renal fibrosis. Differences in microRNA (miRNA) expression of BM-MSC-MVs that correlate with the age of the subjects and the correlation between miRNA expression and the process of renal fibrosis have not been established. The present study aimed to analyze differences in miRNA expression of BM-MSC-MVs between young or older rats and its influence on tumor growth factor-beta 1 (TGF-β1)-mediated epithelial-mesenchymal transition (EMT) of HK2 cells to explore the causes of renal fibrosis in aged tissues.

Methods: miRCURY LNA Array (version 18.0) was used to identify differentially expressed miRNAs in BM-MSC-MVs of 3- and 24-month-old Fisher344 rats. Reverse transcription-polymerase chain reaction was used to verify miRNA levels in BM-MSC-MVs and in the serum of rats. A TGF-β1-mediated EMT model was used to study the effects of BM-MSC-MVs and differentially expressed miRNAs on EMT.

Results: BM-MSCs from older rats showed more severe aging phenotypes compared with those of young rats. In addition, the growth rate and cell migration of BM-MSCs derived from older rats were significantly reduced. In secreted BM-MSC-MVs, the expression of miR-344a, miR-133b-3p, miR-294, miR-423-3p, and miR-872-3p was significantly downregulated in older rats than in younger rats (P < 0.05), and the serum level of these miRNAs exhibited the same patterns. Intervention using BM-MSC-MVs resulted in the weakening of TGF-β1-mediated EMT in the aged rats. MiR-344a, miR-133b-3p, and miR-294 affected TGF-β1-mediated EMT in HK2 cells. Among these, miR-133b-3p and miR-294 significantly inhibited TGF-β1-mediated EMT in HK2 cells (P < 0.05).

Conclusions: In older rats, the inhibitory effect of BM-MSC-MVs on TGF-β1-mediated HK2 cell EMT was weaker than that observed in younger rats. In addition, miR-133b-3p and miR-294, which were downregulated in BM-MSC-MVs of older rats, remarkably inhibited TGF-β1-mediated EMT in HK2 cells, suggesting that these may play a role in the fibrosis of aging renal tissues.

No MeSH data available.


Related in: MedlinePlus

Effect of MVs and miRNAs on the inhibitory effect of TGF-β1 on the EMT in HK2 cells. a, c Western blot analysis of E-cadherin and α-SMA expression in HK2 cells under TGF-β1 stimulation co-cultured with Y-MV /O-MV or pre-transfection with miR-872, miR-344, miR-294, miR-133b -3p, and miR-control mimic. β-actin was used as internal control. b, d Quantification of E-cadherin and α-SMA protein levels in HK2 cells from each experimental group. **P < 0.05; n =3. α-SMA alpha-smooth muscle actin, EMT epithelial-mesenchymal transition, MV microvesicle, TGF-β1 transforming growth factor-beta 1
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Fig3: Effect of MVs and miRNAs on the inhibitory effect of TGF-β1 on the EMT in HK2 cells. a, c Western blot analysis of E-cadherin and α-SMA expression in HK2 cells under TGF-β1 stimulation co-cultured with Y-MV /O-MV or pre-transfection with miR-872, miR-344, miR-294, miR-133b -3p, and miR-control mimic. β-actin was used as internal control. b, d Quantification of E-cadherin and α-SMA protein levels in HK2 cells from each experimental group. **P < 0.05; n =3. α-SMA alpha-smooth muscle actin, EMT epithelial-mesenchymal transition, MV microvesicle, TGF-β1 transforming growth factor-beta 1

Mentions: BM-MSC-MVs derived from younger rats showed significant inhibitory effects on TGF-β1-mediated EMT in HK2 cells. The addition of BM-MSC-MVs from younger rats to TGF-β1 resulted in the stimulation of HK2 cells to reverse the downregulated E-cadherin expression and upregulated α-SMA expression (P < 0.05 for both). On the other hand, BM-MSC-MVs derived from older rats did not show such effects (Fig. 3a, b). These observations of EMT marker expression in HK2 cells subjected to various treatments were further confirmed by immunofluorescence staining (Figs. 4a–d and 5a–d).Fig. 3


Differentially expressed microRNAs in bone marrow mesenchymal stem cell-derived microvesicles in young and older rats and their effect on tumor growth factor-β1-mediated epithelial-mesenchymal transition in HK2 cells.

Wang Y, Fu B, Sun X, Li D, Huang Q, Zhao W, Chen X - Stem Cell Res Ther (2015)

Effect of MVs and miRNAs on the inhibitory effect of TGF-β1 on the EMT in HK2 cells. a, c Western blot analysis of E-cadherin and α-SMA expression in HK2 cells under TGF-β1 stimulation co-cultured with Y-MV /O-MV or pre-transfection with miR-872, miR-344, miR-294, miR-133b -3p, and miR-control mimic. β-actin was used as internal control. b, d Quantification of E-cadherin and α-SMA protein levels in HK2 cells from each experimental group. **P < 0.05; n =3. α-SMA alpha-smooth muscle actin, EMT epithelial-mesenchymal transition, MV microvesicle, TGF-β1 transforming growth factor-beta 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587922&req=5

Fig3: Effect of MVs and miRNAs on the inhibitory effect of TGF-β1 on the EMT in HK2 cells. a, c Western blot analysis of E-cadherin and α-SMA expression in HK2 cells under TGF-β1 stimulation co-cultured with Y-MV /O-MV or pre-transfection with miR-872, miR-344, miR-294, miR-133b -3p, and miR-control mimic. β-actin was used as internal control. b, d Quantification of E-cadherin and α-SMA protein levels in HK2 cells from each experimental group. **P < 0.05; n =3. α-SMA alpha-smooth muscle actin, EMT epithelial-mesenchymal transition, MV microvesicle, TGF-β1 transforming growth factor-beta 1
Mentions: BM-MSC-MVs derived from younger rats showed significant inhibitory effects on TGF-β1-mediated EMT in HK2 cells. The addition of BM-MSC-MVs from younger rats to TGF-β1 resulted in the stimulation of HK2 cells to reverse the downregulated E-cadherin expression and upregulated α-SMA expression (P < 0.05 for both). On the other hand, BM-MSC-MVs derived from older rats did not show such effects (Fig. 3a, b). These observations of EMT marker expression in HK2 cells subjected to various treatments were further confirmed by immunofluorescence staining (Figs. 4a–d and 5a–d).Fig. 3

Bottom Line: A TGF-β1-mediated EMT model was used to study the effects of BM-MSC-MVs and differentially expressed miRNAs on EMT.MiR-344a, miR-133b-3p, and miR-294 affected TGF-β1-mediated EMT in HK2 cells.Among these, miR-133b-3p and miR-294 significantly inhibited TGF-β1-mediated EMT in HK2 cells (P < 0.05).

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, the First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing, Jiangsu, 210029, China. wangwang_890125@sina.com.

ABSTRACT

Introduction: The prevalence of renal fibrosis is higher in older than in younger individuals. Through paracrine activity, bone marrow mesenchymal stem cell-derived microvesicles (BM-MSC-MVs) influence the process of renal fibrosis. Differences in microRNA (miRNA) expression of BM-MSC-MVs that correlate with the age of the subjects and the correlation between miRNA expression and the process of renal fibrosis have not been established. The present study aimed to analyze differences in miRNA expression of BM-MSC-MVs between young or older rats and its influence on tumor growth factor-beta 1 (TGF-β1)-mediated epithelial-mesenchymal transition (EMT) of HK2 cells to explore the causes of renal fibrosis in aged tissues.

Methods: miRCURY LNA Array (version 18.0) was used to identify differentially expressed miRNAs in BM-MSC-MVs of 3- and 24-month-old Fisher344 rats. Reverse transcription-polymerase chain reaction was used to verify miRNA levels in BM-MSC-MVs and in the serum of rats. A TGF-β1-mediated EMT model was used to study the effects of BM-MSC-MVs and differentially expressed miRNAs on EMT.

Results: BM-MSCs from older rats showed more severe aging phenotypes compared with those of young rats. In addition, the growth rate and cell migration of BM-MSCs derived from older rats were significantly reduced. In secreted BM-MSC-MVs, the expression of miR-344a, miR-133b-3p, miR-294, miR-423-3p, and miR-872-3p was significantly downregulated in older rats than in younger rats (P < 0.05), and the serum level of these miRNAs exhibited the same patterns. Intervention using BM-MSC-MVs resulted in the weakening of TGF-β1-mediated EMT in the aged rats. MiR-344a, miR-133b-3p, and miR-294 affected TGF-β1-mediated EMT in HK2 cells. Among these, miR-133b-3p and miR-294 significantly inhibited TGF-β1-mediated EMT in HK2 cells (P < 0.05).

Conclusions: In older rats, the inhibitory effect of BM-MSC-MVs on TGF-β1-mediated HK2 cell EMT was weaker than that observed in younger rats. In addition, miR-133b-3p and miR-294, which were downregulated in BM-MSC-MVs of older rats, remarkably inhibited TGF-β1-mediated EMT in HK2 cells, suggesting that these may play a role in the fibrosis of aging renal tissues.

No MeSH data available.


Related in: MedlinePlus