MicroRNA-29b inhibits TGF-β1-induced fibrosis via regulation of the TGF-β1/Smad pathway in primary human endometrial stromal cells

Abstract

Transforming growth factor (TGF)‑β1 has a key role in the regulation of fibrosis and organ dysfunction. During the pathogenesis and progression of vital organ fibrosis, the microRNA (miR)‑29 family is irregularly downregulated and exogenous supplementation of miR‑29b has a strong anti‑fibrotic capacity. However, whether TGF‑β1 is able to provoke endometrial fibrosis, and the role of miR‑29 in endometrial fibrosis remain unclear. In the present study, RT‑qPCR, immunocytochemistry, western blot analysis, scanning electron microscopy, immunofluorescence staining, cell proliferation assay and flow cytometric analysis were employed. The results demonstrated that the expression levels of collagen, type 1, alpha 1 (COL1A1), α‑smooth muscle actin (α‑SMA) and phosphorylated (p)‑Smad2/3 were increased, whereas miR‑29b and maternally expressed gene 3 (MEG3) were decreased in primary endometrial stromal cells (ESCs) in response to TGF‑β1 stimulation, in a time and dose‑dependent manner. Furthermore, overexpression of miR‑29b markedly reduced the expression levels of COL1A1 and α‑SMA, and decreased the expression and nuclear accumulation of p‑Smad2/3. In addition, ectopic overexpression of miR‑29b increased the expression levels of MEG3, inhibited myofibroblast‑like cell proliferation and induced apoptosis. These findings indicated that miR‑29b may have a significant anti‑fibrotic role, and may attenuate TGF‑β1‑induced fibrosis in ESCs. Therefore, exogenous miR‑29b may serve as a potential therapeutic agent for the treatment of endometrial fibrosis.

Figure 1

Observation and identification of endometrial stromal cells (ESCs) (magnification, ×200). (A) 3^rd and 6^th generation ESCs were observed under an inverted phase contrast microscope. The cells exhibited a fiber-like morphology, and the majority of ESCs formed tightly parallel arrays and were able to grow to confluence. (B) Immunocytochemical staining demonstrated that the ESCs were positively stained for the stromal marker vimentin (brown), and negative for the epithelial cell marker cytokeratin 18. The purity of the stromal cells was >98%. Scale bar, 100 µm.

Figure 2

Pro-fibrotic effects of transforming growth factor (TGF)-β1 in endometrial stromal cells (ESCs). Primary ESCs were treated with TGF-β1 (0, 1, 5 or 10 ng/ml) for 48 h, or TGF-β1 (10 ng/ml) for the indicated time (12, 24, 48 or 72 h) in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum. (A) Time and dose-dependent effects of TGF-β1 on the mRNA expression levels of collagen, type 1, alpha 1 (COL1A1), α-smooth muscle actin (α-SMA), microRNA (miR)-29b and maternally expressed gene 3 (MEG3), as analyzed by reverse transcription-quantitative polymerase chain reaction. (B) Time and dose-dependent effects of TGF-β1 on the protein expression levels of COL1A1, α-SMA and p-Smad2/3 as analyzed by western blotting. (C) Scanning electron microscopy images demonstrated the morphological changes of primary ESCs into spindle-shaped myofibroblast-like cells in response to continuous TGF-β1 stimulation (10 ng/ml) for 4 days. The results are expressed as relative expression against control expression without treatment. Data are presented as the mean ± standard error of the mean. ^*P<0.05 vs. the control group (12 h or 0 ng/ml). Scale bar, 10 or 50 µm.

Figure 3

Anti-fibrotic effects of microRNA (miR)-29b on transforming growth factor (TGF)-β1-treated endometrial stromal cells (ESCs). miR-29b mimics (50 nM) were transfected before or after TGF-β1 (10 ng/ml) treatment of ESCs. (A) In the cells that underwent preventive treatment, exogenous miR-29b significantly decreased the mRNA expression levels of collagen, type 1, α 1 (COL1A1) and α-smooth muscle actin (α-SMA), and increased the expression levels of maternally expressed 3 (MEG3), as compared with the TGF-β1-treated group, as determined by reverse transcription-quantitative polymerase chain reaction analysis. Similar results were observed for the therapeutic treatment. (B) In the cells that underwent preventive treatment, western blot analysis detected a marked downregulation of COL1A1, α-SMA and phosphorylated (p)-Smad2/3 protein expression. (C) In the cells that underwent therapeutic treatment, COL1A1, α-SMA and p-Smad2/3 protein expression levels were decreased, as assessed by western blotting in ESCs transfected with miR-29b mimics following pretreatment with TGF-β1 for 24 h, as compared with the TGF-β1 group. Data are presented as the mean ± standard error of the mean. ^*P<0.05, compared with the control group; ^#P<0. 05 compared with the TGF-β1 group. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

Collagen, type 1, alpha 1 (COL1A1), α-smooth muscle actin (α-SMA) and phosphorylated (p)-Smad2/3 protein expression was detected by immunofluorescence staining. (A) Transforming growth factor (TGF)-β1-treated group exhibited high expression of COL1A1 and α-SMA. Transfection with microRNA (miR)-29b mimics following TGF-β1 stimulation resulted in downregulation of the expression of COL1A1 and α-SMA. (B) TGF-β1-treated group exhibited high expression of intranuclear p-Smad2/3; however, overexpression of miR-29b decreased p-Smad2/3 nuclear translocation and accumulation. Scale bar, 200 µm. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5

Effects of microRNA (miR)-29b on cell proliferation, cell cycle distribution and apoptosis of transforming growth factor (TGF)-β1-treated endometrial stromal cells (ESCs). (A) Effects of miR-29b on apoptosis in TGF-β1-treated ESCs. miR-29b exhibited significant pro-apoptotic effects on activated ESCs, as compared with the control and TGF-β1 groups. (B) Effects of miR-29b on cell cycle distribution in TGF-β1-treated ESCs. Transfection with miR-29b increased the percentage of activated ESCs in G₀/G₁ and G₂/M phases, and decreased the population of cells in S phase, as compared with the control and TGF-β1 groups. (C) Role of miR-29b in regulating TGF-β1-treated ESCs proliferation was determined by Cell Counting kit (CCK)-8 assay. Overexpression of miR-29b significantly inhibited the proliferation of fibrotic ESCs. Data are presented as the mean ± standard error of the mean.^*P<0.05, compared with the control group; ^#P<0. 05, compared with the TGF-β1 group. OD, optical density.