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MicroRNA let-7b-regulated epidermal stem cell proliferation in hypertrophied anal papillae.

Lu H, He X, Wang Q, Zheng D, Han Y, Yang W, Liu T - Mol Med Rep (2015)

Bottom Line: The addition of exogenous microRNA let‑7 resulted in an increased expression level of mature microRNA let‑7b, while the expression of CCND1 and CDK4 was reduced.Epidermal stem cells transfected with microRNA let‑7b were arrested in the G2/M phase and the percentage of cells in S‑phase was significantly reduced.In conclusion, let‑7b expression results in upregulation of the cell cycle-related proteins, CCND1 and CDK4, resulting in the excessive proliferation that leads to the formation of hypertrophic anal papillae.

View Article: PubMed Central - PubMed

Affiliation: Department of Anorectal Dermatology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China.

ABSTRACT
The present study investigated the role of epidermal stem cell-expressed microRNA let-7b in the pathogenesis of hypertrophied anal papillae. Hypertrophied anal papillae were examined for the presence of epidermal stem cells. Epidermal stem cells were identified using flow cytometry and immunofluorescent staining for the cell surface markers, integrin α6 and integrin β1 subunits. Expression levels of microRNA let‑7b in α6+/β1+and α6‑/β1‑cells were compared using reverse transcription‑quantitative polymerase chain reaction and northern blotting. Lentivirus‑mediated expression of microRNA let‑7b in epidermal stem cells was utilized in order to study the effects of this microRNA on the cell cycle proteins, cyclin D1 (CCND1) and cyclin‑dependent kinase 4 (CDK4). MicroRNA let‑7b‑overexpressing cells were examined using flow cytometry, in order to determine the effects of the microRNA on cell cycle progression. α6+/β1+epidermal stem cells were identified in hypertrophic anal papillae. Following isolation and enrichment of the α6+/β1+population, these cells were found to have a rapid rate of proliferation in vitro. The expression of cell cycle‑related proteins was elevated in this population, compared with that in α6‑/β1‑cells. The expression of microRNA let‑7b in α6+/β1+epidermal stem cells was significantly lower than that in α6‑/β1‑cells. Two microRNA let‑7b target genes, CCND1 and CDK4, were found to be upregulated in α6+/β1+cells. When the exogenous precursor, microRNA let‑7, was overexpressed in α6+/β1+ epidermal stem cells, the cell proliferation rate was significantly lower than that in cells expressing microRNA let‑7 containing a mutated seed sequence. The addition of exogenous microRNA let‑7 resulted in an increased expression level of mature microRNA let‑7b, while the expression of CCND1 and CDK4 was reduced. Epidermal stem cells transfected with microRNA let‑7b were arrested in the G2/M phase and the percentage of cells in S‑phase was significantly reduced. In conclusion, let‑7b expression results in upregulation of the cell cycle-related proteins, CCND1 and CDK4, resulting in the excessive proliferation that leads to the formation of hypertrophic anal papillae.

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α6+/β1+EpSCs proliferated more rapidly and expressed lower levels of microRNA let-7b than α6-/β1-cells. (A) The proliferation rates of α6+/β1+EpSCs and α6-/β1-cells were examined 12-72 h after passaging. Between 24-72 h, α6+/β1+EpSCs divided significantly more rapidly than α6-/β1-cells. (B) An miRNA RT-qPCR assay demonstrated that expression of miRNA let-7b was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. (C) Northern blot analysis revealed strong pre-miRNA let-7b and mature microRNA let-7b hybridization signals in α6-/β1-cells at 72 h compared with those α6+/β1+EpSCs. (D) RT-qPCR demonstrated that the mRNA expression of p53 was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. By contrast, mRNA expression of the cell cycle-related factors, CCND1 and CDK4, was significantly higher in α6+/β1+EpSCs at 72 h, compared with that in α6-/β1-cells. (E) Western blotting confirmed that p53 protein expression was significantly reduced in α6+/β1+EpSCs. The expression of CCND1 and CDK4 proteins was significantly elevated in α6+/β1+EpSCs. GAPDH served as a loading control **P<0.01, vs. α6-/β1-cells and *P<0.05, vs. α6-/β1-cells; n=3). EpSCs, Epidermal stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4.
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f3-mmr-12-04-4821: α6+/β1+EpSCs proliferated more rapidly and expressed lower levels of microRNA let-7b than α6-/β1-cells. (A) The proliferation rates of α6+/β1+EpSCs and α6-/β1-cells were examined 12-72 h after passaging. Between 24-72 h, α6+/β1+EpSCs divided significantly more rapidly than α6-/β1-cells. (B) An miRNA RT-qPCR assay demonstrated that expression of miRNA let-7b was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. (C) Northern blot analysis revealed strong pre-miRNA let-7b and mature microRNA let-7b hybridization signals in α6-/β1-cells at 72 h compared with those α6+/β1+EpSCs. (D) RT-qPCR demonstrated that the mRNA expression of p53 was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. By contrast, mRNA expression of the cell cycle-related factors, CCND1 and CDK4, was significantly higher in α6+/β1+EpSCs at 72 h, compared with that in α6-/β1-cells. (E) Western blotting confirmed that p53 protein expression was significantly reduced in α6+/β1+EpSCs. The expression of CCND1 and CDK4 proteins was significantly elevated in α6+/β1+EpSCs. GAPDH served as a loading control **P<0.01, vs. α6-/β1-cells and *P<0.05, vs. α6-/β1-cells; n=3). EpSCs, Epidermal stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4.

Mentions: The proliferation rates of α6+/β1+EpSCs and α6-/β1-cells were examined for up to 72 h following passaging. Measurements were repeated in quintuplicate. No significant differences were observed in the total number of cells between the two groups at 0–12 h (Fig. 3A). However, between 24–72 h, α6+/β1+EpSCs were found to divide significantly more rapidly than α6-/β1-cells (Table III). The miRNA RT-qPCR assay showed that the expression of miRNA let-7b was markedly lower in α6+/β1+EpSCs at 72 h than that in α6-/β1-cells (Fig. 3B). Northern blot analysis demonstrated strong let-7b pre-miRNA and mature miRNA hybridization signals in α6-/β1-cells at 72 h, compared with levels in α6+/β1+EpSCs (Fig. 3C). RT-qPCR and western blotting were used to determine difference in the expression of cell cycle-related proteins in the different groups of cells. RT-qPCR showed that mRNA levels of p53, a protein involved in apoptosis, were markedly lower in α6+/β1+EpSCs at 72 h compared with levels in α6-/β1-cells (Fig. 3D and E). By contrast, mRNA levels of the cell cycle-related proteins, CCND1 and CDK4, were markedly higher in α6+/β1+EpSCs at 72 h than those in α6-/β1-cells (Fig. 3D and E). These findings were confirmed by western blotting (Fig. 3E). These results indicate that miRNA let-7b expression is reduced in α6+/β1+EpSCs, stimulating the expression of cell cycle-related proteins and the proliferation of these cells.


MicroRNA let-7b-regulated epidermal stem cell proliferation in hypertrophied anal papillae.

Lu H, He X, Wang Q, Zheng D, Han Y, Yang W, Liu T - Mol Med Rep (2015)

α6+/β1+EpSCs proliferated more rapidly and expressed lower levels of microRNA let-7b than α6-/β1-cells. (A) The proliferation rates of α6+/β1+EpSCs and α6-/β1-cells were examined 12-72 h after passaging. Between 24-72 h, α6+/β1+EpSCs divided significantly more rapidly than α6-/β1-cells. (B) An miRNA RT-qPCR assay demonstrated that expression of miRNA let-7b was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. (C) Northern blot analysis revealed strong pre-miRNA let-7b and mature microRNA let-7b hybridization signals in α6-/β1-cells at 72 h compared with those α6+/β1+EpSCs. (D) RT-qPCR demonstrated that the mRNA expression of p53 was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. By contrast, mRNA expression of the cell cycle-related factors, CCND1 and CDK4, was significantly higher in α6+/β1+EpSCs at 72 h, compared with that in α6-/β1-cells. (E) Western blotting confirmed that p53 protein expression was significantly reduced in α6+/β1+EpSCs. The expression of CCND1 and CDK4 proteins was significantly elevated in α6+/β1+EpSCs. GAPDH served as a loading control **P<0.01, vs. α6-/β1-cells and *P<0.05, vs. α6-/β1-cells; n=3). EpSCs, Epidermal stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4581746&req=5

f3-mmr-12-04-4821: α6+/β1+EpSCs proliferated more rapidly and expressed lower levels of microRNA let-7b than α6-/β1-cells. (A) The proliferation rates of α6+/β1+EpSCs and α6-/β1-cells were examined 12-72 h after passaging. Between 24-72 h, α6+/β1+EpSCs divided significantly more rapidly than α6-/β1-cells. (B) An miRNA RT-qPCR assay demonstrated that expression of miRNA let-7b was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. (C) Northern blot analysis revealed strong pre-miRNA let-7b and mature microRNA let-7b hybridization signals in α6-/β1-cells at 72 h compared with those α6+/β1+EpSCs. (D) RT-qPCR demonstrated that the mRNA expression of p53 was markedly lower in α6+/β1+EpSCs at 72 h compared with that in α6-/β1-cells. By contrast, mRNA expression of the cell cycle-related factors, CCND1 and CDK4, was significantly higher in α6+/β1+EpSCs at 72 h, compared with that in α6-/β1-cells. (E) Western blotting confirmed that p53 protein expression was significantly reduced in α6+/β1+EpSCs. The expression of CCND1 and CDK4 proteins was significantly elevated in α6+/β1+EpSCs. GAPDH served as a loading control **P<0.01, vs. α6-/β1-cells and *P<0.05, vs. α6-/β1-cells; n=3). EpSCs, Epidermal stem cells; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4.
Mentions: The proliferation rates of α6+/β1+EpSCs and α6-/β1-cells were examined for up to 72 h following passaging. Measurements were repeated in quintuplicate. No significant differences were observed in the total number of cells between the two groups at 0–12 h (Fig. 3A). However, between 24–72 h, α6+/β1+EpSCs were found to divide significantly more rapidly than α6-/β1-cells (Table III). The miRNA RT-qPCR assay showed that the expression of miRNA let-7b was markedly lower in α6+/β1+EpSCs at 72 h than that in α6-/β1-cells (Fig. 3B). Northern blot analysis demonstrated strong let-7b pre-miRNA and mature miRNA hybridization signals in α6-/β1-cells at 72 h, compared with levels in α6+/β1+EpSCs (Fig. 3C). RT-qPCR and western blotting were used to determine difference in the expression of cell cycle-related proteins in the different groups of cells. RT-qPCR showed that mRNA levels of p53, a protein involved in apoptosis, were markedly lower in α6+/β1+EpSCs at 72 h compared with levels in α6-/β1-cells (Fig. 3D and E). By contrast, mRNA levels of the cell cycle-related proteins, CCND1 and CDK4, were markedly higher in α6+/β1+EpSCs at 72 h than those in α6-/β1-cells (Fig. 3D and E). These findings were confirmed by western blotting (Fig. 3E). These results indicate that miRNA let-7b expression is reduced in α6+/β1+EpSCs, stimulating the expression of cell cycle-related proteins and the proliferation of these cells.

Bottom Line: The addition of exogenous microRNA let‑7 resulted in an increased expression level of mature microRNA let‑7b, while the expression of CCND1 and CDK4 was reduced.Epidermal stem cells transfected with microRNA let‑7b were arrested in the G2/M phase and the percentage of cells in S‑phase was significantly reduced.In conclusion, let‑7b expression results in upregulation of the cell cycle-related proteins, CCND1 and CDK4, resulting in the excessive proliferation that leads to the formation of hypertrophic anal papillae.

View Article: PubMed Central - PubMed

Affiliation: Department of Anorectal Dermatology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China.

ABSTRACT
The present study investigated the role of epidermal stem cell-expressed microRNA let-7b in the pathogenesis of hypertrophied anal papillae. Hypertrophied anal papillae were examined for the presence of epidermal stem cells. Epidermal stem cells were identified using flow cytometry and immunofluorescent staining for the cell surface markers, integrin α6 and integrin β1 subunits. Expression levels of microRNA let‑7b in α6+/β1+and α6‑/β1‑cells were compared using reverse transcription‑quantitative polymerase chain reaction and northern blotting. Lentivirus‑mediated expression of microRNA let‑7b in epidermal stem cells was utilized in order to study the effects of this microRNA on the cell cycle proteins, cyclin D1 (CCND1) and cyclin‑dependent kinase 4 (CDK4). MicroRNA let‑7b‑overexpressing cells were examined using flow cytometry, in order to determine the effects of the microRNA on cell cycle progression. α6+/β1+epidermal stem cells were identified in hypertrophic anal papillae. Following isolation and enrichment of the α6+/β1+population, these cells were found to have a rapid rate of proliferation in vitro. The expression of cell cycle‑related proteins was elevated in this population, compared with that in α6‑/β1‑cells. The expression of microRNA let‑7b in α6+/β1+epidermal stem cells was significantly lower than that in α6‑/β1‑cells. Two microRNA let‑7b target genes, CCND1 and CDK4, were found to be upregulated in α6+/β1+cells. When the exogenous precursor, microRNA let‑7, was overexpressed in α6+/β1+ epidermal stem cells, the cell proliferation rate was significantly lower than that in cells expressing microRNA let‑7 containing a mutated seed sequence. The addition of exogenous microRNA let‑7 resulted in an increased expression level of mature microRNA let‑7b, while the expression of CCND1 and CDK4 was reduced. Epidermal stem cells transfected with microRNA let‑7b were arrested in the G2/M phase and the percentage of cells in S‑phase was significantly reduced. In conclusion, let‑7b expression results in upregulation of the cell cycle-related proteins, CCND1 and CDK4, resulting in the excessive proliferation that leads to the formation of hypertrophic anal papillae.

Show MeSH
Related in: MedlinePlus