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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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Characteristics of human hypertrophied anal papillae tissues and integrin α6+/β1+EpSCs. (A) and (B) Human hypertrophied anal papillae tissue. (C) Histology of human hypertrophied anal papillae tissue. (D) Morphology of primary cells isolated and enriched from human hypertrophied anal papillae tissue. (E) Morphology of integrin α6+/β1+EpSCs which were isolated and enriched from human hypertrophied anal papillae tissue. (F) Flow cytometry results, indicating that integrin α6+/β1+EpSCs represented 0.27±0.02% of total primary cells, which were isolated and enriched from human hypertrophied anal papillae tissue following sorting. EpSCs, epidermal stem cell markers.
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f1-mmr-12-04-4821: Characteristics of human hypertrophied anal papillae tissues and integrin α6+/β1+EpSCs. (A) and (B) Human hypertrophied anal papillae tissue. (C) Histology of human hypertrophied anal papillae tissue. (D) Morphology of primary cells isolated and enriched from human hypertrophied anal papillae tissue. (E) Morphology of integrin α6+/β1+EpSCs which were isolated and enriched from human hypertrophied anal papillae tissue. (F) Flow cytometry results, indicating that integrin α6+/β1+EpSCs represented 0.27±0.02% of total primary cells, which were isolated and enriched from human hypertrophied anal papillae tissue following sorting. EpSCs, epidermal stem cell markers.

Mentions: The anal papillae examined in this study arose due to chronic inflammation caused by fibrous connective tissue hyperplasia. The anal papillae were cylindrical or walnut-shaped, with a large upper part and smaller lower part. The smooth surface was milky white, and no bleeding was observed (Fig. 1A and B). Pathological examination demonstrated significant endothelial hyperplasia in the anal papillae, accompanied by infiltration of inflammatory cells and vascular proliferation, although no cell heterogeneity was observed (Fig. 1C). A magnetic-activated cell sorting system was used to isolate and enrich the α6+/β1+ subpopulation from the hypertrophied anal papillae. Following isolation, cells were quantified using FCM. α6+/β1+EpSCs represented 0.27±0.02% of the total population in five primary samples, whereas α6-/β1-cells represented 59.51±8.31% of the total population (Fig. 1D–F). These results demonstrated that α6+/β1+epidermal stem cells (EpSCs), although occurring at a low frequency, may be successfully enriched using magnetic-activated cell sorting.


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)

Characteristics of human hypertrophied anal papillae tissues and integrin α6+/β1+EpSCs. (A) and (B) Human hypertrophied anal papillae tissue. (C) Histology of human hypertrophied anal papillae tissue. (D) Morphology of primary cells isolated and enriched from human hypertrophied anal papillae tissue. (E) Morphology of integrin α6+/β1+EpSCs which were isolated and enriched from human hypertrophied anal papillae tissue. (F) Flow cytometry results, indicating that integrin α6+/β1+EpSCs represented 0.27±0.02% of total primary cells, which were isolated and enriched from human hypertrophied anal papillae tissue following sorting. EpSCs, epidermal stem cell markers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581746&req=5

f1-mmr-12-04-4821: Characteristics of human hypertrophied anal papillae tissues and integrin α6+/β1+EpSCs. (A) and (B) Human hypertrophied anal papillae tissue. (C) Histology of human hypertrophied anal papillae tissue. (D) Morphology of primary cells isolated and enriched from human hypertrophied anal papillae tissue. (E) Morphology of integrin α6+/β1+EpSCs which were isolated and enriched from human hypertrophied anal papillae tissue. (F) Flow cytometry results, indicating that integrin α6+/β1+EpSCs represented 0.27±0.02% of total primary cells, which were isolated and enriched from human hypertrophied anal papillae tissue following sorting. EpSCs, epidermal stem cell markers.
Mentions: The anal papillae examined in this study arose due to chronic inflammation caused by fibrous connective tissue hyperplasia. The anal papillae were cylindrical or walnut-shaped, with a large upper part and smaller lower part. The smooth surface was milky white, and no bleeding was observed (Fig. 1A and B). Pathological examination demonstrated significant endothelial hyperplasia in the anal papillae, accompanied by infiltration of inflammatory cells and vascular proliferation, although no cell heterogeneity was observed (Fig. 1C). A magnetic-activated cell sorting system was used to isolate and enrich the α6+/β1+ subpopulation from the hypertrophied anal papillae. Following isolation, cells were quantified using FCM. α6+/β1+EpSCs represented 0.27±0.02% of the total population in five primary samples, whereas α6-/β1-cells represented 59.51±8.31% of the total population (Fig. 1D–F). These results demonstrated that α6+/β1+epidermal stem cells (EpSCs), although occurring at a low frequency, may be successfully enriched using magnetic-activated cell sorting.

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