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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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Overexpressed exogenous miRNA let-7b decreased proliferation of α6+/β1+EpSCs by interfering with CCND1 expression. (A) Between 48–72 h, α6+/β1+EpSCs transfected with miR let-7b divided significantly more slowly than α6+/β1+EpSCs transfected with miR-Mut (*P<0.05, vs. miR-Mut; n=3). (B) RT-qPCR demonstrated that p53 mRNA expression was markedly higher in α6+/β1+EpSCs transfected with miR let-7b than that in α6+/β1+EpSCs transfected with miR-Mut. By contrast, mRNA expression levels of the cell cycle-related factors, CCND1 and CDK4, were markedly lower in α6+/β1+EpSCs transfected with miR let-7b than in α6+/β1+EpSCs transfected with miR-Mut (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (C) Western blotting confirmed that p53 protein expression was significantly increased in α6+/β1+EpSCs transfected with miR let-7b. The expression of CCND1 and CDK4 proteins was significantly decreased in α6+/β1+EpSCs transfected with miR let-7b (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (D) Northern blotting demonstrated a strong let-7b hybridization signal in α6+/β1+EpSCs transfected with miR let-7 compared with that in α6+/β1+EpSCs transfected with miR-Mut. (E) FCM demonstrated that α6+/β1+EpSCs transfected with miR let-7b were arrested in the G2/M phase, and that the percentage of cells in the S phase was significantly reduced. EpSCs, epidermal stem cells; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4; RT-qPCR, quantitative reverse transcription-polymerase chain reaction; FCM, flow cytometry.
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f4-mmr-12-04-4821: Overexpressed exogenous miRNA let-7b decreased proliferation of α6+/β1+EpSCs by interfering with CCND1 expression. (A) Between 48–72 h, α6+/β1+EpSCs transfected with miR let-7b divided significantly more slowly than α6+/β1+EpSCs transfected with miR-Mut (*P<0.05, vs. miR-Mut; n=3). (B) RT-qPCR demonstrated that p53 mRNA expression was markedly higher in α6+/β1+EpSCs transfected with miR let-7b than that in α6+/β1+EpSCs transfected with miR-Mut. By contrast, mRNA expression levels of the cell cycle-related factors, CCND1 and CDK4, were markedly lower in α6+/β1+EpSCs transfected with miR let-7b than in α6+/β1+EpSCs transfected with miR-Mut (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (C) Western blotting confirmed that p53 protein expression was significantly increased in α6+/β1+EpSCs transfected with miR let-7b. The expression of CCND1 and CDK4 proteins was significantly decreased in α6+/β1+EpSCs transfected with miR let-7b (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (D) Northern blotting demonstrated a strong let-7b hybridization signal in α6+/β1+EpSCs transfected with miR let-7 compared with that in α6+/β1+EpSCs transfected with miR-Mut. (E) FCM demonstrated that α6+/β1+EpSCs transfected with miR let-7b were arrested in the G2/M phase, and that the percentage of cells in the S phase was significantly reduced. EpSCs, epidermal stem cells; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4; RT-qPCR, quantitative reverse transcription-polymerase chain reaction; FCM, flow cytometry.

Mentions: In order to determine whether the addition of exogenous miRNA let-7b influences α6+/β1+EpSC proliferation, recombinant lentiviruses expressing miR-let-7b or miR-Mut were transfected into α6+/β1+EpSCs. Following expression of miRNA let-7b, no significant differences in the number of cells were detected between the two groups from 0-24 h (Fig. 4A). However, between 48–72 h, α6+/β1+EpSCs transfected with miR-let-7b divided significantly less rapidly than α6+/β1+EpSCs transfected with miR-Mut (Table III). The effects of the transfected miRNA on mRNA and protein expression were detected by RT-qPCR and northern blotting, and western blotting, respectively. The RT-qPCR results demonstrated that mRNA expression of p53 was markedly higher in α6+/β1+EpSCs transfected with miR-let-7b than that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4B). By contrast, mRNA expression of the cell cycle-related proteins, CCND1 and CDK4, was lower in α6+/β1+EpSCs transfected with miR-let-7b than that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4B). The western blotting results confirmed that p53 protein expression was significantly increased in α6+/β1+EpSCs transfected with miR-let-7b compared with that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4C). The expression of the CCND1 and CDK4 proteins was significantly decreased in α6+/β1+EpSCs transfected with miR-let-7b compared with that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4E). Northern blotting demonstrated a strong let-7b hybridization signal in α6+/β1+EpSCs transfected with miR-let-7 compared with the signal in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4D). Furthermore, FCM demonstrated significant cell cycle arrest in α6+/β1+EpSCs transfected with miR-let-7b. Compared with α6+/β1+EpSCs transfected with miR-Mut, α6+/β1+EpSCs transfected with miR-let-7b were arrested in the G2/M phase, and the percentage of S-phase cells in this group was significantly decreased (Fig. 4E). These results indicate that proliferation of the α6+/β1+EpSC subpopulation decreased when the expression of cell cycle-related proteins was suppressed by the addition of exogenous let-7b miRNA.


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)

Overexpressed exogenous miRNA let-7b decreased proliferation of α6+/β1+EpSCs by interfering with CCND1 expression. (A) Between 48–72 h, α6+/β1+EpSCs transfected with miR let-7b divided significantly more slowly than α6+/β1+EpSCs transfected with miR-Mut (*P<0.05, vs. miR-Mut; n=3). (B) RT-qPCR demonstrated that p53 mRNA expression was markedly higher in α6+/β1+EpSCs transfected with miR let-7b than that in α6+/β1+EpSCs transfected with miR-Mut. By contrast, mRNA expression levels of the cell cycle-related factors, CCND1 and CDK4, were markedly lower in α6+/β1+EpSCs transfected with miR let-7b than in α6+/β1+EpSCs transfected with miR-Mut (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (C) Western blotting confirmed that p53 protein expression was significantly increased in α6+/β1+EpSCs transfected with miR let-7b. The expression of CCND1 and CDK4 proteins was significantly decreased in α6+/β1+EpSCs transfected with miR let-7b (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (D) Northern blotting demonstrated a strong let-7b hybridization signal in α6+/β1+EpSCs transfected with miR let-7 compared with that in α6+/β1+EpSCs transfected with miR-Mut. (E) FCM demonstrated that α6+/β1+EpSCs transfected with miR let-7b were arrested in the G2/M phase, and that the percentage of cells in the S phase was significantly reduced. EpSCs, epidermal stem cells; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4; RT-qPCR, quantitative reverse transcription-polymerase chain reaction; FCM, flow cytometry.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-mmr-12-04-4821: Overexpressed exogenous miRNA let-7b decreased proliferation of α6+/β1+EpSCs by interfering with CCND1 expression. (A) Between 48–72 h, α6+/β1+EpSCs transfected with miR let-7b divided significantly more slowly than α6+/β1+EpSCs transfected with miR-Mut (*P<0.05, vs. miR-Mut; n=3). (B) RT-qPCR demonstrated that p53 mRNA expression was markedly higher in α6+/β1+EpSCs transfected with miR let-7b than that in α6+/β1+EpSCs transfected with miR-Mut. By contrast, mRNA expression levels of the cell cycle-related factors, CCND1 and CDK4, were markedly lower in α6+/β1+EpSCs transfected with miR let-7b than in α6+/β1+EpSCs transfected with miR-Mut (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (C) Western blotting confirmed that p53 protein expression was significantly increased in α6+/β1+EpSCs transfected with miR let-7b. The expression of CCND1 and CDK4 proteins was significantly decreased in α6+/β1+EpSCs transfected with miR let-7b (**P<0.01, vs. miR-Mut and *P<0.05, vs. miR-Mut; n=3). (D) Northern blotting demonstrated a strong let-7b hybridization signal in α6+/β1+EpSCs transfected with miR let-7 compared with that in α6+/β1+EpSCs transfected with miR-Mut. (E) FCM demonstrated that α6+/β1+EpSCs transfected with miR let-7b were arrested in the G2/M phase, and that the percentage of cells in the S phase was significantly reduced. EpSCs, epidermal stem cells; CCND1, cyclin D1; CDK4, cyclin-dependent kinase 4; RT-qPCR, quantitative reverse transcription-polymerase chain reaction; FCM, flow cytometry.
Mentions: In order to determine whether the addition of exogenous miRNA let-7b influences α6+/β1+EpSC proliferation, recombinant lentiviruses expressing miR-let-7b or miR-Mut were transfected into α6+/β1+EpSCs. Following expression of miRNA let-7b, no significant differences in the number of cells were detected between the two groups from 0-24 h (Fig. 4A). However, between 48–72 h, α6+/β1+EpSCs transfected with miR-let-7b divided significantly less rapidly than α6+/β1+EpSCs transfected with miR-Mut (Table III). The effects of the transfected miRNA on mRNA and protein expression were detected by RT-qPCR and northern blotting, and western blotting, respectively. The RT-qPCR results demonstrated that mRNA expression of p53 was markedly higher in α6+/β1+EpSCs transfected with miR-let-7b than that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4B). By contrast, mRNA expression of the cell cycle-related proteins, CCND1 and CDK4, was lower in α6+/β1+EpSCs transfected with miR-let-7b than that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4B). The western blotting results confirmed that p53 protein expression was significantly increased in α6+/β1+EpSCs transfected with miR-let-7b compared with that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4C). The expression of the CCND1 and CDK4 proteins was significantly decreased in α6+/β1+EpSCs transfected with miR-let-7b compared with that in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4E). Northern blotting demonstrated a strong let-7b hybridization signal in α6+/β1+EpSCs transfected with miR-let-7 compared with the signal in α6+/β1+EpSCs transfected with miR-Mut (Fig. 4D). Furthermore, FCM demonstrated significant cell cycle arrest in α6+/β1+EpSCs transfected with miR-let-7b. Compared with α6+/β1+EpSCs transfected with miR-Mut, α6+/β1+EpSCs transfected with miR-let-7b were arrested in the G2/M phase, and the percentage of S-phase cells in this group was significantly decreased (Fig. 4E). These results indicate that proliferation of the α6+/β1+EpSC subpopulation decreased when the expression of cell cycle-related proteins was suppressed by the addition of exogenous let-7b miRNA.

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