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Expression and preliminary functional profiling of the let-7 family during porcine ovary follicle atresia.

Cao R, Wu WJ, Zhou XL, Xiao P, Wang Y, Liu HL - Mol. Cells (2015)

Bottom Line: In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia.In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways.Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.

No MeSH data available.


Related in: MedlinePlus

Analysis of apoptosis in cultured granulosa cells transfected with let-7 mimics. Granulosa cells transfected with negative control (NC) (A), let-7a/b/c/i mimics (B) or let-7g mimic (C) were stained with Annexin V and PI. The apoptosis level of each group was analyzed by flow cytometry. Analysis of different apoptosis rates in NC, let-7a/b/c/i co-transfection group and let-7g transfection group (D). Data are presented as mean ± SE. A one-way ANOVA test was used for multiple comparisons. P < 0.05 is shown as *. Morphological changes in granulosa cells transfected with NC, let-7a/b/c/i and let-7g mimics (from left to right) were stained with Hoechst33342 and images were obtained under a fluorescence microscope (E) (bar = 20 μm).
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f6-molce-38-4-304: Analysis of apoptosis in cultured granulosa cells transfected with let-7 mimics. Granulosa cells transfected with negative control (NC) (A), let-7a/b/c/i mimics (B) or let-7g mimic (C) were stained with Annexin V and PI. The apoptosis level of each group was analyzed by flow cytometry. Analysis of different apoptosis rates in NC, let-7a/b/c/i co-transfection group and let-7g transfection group (D). Data are presented as mean ± SE. A one-way ANOVA test was used for multiple comparisons. P < 0.05 is shown as *. Morphological changes in granulosa cells transfected with NC, let-7a/b/c/i and let-7g mimics (from left to right) were stained with Hoechst33342 and images were obtained under a fluorescence microscope (E) (bar = 20 μm).

Mentions: To explore the function of let-7 family in cultured granulosa cells, we divided the let-7 family into two groups: let-7a/b/c/i and let-7g. Let-7a, let-7b, let-7c and let-7i mimic were co-transfected into granulosa cells in vitro, while we transfected let-7g alone into cultured granulosa cells; NC (a scrambled sequence) was used as the normalized control. After 72 h incubation, we stained the transfected granulosa cells with Annexin V-FITC and propidium iodide (PI). The fluorescence-activated cell sorting (FACS) analyses are shown in Figs. 6A, 6B and 6C. The extent of apoptosis in granulosa cells transfected with the let-7g mimic increased significantly (Fig. 6D) relative to NC. This suggested that overexpression of let-7g could induce apoptosis in granulosa cells in vitro. In addition, typical apoptotic nuclei including nuclear shrinkage, chromatin condensation and fragmentation were detected by staining with Hoechst33342 when transfected with let-7g (Fig. 6E), which indicated those cells could be in the apoptosis processes.


Expression and preliminary functional profiling of the let-7 family during porcine ovary follicle atresia.

Cao R, Wu WJ, Zhou XL, Xiao P, Wang Y, Liu HL - Mol. Cells (2015)

Analysis of apoptosis in cultured granulosa cells transfected with let-7 mimics. Granulosa cells transfected with negative control (NC) (A), let-7a/b/c/i mimics (B) or let-7g mimic (C) were stained with Annexin V and PI. The apoptosis level of each group was analyzed by flow cytometry. Analysis of different apoptosis rates in NC, let-7a/b/c/i co-transfection group and let-7g transfection group (D). Data are presented as mean ± SE. A one-way ANOVA test was used for multiple comparisons. P < 0.05 is shown as *. Morphological changes in granulosa cells transfected with NC, let-7a/b/c/i and let-7g mimics (from left to right) were stained with Hoechst33342 and images were obtained under a fluorescence microscope (E) (bar = 20 μm).
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Related In: Results  -  Collection

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f6-molce-38-4-304: Analysis of apoptosis in cultured granulosa cells transfected with let-7 mimics. Granulosa cells transfected with negative control (NC) (A), let-7a/b/c/i mimics (B) or let-7g mimic (C) were stained with Annexin V and PI. The apoptosis level of each group was analyzed by flow cytometry. Analysis of different apoptosis rates in NC, let-7a/b/c/i co-transfection group and let-7g transfection group (D). Data are presented as mean ± SE. A one-way ANOVA test was used for multiple comparisons. P < 0.05 is shown as *. Morphological changes in granulosa cells transfected with NC, let-7a/b/c/i and let-7g mimics (from left to right) were stained with Hoechst33342 and images were obtained under a fluorescence microscope (E) (bar = 20 μm).
Mentions: To explore the function of let-7 family in cultured granulosa cells, we divided the let-7 family into two groups: let-7a/b/c/i and let-7g. Let-7a, let-7b, let-7c and let-7i mimic were co-transfected into granulosa cells in vitro, while we transfected let-7g alone into cultured granulosa cells; NC (a scrambled sequence) was used as the normalized control. After 72 h incubation, we stained the transfected granulosa cells with Annexin V-FITC and propidium iodide (PI). The fluorescence-activated cell sorting (FACS) analyses are shown in Figs. 6A, 6B and 6C. The extent of apoptosis in granulosa cells transfected with the let-7g mimic increased significantly (Fig. 6D) relative to NC. This suggested that overexpression of let-7g could induce apoptosis in granulosa cells in vitro. In addition, typical apoptotic nuclei including nuclear shrinkage, chromatin condensation and fragmentation were detected by staining with Hoechst33342 when transfected with let-7g (Fig. 6E), which indicated those cells could be in the apoptosis processes.

Bottom Line: In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia.In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways.Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Genetics, Breeding and Reproduction, College of Animal Science and Technology, Nanjing Agricultural University, Nanjing, 210095, China.

ABSTRACT
Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.

No MeSH data available.


Related in: MedlinePlus