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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

Specific, quantitative detection of let-7 family by stem-loop RT-PCR and sequencing. Let-7 miRNA sequences were amplified by PCR and the PCR products were fractionated by electrophoresis on 4% agarose gels. Each band represented one member of the let-7 family. Let-7 family miRNAs were cloned into vector pMD18-T and their sequences determined (B–F).
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f3-molce-38-4-304: Specific, quantitative detection of let-7 family by stem-loop RT-PCR and sequencing. Let-7 miRNA sequences were amplified by PCR and the PCR products were fractionated by electrophoresis on 4% agarose gels. Each band represented one member of the let-7 family. Let-7 family miRNAs were cloned into vector pMD18-T and their sequences determined (B–F).

Mentions: To evaluate the specificity of Stem-loop RT-PCR for the let-7 family, we chose let-7a/b/c/g/i as candidates and PCR was conducted to amplify miRNA cDNA. Agarose gel electrophoresis confirmed the length of the PCR products (64 bp). As shown in Fig. 3A, all the PCR products were slightly above 60 bp, without hairpins or dimers. Furthermore, TA cloning and sequencing confirmed the let-7 family mature sequences. In eight successful sequencing samples, seven samples’ sequences matched with let-7a mature sequences (Fig. 3B). Let-7b was successfully identified in six out of 11 sequenced samples (Fig. 3C), let-7c was successfully identified in eight out of nine sequenced samples (Fig. 3D), let-7i was successfully identified in six out of eight sequenced samples (Fig. 3E) and let-7g was successfully identified in 19 out of 21 sequenced samples (Fig. 3F). All the unmatched sequences did not match the other members of let-7 family.


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)

Specific, quantitative detection of let-7 family by stem-loop RT-PCR and sequencing. Let-7 miRNA sequences were amplified by PCR and the PCR products were fractionated by electrophoresis on 4% agarose gels. Each band represented one member of the let-7 family. Let-7 family miRNAs were cloned into vector pMD18-T and their sequences determined (B–F).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4400304&req=5

f3-molce-38-4-304: Specific, quantitative detection of let-7 family by stem-loop RT-PCR and sequencing. Let-7 miRNA sequences were amplified by PCR and the PCR products were fractionated by electrophoresis on 4% agarose gels. Each band represented one member of the let-7 family. Let-7 family miRNAs were cloned into vector pMD18-T and their sequences determined (B–F).
Mentions: To evaluate the specificity of Stem-loop RT-PCR for the let-7 family, we chose let-7a/b/c/g/i as candidates and PCR was conducted to amplify miRNA cDNA. Agarose gel electrophoresis confirmed the length of the PCR products (64 bp). As shown in Fig. 3A, all the PCR products were slightly above 60 bp, without hairpins or dimers. Furthermore, TA cloning and sequencing confirmed the let-7 family mature sequences. In eight successful sequencing samples, seven samples’ sequences matched with let-7a mature sequences (Fig. 3B). Let-7b was successfully identified in six out of 11 sequenced samples (Fig. 3C), let-7c was successfully identified in eight out of nine sequenced samples (Fig. 3D), let-7i was successfully identified in six out of eight sequenced samples (Fig. 3E) and let-7g was successfully identified in 19 out of 21 sequenced samples (Fig. 3F). All the unmatched sequences did not match the other members of let-7 family.

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