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Expression of microRNA‑26b and identification of its target gene EphA2 in pituitary tissues in Yanbian cattle.

Yuan B, Yu WY, Dai LS, Gao Y, Ding Y, Yu XF, Chen J, Zhang JB - Mol Med Rep (2015)

Bottom Line: The results of a Luciferase reporter system assay revealed that miR‑26b is able to suppress EphA2 expression at the transcription level.Following the site‑directed mutagenesis of plasmid EphA2 3'‑UTR pmirGLO‑MUT‑ and miR‑26b mimic‑transfected HeLa cells, the dual‑luciferase reporter gene assay revealed that there were three consecutive nucleotide mutations in the 3'‑UTR, binding with the predicted seed region.This may have caused the miR‑26b inhibition of luciferase activity to decrease from 60% in the wild‑type to 26%, suggesting that miR‑26b achieved its function via binding with the TACTTGAA sequence of the 3'‑UTR in EphA2.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Center, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, P.R. China.

ABSTRACT
microRNAs (miRNAs/miRs) are a class of single‑stranded non‑coding RNA molecules of 19‑24 nucleotides (nt) in length. They are widely expressed in animals, plants, bacteria and viruses. Via specific mRNA complementary pairing of target genes, miRNAs are able to regulate the expression of mRNA levels or inhibit protein translation following transcription. miRNA expression has a time‑ and space specificity, and it is involved in cell proliferation and differentiation, apoptosis, development, tumor metastasis occurrence and other biological processes. miR‑26b is an miRNA of 22 nt and is important in the regulation of cellular processes. With the advancement of molecular biology techniques in recent years, there have been extensive investigations into miR‑26b. Numerous studies have observed that miR‑26b is involved in early embryonic development, cell proliferation regulation, pituitary hormone secretion and other physiological activities. miRNAs are associated with the function of propagation. The present study used reverse transcription quantitative polymerase chain reaction to detect the relative expression levels of miR‑26b in the pituitary tissue of Yanbian cattle at different developmental stages. The 2‑∆∆Ct method was used to calculate the relative gene expression levels. The miRNA target gene database TargetScan and RNA22 were used for prediction of the miR‑26b target gene and selective recognition was also performed. The results demonstrated that miR‑26b is expressed in the pituitary tissues of Yanbian cattle at 6 and 24 months of age. The relative expression levels of miR‑26b in the pituitary tissues of 24‑month‑old Yanbian cattle were 2.41 times that of those in the six‑month‑old Yanbian cattle, demonstrating significant differences in the relative expression (P<0.01). The relative expression of the candidate target genes, EphA2 and miR‑26b, exhibited the opposite expression pattern. The relative expression levels in the pituitary tissues of six‑month‑old Yanbian cattle were 3.34 times that of those in 24‑month‑old Yanbian cattle (P<0.01). There are miR‑26b binding sites in the 3'‑untranslated region (3'‑UTR) of EphA2 in bovine, human, murine and other mammalian mRNAs, suggesting that the EphA2 gene may be a target gene of miR‑26b. The results of a Luciferase reporter system assay revealed that miR‑26b is able to suppress EphA2 expression at the transcription level. Following the site‑directed mutagenesis of plasmid EphA2 3'‑UTR pmirGLO‑MUT‑ and miR‑26b mimic‑transfected HeLa cells, the dual‑luciferase reporter gene assay revealed that there were three consecutive nucleotide mutations in the 3'‑UTR, binding with the predicted seed region. This may have caused the miR‑26b inhibition of luciferase activity to decrease from 60% in the wild‑type to 26%, suggesting that miR‑26b achieved its function via binding with the TACTTGAA sequence of the 3'‑UTR in EphA2. In conclusion, the present study successfully assessed the expression pattern of miR‑26b in the pituitary tissue of Yanbian cattle, and also confirmed that EphA2 was a target gene of miR‑26b in Yanbian cattle in vitro. The present study provided the theoretical basis to further investigate the role of miR‑26b in early embryonic development, pituitary hormone secretion and other reproductive functions.

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Identification of miR-26b target sites in EphA2. 1 represents EphA2 and 3′-UTR pmirGLO and the miR-26b mimics co-transfected HeLa cells. 2 represents EphA2 3′-UTR pmirGLO and negative control co-transfected Hela cells. Values are expressed as the mean ± standard error (n=3). **P<0.01. UTR, untranslated region; miR, microRNA.
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f15-mmr-12-04-5753: Identification of miR-26b target sites in EphA2. 1 represents EphA2 and 3′-UTR pmirGLO and the miR-26b mimics co-transfected HeLa cells. 2 represents EphA2 3′-UTR pmirGLO and negative control co-transfected Hela cells. Values are expressed as the mean ± standard error (n=3). **P<0.01. UTR, untranslated region; miR, microRNA.

Mentions: The dual-luciferase gene reporter assay demonstrated that the seed region of miR-26b specifically binds with the mutation in the 3′-UTR of candidate target gene, causing miR-26b inhibition of the luciferase activity to decrease 60% (100–40%; Fig. 14) and 26% (100–74%; Fig. 15) in the wild-type. This suggested that the predicted binding sites were miR-26b activation sites. The cell luciferase relative expression results following transfection are shown in Fig. 15. The results suggested that the miR-26b seed sequence AUGAACUU exerted a function via binding with the TACTTGAA sequence of the 3′-UTR in EphA2. Therefore, it was hypothesized that the TACTTGAA sequence in the 3′-UTR of EphA2 was the target sequence of miR-26b.


Expression of microRNA‑26b and identification of its target gene EphA2 in pituitary tissues in Yanbian cattle.

Yuan B, Yu WY, Dai LS, Gao Y, Ding Y, Yu XF, Chen J, Zhang JB - Mol Med Rep (2015)

Identification of miR-26b target sites in EphA2. 1 represents EphA2 and 3′-UTR pmirGLO and the miR-26b mimics co-transfected HeLa cells. 2 represents EphA2 3′-UTR pmirGLO and negative control co-transfected Hela cells. Values are expressed as the mean ± standard error (n=3). **P<0.01. UTR, untranslated region; miR, microRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581756&req=5

f15-mmr-12-04-5753: Identification of miR-26b target sites in EphA2. 1 represents EphA2 and 3′-UTR pmirGLO and the miR-26b mimics co-transfected HeLa cells. 2 represents EphA2 3′-UTR pmirGLO and negative control co-transfected Hela cells. Values are expressed as the mean ± standard error (n=3). **P<0.01. UTR, untranslated region; miR, microRNA.
Mentions: The dual-luciferase gene reporter assay demonstrated that the seed region of miR-26b specifically binds with the mutation in the 3′-UTR of candidate target gene, causing miR-26b inhibition of the luciferase activity to decrease 60% (100–40%; Fig. 14) and 26% (100–74%; Fig. 15) in the wild-type. This suggested that the predicted binding sites were miR-26b activation sites. The cell luciferase relative expression results following transfection are shown in Fig. 15. The results suggested that the miR-26b seed sequence AUGAACUU exerted a function via binding with the TACTTGAA sequence of the 3′-UTR in EphA2. Therefore, it was hypothesized that the TACTTGAA sequence in the 3′-UTR of EphA2 was the target sequence of miR-26b.

Bottom Line: The results of a Luciferase reporter system assay revealed that miR‑26b is able to suppress EphA2 expression at the transcription level.Following the site‑directed mutagenesis of plasmid EphA2 3'‑UTR pmirGLO‑MUT‑ and miR‑26b mimic‑transfected HeLa cells, the dual‑luciferase reporter gene assay revealed that there were three consecutive nucleotide mutations in the 3'‑UTR, binding with the predicted seed region.This may have caused the miR‑26b inhibition of luciferase activity to decrease from 60% in the wild‑type to 26%, suggesting that miR‑26b achieved its function via binding with the TACTTGAA sequence of the 3'‑UTR in EphA2.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Center, College of Animal Sciences, Jilin University, Changchun, Jilin 130062, P.R. China.

ABSTRACT
microRNAs (miRNAs/miRs) are a class of single‑stranded non‑coding RNA molecules of 19‑24 nucleotides (nt) in length. They are widely expressed in animals, plants, bacteria and viruses. Via specific mRNA complementary pairing of target genes, miRNAs are able to regulate the expression of mRNA levels or inhibit protein translation following transcription. miRNA expression has a time‑ and space specificity, and it is involved in cell proliferation and differentiation, apoptosis, development, tumor metastasis occurrence and other biological processes. miR‑26b is an miRNA of 22 nt and is important in the regulation of cellular processes. With the advancement of molecular biology techniques in recent years, there have been extensive investigations into miR‑26b. Numerous studies have observed that miR‑26b is involved in early embryonic development, cell proliferation regulation, pituitary hormone secretion and other physiological activities. miRNAs are associated with the function of propagation. The present study used reverse transcription quantitative polymerase chain reaction to detect the relative expression levels of miR‑26b in the pituitary tissue of Yanbian cattle at different developmental stages. The 2‑∆∆Ct method was used to calculate the relative gene expression levels. The miRNA target gene database TargetScan and RNA22 were used for prediction of the miR‑26b target gene and selective recognition was also performed. The results demonstrated that miR‑26b is expressed in the pituitary tissues of Yanbian cattle at 6 and 24 months of age. The relative expression levels of miR‑26b in the pituitary tissues of 24‑month‑old Yanbian cattle were 2.41 times that of those in the six‑month‑old Yanbian cattle, demonstrating significant differences in the relative expression (P<0.01). The relative expression of the candidate target genes, EphA2 and miR‑26b, exhibited the opposite expression pattern. The relative expression levels in the pituitary tissues of six‑month‑old Yanbian cattle were 3.34 times that of those in 24‑month‑old Yanbian cattle (P<0.01). There are miR‑26b binding sites in the 3'‑untranslated region (3'‑UTR) of EphA2 in bovine, human, murine and other mammalian mRNAs, suggesting that the EphA2 gene may be a target gene of miR‑26b. The results of a Luciferase reporter system assay revealed that miR‑26b is able to suppress EphA2 expression at the transcription level. Following the site‑directed mutagenesis of plasmid EphA2 3'‑UTR pmirGLO‑MUT‑ and miR‑26b mimic‑transfected HeLa cells, the dual‑luciferase reporter gene assay revealed that there were three consecutive nucleotide mutations in the 3'‑UTR, binding with the predicted seed region. This may have caused the miR‑26b inhibition of luciferase activity to decrease from 60% in the wild‑type to 26%, suggesting that miR‑26b achieved its function via binding with the TACTTGAA sequence of the 3'‑UTR in EphA2. In conclusion, the present study successfully assessed the expression pattern of miR‑26b in the pituitary tissue of Yanbian cattle, and also confirmed that EphA2 was a target gene of miR‑26b in Yanbian cattle in vitro. The present study provided the theoretical basis to further investigate the role of miR‑26b in early embryonic development, pituitary hormone secretion and other reproductive functions.

Show MeSH
Related in: MedlinePlus