Identification of target genes for adenohypophysis-prefer miR-7 and miR-375 in cattle.
In this study, expression levels of miRNAs (miRNAs), miR-375 and miR-7, were detected in different tissues of cattle to determine whether adenohypophysis-prefer or exclusively expressed miRNAs, and target genes could be predicted by TargetScan, RNA22, and other software. Target genes related to pituitary function or reproductive traits were identified using a dual-luciferase assay. miR-375 and miR-7 were expressed differently in various tissues. miR-375 and miR-7 showed higher expression in the adenohypophysis, and there was a significant difference compared with expression in other tissues (P < 0.01). The binding sites for miR-7 were the mRNAs of bone morphogenetic protein receptor type II (BMPR2), prostaglandin F2 receptor negative regulator, gonadotropin-releasing hormone receptor, follicle-stimulating hormoneβ, somatostatin receptor 1, and interleukin-1β by bioinformatic analysis; similarly, the mRNAs of BMPR2 and leptin contained binding sites for miR-375, suggesting that these genes are affected by miR-7 or miR-375. Dual-luciferase reporter assays showed that miR-7 regulated prostaglandin F2 receptor negative regulator expression, while miR-375 regulated BMPR2 expression. The mutated plasmid and miRNA mimics were used to co-transfect NIH3T3 cells; luciferase reporter assays showed that the inhibition of luciferase activity in the wild-type cells dramatically decreased from 75 to 26% with a 3-5-nucleotide mismatch mutation into the seed region of miR-7. miR-375 had nearly lost the ability to inhibit luciferase activity, suggesting that GTCTTCC is the site of interaction between miR-7 and the prostaglandin F2 receptor negative regulator sequence and that GAACAAA is the site of interaction between miR-375 and the BMPR2 sequence.
College of Animal Sciences, Jilin University, Changchun, China.
B
B
Yuan
College of Animal Sciences, Jilin University, Changchun, China.
G J
GJ
Sun
Branch of Animal Husbandry, Jilin Academy of Agricultural Science, Gongzhuling, China.
G L
GL
Zhang
Branch of Animal Husbandry, Jilin Academy of Agricultural Science, Gongzhuling, China.
J
J
Wu
College of Animal Sciences, Jilin University, Changchun, China.
C
C
Xu
College of Animal Sciences, Jilin University, Changchun, China.
L S
LS
Dai
College of Animal Sciences, Jilin University, Changchun, China.
J
J
Chen
College of Animal Sciences, Jilin University, Changchun, China.
X F
XF
Yu
College of Animal Sciences, Jilin University, Changchun, China.
Z H
ZH
Zhao
College of Animal Sciences, Jilin University, Changchun, China.
J B
JB
Zhang
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D015870:Gene Expression; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D035683:MicroRNAs / Q000737:chemistry / Q000235:genetics*; D009690:Nucleic Acid Conformation; D009928:Organ Specificity / Q000235:genetics; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*
D035683:MicroRNAs; D012333:RNA, Messenger