Salaklar Derneği, Sözen'i yılın adamı seçti

Taha Toros Arşivi, Dosya No: 207-Nurettin-Gürol-Metin SözenUnutma İstanbul projesi İstanbul Kalkınma Ajansı'nın 2016 yılı "Yenilikçi ve Yaratıcı İstanbul Mali Destek Programı" kapsamında desteklenmiştir. Proje No: TR10/16/YNY/010

Differentially Expressed miRNAs after GnRH Treatment and Their Potential Roles in FSH Regulation in Porcine Anterior Pituitary Cell

<div><p>Hypothalamic gonadotropin-releasing hormone (GnRH) is a major regulator of follicle-stimulating hormone (FSH) secretion in gonadotrope cell in the anterior pituitary gland. microRNAs (miRNAs) are small RNA molecules that control gene expression by imperfect binding to the 3′-untranslated region (3′-UTR) of mRNA at the post-transcriptional level. It has been proven that miRNAs play an important role in hormone response and/or regulation. However, little is known about miRNAs in the regulation of FSH secretion. In this study, primary anterior pituitary cells were treated with 100 nM GnRH. The supernatant of pituitary cell was collected for FSH determination by enzyme-linked immunosorbent assay (ELISA) at 3 hours and 6 hours post GnRH treatment respectively. Results revealed that GnRH significantly promoted FSH secretion at 3 h and 6 h post-treatment by 1.40-fold and 1.80-fold, respectively. FSHβ mRNA at 6 h post GnRH treatment significantly increased by 1.60-fold. At 6 hours, cells were collected for miRNA expression profile analysis using MiRCURY LNA Array and quantitative PCR (qPCR). Consequently, 21 up-regulated and 10 down-regulated miRNAs were identified, and qPCR verification of 10 randomly selected miRNAs showed a strong correlation with microarray results. Chromosome location analysis indicated that 8 miRNAs were mapped to chromosome 12 and 4 miRNAs to chromosome X. Target and pathway analysis showed that some miRNAs may be associated with GnRH regulation pathways. In addition, In-depth analysis indicated that 10 up-regulated and 3 down-regulated miRNAs probably target FSHβ mRNA 3′-UTR directly, including miR-361-3p, a highly conserved X-linked miRNA. Most importantly, functional experimental results showed that miR-361-3p was involved in FSH secretion regulation, and up-regulated miR-361-3p expression inhibited FSH secretion, while down-regulated miR-361-3p expression promoted FSH secretion in pig pituitary cell model. These differentially expressed miRNAs resolved in this study provide the first guide for post-transcriptional regulation of pituitary gonadotrope FSH secretion in pig, as well as in other mammals.</p> </div

GnRH promoted FSH secretion at hormone and mRNA transcription.

<p><b>A.</b> at 3 h post GnRH treatment, FSH concentration (mIU/mg total protein) in GnRH treatment group (180.00±41.08) was higher than that in the control group (128.11±7.67).At 6 h, FSH concentration in GnRH treatment was still higher relative to control group (264.74±60.92 vs. 147.7±21.44). Statistical significance was determined by ANOVA, followed by Tukey multiple comparisons test, p<0.05 was considered significant. <b>B.</b> GnRH increased FSHβ mRNA expression. Statistical significance was determined by Student's t test, p<0.05 was considered significant. Columns are means ± SD. The panels with different letters were considered statistically significant (p<0.05), N = 6.</p

Down-regulated miRNA profiles of porcine pituitary cell after GnRH treatment.

<p>Down-regulated miRNA profiles of porcine pituitary cell after GnRH treatment.</p

Predicted miRNAs against FSHβ, CREB, c-Fos and c-Jun.

<p>Round boxes (let-7a,let-7c,miR-22-5p,miR-19b,miR-181d-5p,miR-338,miR-423-3p,miR-133b, miR-206,miR-1307, miR-130b,miR-425-3p, and miR-532-5p) represent up-regulated miRNAs, gray octangle boxes (miR-30e-3p, miR-320, miR-324, miR-361-3p, miR-152, miR-708-5p, and miR-22-3p) represent down-regulated miRNAs, black hexagon boxes(FSHβ, CREB, c-Jun, c-Fos) indicate target genes. Arrows represent transcription factor-gene regulation, while dark lines represent miRNA regulation on mRNA except c-Jun and c-Fos.</p

Relative expression of 10 differentially expressed miRNAs by revealed by quantitative PCR.

<p>ssc-let-7c, ssc-miR-361-5p, ssc-423-3p, ssc-miR-425-3p and ssc-miR-451 were up-regulated, while ssc-miR-30e-3p, ssc-miR-320, ssc-miR-324 and ssc-miR-361-3p were down-regulated after GnRH challenge. ssc-miR-708-5p was not significantly changed. Data in columns is means ± SD. statistical significance was determined by Student's t test, p<0.05 was considered significant. The panels with different letters were considered statistically significant (p<0.05), N = 6.</p

ssc-miR-361-3p involved in FSH secretion regulation.

<p><b>A.</b> ssc-miR-361-3p mimics (mimics) and duplex negative control (NC) were separately transfected to primary pig pituitary cell, cell supernatant were collected for FSH determination at 24 h after transfection, ssc-miR-361-3p mimics led to significant decrease of FSH secretion by 14.10% (140.00±11.45 vs. 162.94±5.01 p<0.05). <b>B.</b> Transfection of miR-361-3p mimics increased miR-361-3p level in pituitary cell by 3.22-folds (p<0.05). <b>C.</b> miR-361-3p inhibitors increased FSH secretion by 15.07% compared with inhibitor negative control (I-NC) (163.31±7.04 vs. 141.91±7.81). <b>D.</b> Transfection of miR-361-3p inhibitors exhibited 80.87% decline of miR-361-3p level relative to I-NC (p<0.05). Data in columns is means ±SD, N = 6. Statistical significance between two groups was determined by Student's t test. The panels with different letters were considered statistically significant (p<0.05).</p

Possible pathways affected by differentially expressed miRNAs.

<p>Possible pathways affected by differentially expressed miRNAs.</p

Predicted targets of miRNAs in GnRH signaling pathway.

<p>This figure was drawn according to KEGG pathway (ssc4912), red arrow near the box (general on the right) represents up-regulated miRNAs while blue arrow represents down-regulated miRNAs. The number near the arrow indicates number of miRNAs targeting this gene or its family. For example, 10 up-regulated miRNAs and 3 down-regulated miRNAs were predicted to target FSHβ mRNA 3′-UTR.TF indicates transcription factor involved in FSHβ transcription.</p

Up-regulated miRNA profiles of porcine pituitary cell after GnRH treatment.

<p>Up-regulated miRNA profiles of porcine pituitary cell after GnRH treatment.</p