Abstract
Nutrients are utilized and re-constructed by endodermal epithelial cells (EECs) of yolk sac membrane (YSM) in avian species during embryonic development. Sterol O-acyltransferase 1 (SOAT1) is the key enzyme to convert cholesterol to cholesteryl ester for delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Presence of microRNAs (miRNAs) are observed in the embryonic liver and muscle during avian embryogenesis. However, the expression of miRNAs in YSM during embryogenesis and the involvement of miRNAs in lipid utilization are not known. Using a miRNA sequencing technique, we found several miRNA candidates and confirmed their expression patterns individually by real time PCR. MiRNA candidates were selected based on the expression pattern and their possible roles in inhibiting transforming growth factor beta receptor type 1 (TGFBR1) that would regulate the function of SOAT1. Similar to SOAT1 mRNA, the gga-miR-181a-5p expression was gradually elevated during embryonic development. However, the expression of gga-miR-429-3p in YSM was gradually decreased during embryonic development. The inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) were demonstrated by transient miRNA transfections in EECs. We also found that mutated TGFBR1 3'UTR prevented the direct pairings of gga-miR-181a-5p and gga-miR-429-3p. Treatment of TGFBR1 inhibitor, LY364947, further decreased SOAT1 transcription. Similar results were also observed by the miRNA transfection studies. The results showed the vital participations of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFβ pathway, and affecting downstream SOAT1 expression and function in the YSM. This is indicative of possible regulation of avian yolk lipid utilization by changing YSM miRNA expressions.
Highlights
Sterol-O acyltransferase 1 (SOAT1), named ACAT1, is the key enzyme to catalyze cholesterol conversion into cholesteryl ester, by adding fatty acyl coenzyme A; a less polar molecule is produced [1]
The microRNA sequencing of yolk sac membranes (YSM) during Japanese quail (Coturnix coturnix) embryonic development was analyzed by PhalanxBio Inc. (Hsinchu, Taiwan)
For a better understanding of the overall miRNA expression profiles, samples of Yolk sac membrane (YSM) were collected at embryonic day 5 (ED5), ED10, ED15, and post-hatch day 2 (PH2); one sample was used at each time point
Summary
SOAT1 (sterol-O acyltransferase 1), named ACAT1 (acyl-Coenzyme A: cholesterol acyltransferase 1), is the key enzyme to catalyze cholesterol conversion into cholesteryl ester, by adding fatty acyl coenzyme A; a less polar molecule is produced [1]. Yolk sac membrane (YSM), a three-layer extraembryonic tissue, serves crucial roles for avian embryos during the entirety of embryonic development. SOAT1 activity in endodermal epithelial cells (EECs, the third layer of YSM) was activated by specific nutrients and hormones through the cAMP-dependent PKA signaling pathway, and accumulated more cholesterol ester in EECs [2]. The diversity of bio-functions and involvement of non-coding RNAs has raised considerable issues. The functions and regulations of miRNAs have been examined in mammalian species for decades. Mature miRNAs are paired to 3’ untranslated regions (UTR) or 5’UTR by identifying seed regions of target genes [4]
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