日本鵪鶉卵黃囊膜內胚層上皮細胞模式探討小分子miR-181a-5p/ miR-429-3p經TGFβ pathway降低固醇轉醯酶1表現與活性

Abstract

在家禽胚胎發育過程，卵黃為重要營養來源。其中固醇O–醯基轉移酶1（Sterol O-acyltransferase 1, SOAT1）將游離膽固醇酯化成膽固醇酯。於家禽胚胎發育後期，卵黃囊膜中SOAT1 mRNA濃度與酶活性皆隨時間顯著增高，且卵黃囊膜的膽固醇酯濃度也極高，推論膽固醇酯化與利用卵黃脂質機制息息相關。小片段核糖核酸（microRNAs, miRNAs）被認為是影響mRNA穩定性而降低其轉錄與轉譯的原因之一。發育中的家禽胚胎肝臟與肌肉皆發現有miRNAs參與調節代謝，但miRNAs對於卵黃囊膜的影響或參與其中脂質代謝的角色卻仍未知。因此利用miRNA sequencing分析入孵後不同胚胎時期卵黃囊膜中miRNA，尋找可能藉由抑制乙型轉化生長因子受體1型（transforming growth factor beta receptor type 1, TGFBR1）去影響SOAT1表現的候選miRNA，並以real time PCR確認這些miRNAs於胚胎發育各時期之表現量。研究發現gga-miR-181a-5p表現量與發育時期SOAT1 mRNA變化模式相近、gga-miR-429-3p則與其相反。在鵪鶉卵黃囊膜內胚層上皮細胞的試驗中發現，轉染5 nM的gga-miR-181a-5p、gga-miR-133a-5p與gga-miR-429-3p有效降低SOAT1、TGFBR1 mRNA表現量。通常miRNA以seed region辨識並結合到目標基因3’UTR，作為miRNA直接影響目標的證據之一。本試驗確認gga-miR-181a-5p與gga-miR-429-3p能結合至TGFBR1 3’UTR，而在突變miRNA seed region之後，gga-miR-181a-5p與gga-miR-429-3p皆失去與3’UTR之結合能力。此二miRNA轉染會顯著的降低SOAT1與TGFBR1蛋白質表現量。此外，TGFBR1抑制劑LY364947，也能有效降低SOAT1 mRNA表現量，與miRNA轉染試驗結果相符。這些結果顯示gga-miR-181a-5p與gga-miR-429-3p可能都是藉由調控TGFβ訊息傳導路徑，去影響卵黃囊膜內胚層上皮細胞內的SOAT1基因表現與其功能。所以在家禽胚胎發育期間，藉由調控卵黃囊膜中某些miRNAs表現量，就可影響卵黃囊膜對於脂質的利用能力，此種特性可能可以利用來改進禽胚的發育和孵化率。

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 to facilitate the delivery to growing embryos. During embryonic development, yolk absorption is concomitant with significant changes of SOAT1 mRNA concentration and enzyme activity in YSM. Expression of microRNAs (miRNAs) occurs 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, I 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 respect to transforming growth factor beta receptor type 1 (TGFBR1) that regulates the function of SOAT1. Similar to SOAT1 mRNA, gga-miR-181a-5p expression was gradually elevated during embryonic development, whereas of gga-miR-429-3p expression in YSM was gradually decreased during embryonic development. Inhibitory effects of gga-miR-181a-5p or gga-miR-429-3p on the potential targets (SOAT1 and TGFBR1) at both in transcript and protein level were further observed by transient miRNA transfections in EECs. Mutated TGFBR1 3’UTR failed to 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 a vital role of gga-miR-181a-5p and gga-miR-429-3p in regulating TGFβ pathway, further affecting downstream SOAT1 expression and function in the YSM, and therefore suggesting a critical miRNA-mRNA regulation of avian yolk lipid utilization in YSM.