miR-221/222在胎盤所扮演的角色

Abstract

微核醣核酸 (micro RNA) 是小的、非編碼的單股核醣核酸，它們在細胞的增生、分化與凋亡，以及胚胎發育和生理過程中扮演著重要的調節角色。細胞的微核醣核酸表達若失衡，已被證實會藉由影響其下游調控基因的表達而造成細胞型態、特徵或功能改變。本實驗室為深入探討 miR-221/222 在細胞和動物生理功能或病理過程所扮演的角色，已建立 miR-221/222 基因剔除小鼠。而鑒於miR-221和miR-222在胎盤的高表達量及其可能標的基因p27 和 p57，可調控胎盤之血管新生，因此，胎盤表達的 miR-221 和 miR-222是否可藉由抑制p27及p57而影響胎盤發育，值得深入探討。 根據配種經驗，miR-221/222 基因剔除小鼠之生育及繁殖力、生產胎數以及胎兒的生長等，皆和野生型小鼠相似，顯示可能需在特定環境刺激下，miR-221/222才彰顯其功能。過去文獻指出懷孕母鼠餵食高鹽飲水會導致胎盤功能受損以及胎兒發育不良，因此，本研究利用此模式探討 miR-221 及 miR-222 在此病理過程中可能扮演的角色。實驗方法是在小鼠懷孕E8.5開始餵食2.7%NaCl高鹽飲水，同時測量懷孕母鼠之體重變化，並於懷孕中期 (E13.5) 或晚期 (E18.5) 犧牲，收集胎兒與胎盤，除測量其大小和重量、記錄其形狀和表徵外，亦利用即時定量PCR及西方墨點法，分析RNA和蛋白質的表達量，另透過H E染色觀察胎盤的結構，以及以Ki67免疫染色和TUNEL assay分析細胞之增生及凋亡情形。 研究結果顯示，相較於正常飲水之對照組，給予野生型孕鼠高鹽飲水會造成孕鼠體重增加遲緩、胎兒發育異常比例提高(1%增加至54%)、胎兒體長和體重皆明顯下降、胎盤重量大幅減輕且其血管組織結構異常，而miR-221/222 基因剔除孕鼠餵食高鹽飲水和正常飲水相比，也和上述野生型孕鼠具類似現象。然而餵食高鹽飲水的野生型孕鼠相較於 miR-221/222 基因剔除孕鼠，胎兒發育異常的比例、胎兒體重和體長、胎盤重量異常程度皆顯著較為嚴重，此結果顯示胎盤失去 miR-221/222的表達，可有效減緩高鹽飲水所導致的胎兒和胎盤異常病徵。在胎盤之RNA與蛋白質表現方面，miR-221/222基因剔除孕鼠和野生型孕鼠相比，p57 mRNA表達量明顯上升，而野生型與基因剔除小鼠經餵食鹽水後，皆會誘導miR-221/222 RNA、p57蛋白質表現量上升和 p57 mRNA 表現量下降，推論 p57之表達，可能並非受 miR-221/222 單一因子之調控。另外，本研究發現高鹽餵食會導致胎盤組織結構鬆散、細胞分佈異常和壞死增加、胎盤細胞增生減少和凋亡增加，而缺乏 miR-221/222 並未增加胎盤細胞之增生或減緩其凋亡，因此推論，miR-221/222 可能非透過改變胎盤細胞之增生和凋亡而影響胎盤發育，較可能是造成胎盤血管生成異常，導致胎盤血液灌注量不足，進而影響胎盤組織的發育，最後造成胎兒發育或表徵的異常。

MicroRNA (miRNA) is a class of endogenous, single-stranded, non-coding RNA with 20~24 nucleotides. In addition to regulate cellular functions and normal development, these small non-coding RNAs also participate in the pathogenesis of many diseases. With the same seed sequence and very close to each other, a knockout (KO) mouse with the deletion of two miRNAs, miR-221 and miR-222, was created. Because miR-221 and miR-222 are highly expressed in placenta, and their possible target gene, p27 and p57, are functioned in the placental angiogenesis. Whether miR-221 and miR-222 play their roles in the placental development through down-regulating the expression of p27 and p57 needs further investigations. Because the fetal growth and live-birth of miR-221/222 knockout mice is very similar to those of wild-type mice, it was proposed that miR-221/222 may only exert their physiological function upon certain stimulations. Thus, a high salt water-induced model was subject to explore how miR-221/222 functions on the development of fetus and placenta. Pregnant mice were feed with 2.7% NaCl high salt water starting from E8.5 to E13.5 (mid-pregnancy) or E18.5 (late-pregnancy). The maternal body weights were measured daily till sacrificed. The morphology, size and weight of collected fetus and placenta were examined and quantified. The level of RNA and protein were determined by quantitative real-time PCR and western blot, respectively. The placental structure, cells proliferation and apoptosis were analyzed by H E stain, Ki67 immunohistochemical stain and TUNEL assay. The results indicated that, when compare to control pregnant mice, the gain of maternal body weight was lower both in wild type and knockout pregnant mice feed with high salt water. The fetal abnormality was raised from 1% to 54% in wild type pregnant mice feed with high salt water. The size and weight of fetus and placenta also decreased significantly in wild type pregnant mice feed with high salt water. And the fetal defects were less severe in miR-221/222 knockout fetus, indicating that miR-221/222 may play some roles in promoting the clinical representations during high-salt water treatment. In another word, these phenomena can be rescued by miR-221/222 knockout, or the clinical symptoms induced by high-salt water can be reversed through reducing miR-221/222 expression. About the RNA and protein expression, p57 mRNA level was increased by knockout of miR-221/222. But miR-221/222 and p57 protein level were increased and p57 mRNA level declined both in wild-type and knockout mice drinking high salt water. These results may indicate that there were some other genes or factors involved in regulating p57 expression, either in the presence of miR-221/222 or not. Furthermore, H E and immunohistochemical stains demonstrate that abnormal distribution, loose structure, more necrosis and apoptosis, less cell proliferation was investigated in placenta treated with high salt water. Although these defects were not rescued in the absence of miR-221/222, these results suggest another possibility that miR-221/222 may have some other impacts on angiogenesis, which leading to decreased placental blood perfusion, and thereby affecting the development of fetuses.