探討微小 RNA let-7g 與氧化性低密度脂蛋白接受器 ( LOX-1) 之間的負回饋調控機制

MicroRNA 是一種內生性且不轉錄出任何蛋白質產物的微小 RNA，經轉譯後修飾作用來調控基因的表現。文獻報導證實 microRNA 與動脈硬化的發生有關。 Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) 是一種細胞膜蛋白，為氧化性低密度脂蛋白 (oxLDL) 的接受器，負責將 oxLDL 胞飲 (endocytosis) 入細胞內，並產生一連串與動脈硬化發生相關的訊息傳遞。已知 LOX-1 受到許多轉錄因子調節表現，然而 LOX-1 的表現是否被 microRNA 調控仍未被釐清。在本論文中，我們利用 oxLDL 刺激初代人類主動脈血管平滑肌細胞後，發現到 LOX-1 的表現會隨之增加。若將 LOX-1 過度表現可促進 oxLDL 誘導的細胞增生及移動，而 LOX-1 抑制表現則會減少 oxLDL 誘導的細胞增生 (proliferation) 及移動(migration)。我們進一步發現腫瘤抑制型的 microRNA let-7g，其表現量在 oxLDL 的處理後有顯著下降的趨勢。此外， let-7g 也被預測可結合上 LOX-1 mRNA 的 3’UTR，我們也利用 3’UTR luciferase reporter assay 證實 LOX-1 是 let-7g 的標靶基因。已知 OCT-1 是一種抑制型的轉錄因子並可受到 oxLDL 透過 LOX-1 的調控而表現上升。我們利用 promoter assay 以及 chromatin immunoprecipitation 證明 OCT-1 會結合到 let-7g promoter 並抑制其基因的表現。利用 shRNA 抑制 OCT-1 表現也明顯地回復 let-7g 表現並伴隨 LOX-1 表現下降。已知細胞內的鈣離子及其下游調控因子 Protein kinase C (PKC) 是 OCT-1 的上游調節者。我們利用 Chelerythrine (PKC抑制劑) 或是MAPTAM (鈣離子螯合劑) 可有效的抑制 oxLDL 對 let-7g/LOX-1/OCT-1 的基因表現。此外，細胞轉染 let-7g mimic 後也可明顯地影響 oxLDL 調節 LOX-1/OCT-1 表現、細胞增生及細胞移動。在動物實驗中，我們也觀察到餵食高脂質食物的 C57BL/6J 小鼠其 let-7g 表現量偏低及 LOX-1/OCT-1 高度表現的現象。由上述結果得知，oxLDL 會透過 LOX-1/Ca2+/PKC 的路徑來促進 OCT-1 表現並進而抑制 let-7g 的生成。而 let-7g 的存在也可負回饋抑制 LOX-1 的表現。這些結果指出 let-7g 與 LOX-1 之間形成一個負回饋調控機制並且在動脈硬化形成的過程中扮演一個重要的角色。

關鍵字

氧化性低密度脂蛋白接受器；微小核醣核酸let-7g ；動脈硬化；氧化低密度脂蛋白

並列摘要

MicroRNAs (miRNAs), endogenous small non-coding RNAs, regulate cellular gene expression on the post-transcriptional level and are functionally linked to atherosclerosis. Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) acts as a surface scavenger receptor by mediating the uptake of oxidized low-density lipoprotein (oxLDL) and also plays a proatherogenic role in atherosclerosis. It is not clear whether LOX-1 expression can be regulated by miRNAs. In the present study, a dose-dependent increase of LOX-1 was observed after oxLDL treatment. Over-expression or knockdown of LOX-1 expression affected oxLDL–mediated proliferation and migration of primary human aortic smooth muscle cells (HASMC). We further showed that a tumor suppressor miRNA, let-7g, was down-regulated by oxLDL in a dose-dependent manner. We also confirmed let-7g binds to the 3'-untranslated region (UTR) of LOX-1 mRNA by using 3’-UTR luciferase reporter assay. LOX-1 was identified as a direct target of let-7g. OCT-1 was a well-known transcriptional repressor and could be up-regulated by oxLDL via LOX-1 receptor. We demonstrated that OCT-1 can bind to let-7g promoter to inhibit gene expression by using the promoter assay and chromatin immunoprecipitation. Knockdown of OCT-1 expression abolished oxLDL-reduced let-7g expression accompanied with LOX-1 down-regulation. Intracellular Ca2+-activated PKC was a known upstream regulator of OCT-1. Either Chelerythrine (PKC inhibitor) or MAPTAM (Ca2+ chelator) could significantly attenuate oxLDL-mediated let-7g down-regulation and LOX-1/OCT-1 up-regulation. Besides, oxLDL-regulated LOX-1/OCT-1 expression, cell proliferation, and migration could be inhibited after let-7g mimic transfection. Our in vivo studies show both let-7g down-regulation and LOX-1/OCT-1 up-regulation in C57BL/6J mice fed with high fat diet. Taken together, reduced let-7g and enhanced LOX-1 expression are found in oxLDL-induced HASMC proliferation and migration. LOX-1 is also confirmed as a target of let-7g. OxLDL-mediated OCT-1 up-regulation inhibits let-7g expression via LOX-1-Ca2+-PKC pathway. These results suggest that let-7g-targeted LOX-1 is a feedback regulation and plays an important role in arteriosclerosis.

並列關鍵字

MicroRNA ； LOX-1 ； let7-g ； oxLDL ； arteriosclerosis

參考文獻

1. Sacco, R.L., The new American Heart Association 2020 goal: achieving ideal cardiovascular health. J Cardiovasc Med (Hagerstown), 2011. 12(4): p. 255-7.