DHX30對內皮細胞中MCPIP1的影響

Abstract

Monocyte chemotactic protein-1 induced protein 1 (MCPIP1)，又稱RENASE-1，是一種參與發炎反應和血管新生等生理機制的鋅指核糖核酸酶。它能能夠與目標mRNA 3’UTR的莖-環結構結合，進而降解目標mRNA和抑制其轉譯，但對於MCPIP1如何選擇目標mRNA的機制仍不清楚。先前研究發現MCPIP1的交互作用蛋白up-frameshift protein 1 (UPF1)可促進MCPIP1辨認轉譯活躍的mRNA，強調交互作用的蛋白對MCPIP1選擇特定目標mRNA的重要性。我們實驗室先前透過質譜分析找到了一個潛在的MCPIP1交互作用蛋白－ATP-dependent RNA helicase－DHX30。為了探討DHX30對MCPIP1功能的重要性，我們先從兩個蛋白是否會互相影響彼此蛋白的量開始檢驗。 目前已知MCPIP1會參與血管新生，然而其是促進還是抑制血管新生仍存在爭議。因此，我們使用胰島內皮(MS1)細胞探討DHX30在MCPIP1功能中的角色，並研究與血管新生的關聯。初步的數據顯示調降Mcpip1不影響DHX30 mRNA和蛋白表現量，但調降Dhx30則會使MS1細胞中MCPIP1蛋白表現量上升。透過cycloheximide此蛋白合成抑制劑的實驗測試MCPIP1蛋白降解速率，我們觀察到在Dhx30調降的MS1細胞中，MCPIP1蛋白的降解速率較快，代表MCPIP1蛋白含量的增加可能是因其蛋白合成增加所致。由於MCPIP1的mRNA也是MCPIP1 的目標mRNA之一，因此我們推論DHX30與MCPIP1協同作用來抑制MCPIP1目標基因的表現。RNA免疫沉澱的實驗結果顯示調降Dhx30不影響MCPIP1和目標mRNA的結合能力，這表明DHX30抑制MCPIP1蛋白的表現應該發生在MCPIP1蛋白和降解目標mRNA的結合之後。儘管仍需要進一步的檢測，我們的研究結果顯示MCPIP1和DHX30之間存在功能性相互作用。這樣的研究結果也為MCPIP1在血管生成中潛在機制提供了一個新的視角。

Monocyte chemotactic protein-1 induced protein 1 (MCPIP1), also known as REGNASE-1, is a zinc finger ribonuclease involved in several physiological mechanisms, such as inflammation and angiogenesis. Although MCPIP1 is recognized for its ability to target the stem-loop structure of mRNA 3’UTR, resulting in both degradation of targeted mRNA and inhibition of targeted mRNA translation, the specificity in the selection of the targeted mRNA remains largely unknown. A previous study has shown that up-frameshift protein 1 (UPF1), a protein interactor of MCPIP1, facilitates MCPIP1 in recognizing translationally active mRNA, highlighting the importance of protein interactors in the targeted mRNA selection of MCPIP1. Our lab previously identified an ATP-dependent RNA helicase, DHX30, as a candidate interactor of MCPIP1 via mass spectrometry analysis. To investigate the role of DHX30 in MCPIP1 function, we aim to determine whether DHX30 and MCPIP1 mutually influence each other's protein expression. While MCPIP1 is known to participate in angiogenesis, the question of whether MCPIP1 promotes or inhibits angiogenesis is still controversial. Thus, we utilize mouse pancreatic islet endothelial (MS1) cells with the goal of not only understanding the role of DHX30 in MCPIP1 function but also extrapolating our findings to angiogenesis. Despite our preliminary data show Mcpip1 knockdown has no effect on DHX30 mRNA and protein levels, we find Dhx30 knockdown results in up-regulated protein levels of MCPIP1 in MS1 cells. Measuring the decay rate of the MCPIP1 protein via cycloheximide treatment reveals a higher degradation rate in Dhx30 knockdown cells, suggesting that the increased steady-state level of MCPIP1 protein is contributed from increasing its protein synthesis. Since Mcpip1 mRNA is also a target of the MCPIP1 protein, we hypothesize that DHX30 works together with MCPIP1 to repress MCPIP1-targeted gene expression. The results of the RNA immunoprecipitation experiments showed that Dhx30 knockdown does not affect the binding ability of MCPIP1 to its target mRNA. This suggests that DHX30's role in repressing MCPIP1 protein expression occurs downstream of MCPIP1's recognition of its target mRNAs, potentially similar to how UPF1 regulates targeted mRNA decay. Although further testing is needed, our findings suggest a functional interaction between MCPIP1 and DHX30. It provides a new perspective to uncover the potential mechanism of MCPIP1 in angiogenesis.