在努南氏症(Noonan syndrome, NS)的病人中，先天性心臟異常是主要的臨床表徵，包含了肺動脈瓣狹窄(pulmonary valve stenosis, PVS), 心房中隔缺損 (atrial septal defect, ASD), 以及肥厚性心肌症(hypertrophic cardiomyopathy, HCM)。目前我們對於一些致病基因與其造成的臨床心臟相關病癥之間的關聯性尚待釐清。雖然有部分努南氏症致病基因已證實和活化RAS-ERK 路徑有關，活化機制的啟動如何影響細胞轉錄調控依舊不清楚。先前我們已發現NS致病基因RASA2的突變造成磷酸化的ERK1/2表現量上升，在本次研究中我們主要想探討RASA2的調控角色，以及帶有病患特定突變的RASA2如何影響細胞的基因表現。
首先，我們研究其他可能活化ERK1/2的訊息傳遞路徑如何被活化。RASA2為GAP1m家族的一員，目前已知GAP1m家族和RAP-GAP功能有關，而Rap1對心血管發育以及血管新生過程十分重要。我們經由RAP1活性測定實驗結果發現突變的RASA2喪失RAP1-GAP的功能，顯示病患特定突變RASA2可能不會藉由RAP1活化ERK1/2。而在帶有RASA2突變的細胞中，經由EGF刺激後被活化的ERK1/2則是由RAS調控。
接著我們做了RNA-sequencing，結果顯示野生型和病患特定突變RASA2的mRNA表現量有顯著變化。我們利用生物資訊分析技術mRNA表現量差異最顯著的基因，並用其他實驗來確認結果。我們使用基因組富集分析(GSEA)來分析 RNA-seq的實驗結果，以此找出不同基因集 (gene sets) 之間的關聯性以及在富集基因集中貢獻程度較高的基因。我們從ranking list中選出表現量差異最大的一些基因，而會產出angiomotin的基因AMOT在RASA2過度表現的HEK293細胞模式和CRISPR基因編輯的HEK293細胞模式中， 無論是mRNA或蛋白質的表現都有被調控。AMOT屬於motin家族的一員，它在內皮細胞相關調控功能扮演重要角色，包含參與血管新生與影響細胞遷移等等。我們發現帶有RASA2突變的細胞遷移能力下降。敲除AMOT會導致帶有RASA2突變的細胞遷移能力下降更加明顯，而在CRISPR基因編輯的RASA2突變細胞過度表現AMOT-p80異構物則可以稍微挽救細胞的遷移能力。綜合以上結果，我們推測突變的RASA2影響正常的細胞遷移能力部分受到AMOT負調控的作用。此外，帶有RASA2突變的人類臍帶靜脈內皮細胞(HUVEC)的血管生成功能較差，表示突變的RASA2在內皮細胞的血管生成功能十分重要。

Congenital cardiac abnormalities are one of the predominant clinical features in Noonan syndrome (NS). These clinical defects include pulmonary valve stenosis (PVS), atrial septal defect (ASD) and hypertrophic cardiomyopathy (HCM). The relationship between some disease-causing genes and clinical cardiac phenotypes remains to be determined. Though several NS causing genes have shown to activate the RAS-ERK pathway, the impact of the activation on the transcriptional program is still unclear. Previously, we showed that NS-associated RASA2 mutations caused increased level of phosphorylated-ERK1/2. In this study, we aim to investigate the regulatory role of RASA2 and the influence of patient-specific RASA2 mutations in gene expression in the cells.
We first investigated the activation of alternative signaling pathways that may also activate ERK1/2. RASA2 belongs to GAP1m family which has been linked to RAP-GAP function. RAP1 is also important to cardiovascular vasculogenesis and angiogenesis. We showed that mutant RASA2 loss the RAP1-GAP function through RAP1 activity assay, suggesting that NS-associated RASA2 mutations may not activate ERK1/2 through RAP1. Instead, the activation of ERK1/2 in response to EGF stimulation in cells expressing RASA2 mutant is likely through RAS.
Next, we performed RNA-sequencing in order to reveal significant changes of mRNA expression level between wild-type and patient-specific mutant RASA2. Genes that had the most significant mRNA expression level changes are identified by bioinformatics methods and confirmed with functional assays. Gene set enrichment analysis (GSEA) identified the correlation between gene sets and the genes that contribute most to the enrichment gene sets. Among the genes from the top of the ranking list, AMOT, which encodes angiomotin, was downregulated at the mRNA and protein level in both RASA2-mutant inducible cell lines and CRISPR-edited cells. Angiomotin belongs to the motin family, which has critical effects on endothelial cell functions, including angiogenesis and cell migration. Cell migration ability was decreased in RASA2 mutant cells. Knocking-down of AMOT decreased cell migration ability further more in RASA2 mutant cells, while overexpressing AMOT-p80 isoform slightly rescued the cell migration ability in CRISPR-edited RASA2 mutant cells. These results suggest that RASA2 mutation impairs the cell migration, partially through down-regulation of AMOT. In addition, poor angiogenesis ability was seen in CRISPR-edited RASA2-RC mutant HUVEC cells, suggesting that RASA2 is important for tube formation ability in endothelial cells. Taken together, mutant RASA2 interacted with RAS rather than RAP1, and through transcriptional regulation, decreased AMOT lowered cell migration ability in vitro.

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