Nuclear respiratory factor 1 (簡稱NRF-1)，是人類基因組中重要的轉錄因子。先前研究證實NRF-1會調控許多負責能量傳遞及發育的基因。近年來，我們實驗室發現NRF-1有調控神經突生長的新功能。我們利用生物資訊方法發現約有兩千一百多個基因的啟動子區域含有預測的NRF-1 responsive element（簡稱NRE）。本篇研究針對其中十個含有典型且高度保留性NRE的基因，並依照Gene Ontology分為四個類別：(1) 訊息傳導相關- SPRY4、PIP5K1A 及 USP10; (2) 調控轉錄相關- NR1D1 及 GTF2F2; (3) 細胞週期相關- SKA3 及 SUV39H2; (4) 功能尚未明確- MPP5、CCDC75 及 C1orf128。我們假設這十個基因會被NRF-1所調控並參與在NRF-1所調控的神經突生長功能。利用electrophoretic mobility shift assay實驗證實，在試管中NRF-1可以鍵結這十個基因的NRE。而在chromatin immunoprecipitation assay則證實在細胞內，這十個基因中的九個基因的NRE可以被NRF-1所鍵結。在人類神經纖維母細胞株IMR-32中利用shRNA減少NRF-1 mRNA的表現後，以即時定量及半定量聚合酶連鎖反應偵測目標基因mRNA表現量，發現SPRY4、USP10、SKA3、MPP5 及 CCDC75表現量減低、NR1D1 和 GTF2F2表現量升高，而PIP5K1A、SUV39H2 與 C1orf128 表現量則不變，證實NRF-1可以調控SPRY4、USP10、SKA3、MPP5、CCDC75、NR1D1 和 GTF2F2這七個基因的mRNA表現量。當IMR-32細胞同時以帶有目標基因shRNA的重組慢病毒感染及轉殖NRF-1處理後，在型態上發現轉殖NRF-1後所增長的最長神經突的平均長度會因為感染SPRY4、USP10、GTF2F2、SKA3、MPP5 及 CCDC75 基因的shRNA重組慢病毒而減低，可是在NR1D1基因上並沒有發生。以上這些結果指出參與在訊息傳導、調控轉錄、細胞週期相關的基因群會影響轉錄因子NRF-1所調控的神經突生長功能並可以幫助我們聯繫起NRF-1調控神經突生長的分子網絡。

Nuclear respiratory factor 1 (NRF-1) is one of the major transcription factors in the human genome. NRF-1 is known to regulate genes responsible for energy transduction and development. Recently, our lab identified a novel function of NRF-1 in neurite outgrowth. Using bioinformatic analyses, we found that ~2,100 genes have putative NRF-1 responsive element (NRE) in the promoter region. This study focus on 10 genes which NRE is typical and highly conserved between the human and mouse. These 10 genes are grouped into 4 categories according to the Gene Ontology: (1) signal transduction- SPRY4, PIP5K1A and USP10; (2) regulation of transcription- NR1D1 and GTF2F2; (3) cell cycle- SKA3 and SUV39H2; (4) unclear function- MPP5, CCDC75 and C1orf128. We hypothesized that these 10 genes are regulated by NRF-1 and mediate its function in neurite outgrowth. Electrophoretic mobility shift assays showed that the NREs of all these 10 genes are bound by NRF-1 in vitro. Chromatin immunoprecipitation assay confirmed that nine NREs are bound by NRF-1 in vivo. Real-time and semi-quantitative RT-PCR demonstrated that, when knockdown of NRF-1 in neuroblastoma IMR-32 cells, the mRNA levels of SPRY4, USP10, SKA3, MPP5 and CCDC75 were decreased, those of NR1D1 and GTF2F2 were increased, and those of PIP5K1A, SUV39H2 and C1orf128 were not changed. When IMR-32 cells co-treated with infection of recombinant lentivirus containing shRNA and transfection of full-length NRF-1, we found that the NRF-1-increased average lengths of longest neurite are decreased by lentivirus containing shRNA of SPRY4, USP10, GTF2F2, SKA3, MPP5 and CCDC75 genes, but not of NR1D1. These results suggest that genes involve in signal transduction, regulation of transcription and cell cycle mediating the function of NRF-1 in neurite outgrowth and help us to map the molecular network responsible for the function of NRF-1 in neurite outgrowth.

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