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標題: | 一個新穎的lncRNA參與LPS誘導IL-12p40表現 Identification of a novel long non-coding RNA important for LPS induction of IL-12p40 expression |
作者: | 賴政儂 Cheng-Nong Lai |
指導教授: | 莊志立 Jyh-Lyh Juang |
關鍵字: | IL-12p40,lncRNA,NF-κB,單核球,M1巨噬細胞, IL-12p40,lncRNA,NF-κB,monocyte,M1 macrophage, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 細胞激素12 (Interleukin 12,IL-12) ,它是由IL-12p40和IL-12p35組成的異型雙體蛋白質,是連接先天性免疫和後天性免疫的重要橋梁。因為IL-12p40是導致免疫反應失調的重要因子,因此這個基因的表達必須受到嚴密調控,假如其表現量不足則免疫力太低,將導致個體對抵抗病原感染的能力下降,若太多則免疫反應過度將造成組織、器官的傷害或衰竭。然而,目前對於調控IL-12p40表現的了解仍相當有限。在本研究中,我們發現一個潛在的lncRNA (Long non‐coding RNA) LNC-IL12B座落於IL-12p40基因的上游,且其轉錄方向與IL-12p40相反。經由生物資訊分析發現,該lncRNA的5’端位置富含GC鹼基,而其3’ 尾端具有多腺苷酸序列 (Polyadenylation sequence) 。此外,經由跨物種的序列比對發現LNC-IL12B保守地存在於哺乳動物中;這演化上的保守性,顯示LNC-IL12B可能在動物體中扮演一重要的角色。過去文獻指出,轉錄方向相反的兩個相鄰基因常有被共同調控其表現的情形。因此我們利用NCBI生物計畫資料庫進行分析,探討LNC-IL12B 和IL-12p40這兩個基因,在不同組織中表現的形式。結果發現這兩個基因,皆主要表現在淋巴組織中,如脾臟、淋巴結和骨髓。除此之外,它們在其它的組織中也呈現相似的表現趨勢,並且呈現顯著的統計正相關性,顯示這兩個基因的表現,可能受到同樣的調控機制影響。為了探討這個假設,我們在THP-1單核球細胞中,以內毒素LPS誘導IL-12p40的表現,藉此觀察LNC-IL12B表現是否也隨之變化。果然如預期,結果顯示LNC-IL12B 和IL-12p40呈現相似的表現趨勢,兩者皆隨著時間進程而有相似的表現量變化,並且兩者的表現量呈現高度正相關性,這結果證實兩個基因確實受到相似的調控機制影響。另外,因為過去文獻指出,有些lncRNA具有調控鄰近基因表現的能力,因此我們假設LNC-IL12B可能參與調控IL-12p40基因的表現。為測試此一假設,我們以siRNA降低LNC-IL12B的表現量,發現這能夠抑制LPS所誘導的IL-12p40的表現量。另一方面,我們以慢病毒過量表現LNC-IL12B,也發現這能增強IL-12p40的表現量。因此,我們認為LNC-IL12B在單核球中參與調控IL-12p40的表現。因為LPS是透過活化轉錄因子NF-κB來誘導IL-12p40表現,所以我們進一步想了解LNC-IL12B是否透過活化NF-κB來影響IL-12p40表現。結果發現以siRNA降低LNC-IL12B,也能減少NF-κB的活化,這顯示LNC-IL12B能透過影響NF-κB來調控IL-12p40表現。此外M1巨噬細胞是種能持續表現IL-12p40的免疫細胞,我們也想知道是否LNC-IL12B能在該細胞中影響IL-12p40。結果也發現,過量表現LNC-IL12B能在M1巨噬細胞促進IL-12p40的表現,此結果顯示LNC-IL12B也能調控M1巨噬細胞中的IL-12p40。總結本研究的結果,我們發現一個新穎的lncRNA LNC-IL12B,它能夠透過NF-κB來調控IL-12p40的表現。本研究的貢獻,在於發現一個新的調控IL-12p40的因子,其未來可能成為調節IL-12p40的新穎標的。未來能夠測試LNC-IL12B是否可以應用於,降低癌症轉移、提高化療耐受性、改善慢性發炎和自體免疫疾病的藥物標靶。 Interleukin 12 (IL-12) is heterodimeric cytokine composed of IL-12p35 and IL-12p40 subunits functioning in stimulating both innate and adaptive responses upon pathogenic challenge. IL-12p40 is also a common component for another heterodimeric cytokine IL-23, which also functions as a key mediator of inflammation. Thus, the expression of IL-12p40 is tightly regulated in immune cells and the impaired regulation of IL-12p40 expression will result in an insufficient cellular immune response to infection. However, the current understanding of the transcriptional regulation of IL-12p40 is still very limited. In this study, we identified a potential long non-coding RNA (LncRNA) gene located adjacent to the gene locus (in the 5q33.3 region of human chromosome 5) for IL-12p40 in a head-to-head (H2H) orientation, hereafter called LNC-IL12B. Our bioinformatic analysis suggested that this gene contains a 5’- GC-rich region and a poly-A tail, but it lacks a protein-coding sequence, as evidenced by our sequence analysis and experimental results of RT-PCR analysis. Cross-species DNA sequence comparison shows that LNC-IL12B is evolutionarily conserved in the mammals, but not present in other vertebrates. Since it has been reported that the H2H paired genes are prone to a co-regulation and co-function in the same signaling pathway, we analyzed the tissue expression profiles of LNC-IL12B and IL-12p40 genes by using the NCBI BioProject database. Indeed, our results suggested that these two genes showed very similar expression patterns in different human tissues with their highest expression levels in the lymphatic tissue of spleen, lymph node, and bone marrow. To test whether these two genes to be co-regulated and co-expressed under certain experimental conditions, we treated THP-1 monocyte cells with LPS (Lipopolysaccharide) to induce IL-12p40 expression and examined the expression pattern of LNC-IL12B expression during the time course of treatment. Indeed, LNC-IL12B showed very similar time-dependent expression patterns as IL-12p40. Prompted by this result, we hypothesized that LNC-IL12B may be involved in the LPS-induced IL-12p40 expression. Supporting this hypothesis, we showed that the siRNA knockdown of LNC-IL12B expression suppressed the LPS-induced IL-12p40 expression in THP-1 monocyte. In contrast, overexpression of LNC-IL12B increased the IL-12p40 expression, suggesting a role of LNC-IL12B in modulating the LPS-induced IL-12p40 expression in immune cells. Furthermore, since the LPS-induced IL-12p40 expression is known to be regulated by NF-κB, we used siRNA knocked down LNC-IL12B in THP-1 monocytes treated with LPS and found that NF-κB signaling was markedly suppressed. In contrast, overexpression of LNC-IL12B in monocytes increased NF-κB protein activity upon LPS treatment. These results suggested that LNC-IL12B may mediate IL-12p40 upregulation via NF-κB signaling pathway. Taken together, our results indicate that we have identified a novel lncRNA, LNC-IL12B, which plays a key role in modulating the LPS-induced NF-κB signaling in promoting the IL-12p40 expression in monocytes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/78827 |
DOI: | 10.6342/NTU201900366 |
全文授權: | 未授權 |
電子全文公開日期: | 2024-02-15 |
顯示於系所單位: | 分子與細胞生物學研究所 |
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