第一型主要組織相容性複合體抗原相關鏈B 的mRNA 三端未轉譯區對其蛋白表現的調控

人類第一型主要組織相容性複合體抗原相關鏈B (human major histocompatibility complex class I related chain B, MICB) 基因轉譯成細胞膜上的醣修飾蛋白，且為自然殺手細胞 (natural killer cell) 受體 (receptor) NKG2D的配體 (ligand)。當MICB與NKG2D結合時，會活化自然殺手細胞毒殺的活性。MICB與其有相似功能的第一型主要組織相容性複合體抗原相關鏈A (MICA) 會在受外在壓力刺激時表現，例如受熱或是病毒感染；也會選擇性的表現在癌化的細胞上。然而，不同於MICA的是，MICB mRNA轉錄體 (transcript) 較不穩定，進而可能造成MICB蛋白質在細胞表面的表現量較低。在目前的研究，我已經確定內生性的MICB mRNA降解的速度比MICA mRNA快。接下來，既然MICB跟MICA mRNA序列上最大的差異為MICB mRNA的三端未轉譯區(3’untranslated region, 3’UTR) (1229bp) 較MICA mRNA 3’UTR (175 bp) 長，所以我假設MICB mRNA的穩定性是經由其3’UTR所調控。我用即時聚合酶連鎖反應 (quantitative real-time PCR) 來偵測包含不同MICB 3’UTR的突變體 (mutant)，發現MICB mRNA 3’UTR的長度與與其穩定性成反比，而且MICB 3’UTR靠近轉譯區 (coding region) 的部分可能在調節功能上扮演重要的角色。用流式細胞儀 (flow cytometry) 及西方墨點法 (Western blotting) 觀察不同突變體的蛋白質在COS-7及HEK293FT細胞表現也得到相似的結果。這些結果意味著在MICB mRNA 3’UTR bp 194~361可能有一個潛在的調節區來調控MICB蛋白質的表現。最後，我將MICB 3’UTR全長接在另一種報導基因(reporter gene)–綠色螢光蛋白 (eGFP) 的轉譯區之後來觀察MICB 3’UTR對eGFP表現量的影響。結果發現，MICB 3’UTR同樣可降低大約一半的eGFP蛋白質表現，進而暗示MICB 3’UTR的調節功能在不同基因的保留性。若能解釋調控MICB mRNA穩定性的機轉，則可提高我們對MICB蛋白質後轉譯修飾的機轉的了解。這些資訊可提供一些方法來增加MICB mRNA的穩定性及提高MICB蛋白質在細胞的表現，進而促進自然殺手細胞毒殺標的細胞 (例如癌細胞) 的能力。

關鍵字

MICB ； 3'UTR ； NKG2D

並列摘要

The human major histocompatibility complex (MHC) class I related chain B (MICB) gene encodes a membrane-bound glycosylated protein which is a ligand for the natural killer (NK) cell receptor NKG2D. Binding of MICB to NKG2D activates the cytolytic response of NK cells. MICB, like its close functional homolog MICA, is expressed selectively by cells undergoing malignant transformation or exposed to stress such as heat shock or viral infection. Nevertheless, unlike MICA, MICB mRNA transcripts are unstable, which may contribute to the low level of MICB protein expression in cells. In the present study, I has determined that endogenous MICB mRNA degrades 8-times faster than MICA mRNA. Next, since MICB mRNA carries a much longer 3’ untranslated region (UTR) (1229bp) than MICA mRNA (175bp), I hypothesized that the stability of MICB mRNA was affected by its unusually long 3’UTR. Real-time RT-PCR analysis of COS-7 cells transfected with a series of MICB 3’UTR deletion mutants showed an inverse correlation between the length of MICB 3’UTR and MICB mRNA stability where the proximal end of MICB 3’UTR appeared to play a critical role. These findings were corroborated by the results of flow cytometric and Western blot analyses of MICB protein expression in COS-7 and HEK293FT cells transfected with MICB 3’UTR deletion mutants. As a result, a potential regulatory region between bp194 and bp361 of the proximal MICB 3’ UTR was identified. Lastly, the conservation of the MICB mRNA 3’UTR regulatory function was indicated by the markedly reduced expression of enhanced green fluorescence protein (eGFP) encoded by the eGFP construct engineered with the 1152 bp MICB 3’ UTR. Elucidation of the role of MICB 3’ UTR in the regulation of MICB expression will improve our understanding of how the status of MICB protein is altered in cells by a post-transcriptional mechanism. This information may suggest ways to increase MICB mRNA stability and protein production in cells, making them a better cytolytic target for NK cells.

並列關鍵字

無資料

參考文獻

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