探討人類可轉錄假基因的調控及功能分析

Abstract

假基因 (pseudogene)主要被認為是演化過程中所產生的垃圾DNA序列。然而，近年來研究發現假基因-特別是可轉錄的假基因，可能經由產生內生性小的干擾RNA、反股RNA或抑制RNA，以扮演調控基因的角色。在老鼠及果蠅已被証實轉錄假基因可以產生內生性小的干擾RNA進而調控蛋白質基因的表現，但是在人類此機制未知。因此，本研究主要目的是系統化探討人類可轉錄假基因的二個機制：產生內生性小的干擾RNA以及miRNA 誘餌的功能。為了系統化的處理及更新這些結果及相關的資訊，我們也建立一個新頴整合性的資炓庫-pseudoMap，做為研究人類可轉錄假基因的研究平台。由生物資訊所導出的一個假基因TPG-PPM1K-經由PPM1K部分反轉錄所產生的轉錄假基因，因反向重覆序列建構成髮夾式結構而產生二個內生性小的干擾RNA進而調控許多細胞相關基因，也包含了NEK8。41對肝癌及其周邊非癌化組織研究顯示，由TPG-PPM1K所產生的二個內生性小的干擾RNA及其對應的正常蛋白質基因，在癌組織的表現均低於非癌化組織的表現，而此現象相反於所預測的esiRNA1的調控基因NEK8的表現。除此之外，NEK8 及 PPM1K 在過度表現TPG-PPM1K的載體比將esiRNA1刪除的載體表現還低。甚至於若將NEK8 表現在已轉染TPG-PPM1K的載體，顯示NEK8可抵消TPG-PPM1K對細胞成長的抑制。就我們所知，這是第一個實驗証實人類可轉錄假基因所產生的內生性小的干擾RNA調控肝癌細胞的表現。也同時驗証生物資訊預測的可行性。經由此研究，可以對人類可轉錄假基因的功能、調控，以及與其他蛋白質基因的關係有更完整、深入的認識。

Pseudogenes have been mainly considered as “junk” DNA, failed copies of genes that arise during the evolution of genomes. However, recent studies indicate that pseudogenes, especially the transcribed ones, may function as gene regulators through generation of endogenous small interfering RNAs (esiRNAs), antisense RNAs, or RNA decoys. In mice and flies, pseudogene transcripts can be processed into esiRNAs that regulate protein-coding genes, but this mechanism in human remains unknown. Therefore, the aim of this work is systematically to demonstrate two mechanisms of human transcribed pseudogenes (TPGs): encoding esiRNAs and decoying miRNAs that target the parental gene. To enable the systematic compilation and updating of these results and additional information, we have developed a database, pseudoMap, an innovative and comprehensive resource to study human TPGs. A genome-wide survey revealed a TPG-PPM1K, a partial retrotranscript from PPM1K (protein phosphatase, Mg2+/Mn2+ dependent, 1K), containing inverted repeats capable of folding into hairpin structures that can be processed into two esiRNAs; these esiRNAs potentially target many cellular genes, including NEK8. In 41 paired surgical specimens, we found significantly reduced expression of two predicted TPG-PPM1K-specific esiRNAs, and the cognate gene PPM1K, in hepatocellular carcinoma (HCC) compared to matched non-tumor tissues, whereas the expression of target gene NEK8 was increased in tumors. Additionally, NEK8 and PPM1K were down-regulated in stably transfected TPG-PPM1K-overexpressing cells, but not in cells transfected with an esiRNA1-deletion mutant of TPG-PPM1K. Furthermore, expression of NEK8 in TPG-PPM1K-transfected cells demonstrated that NEK8 can counteract the growth inhibitory effects of TPG-PPM1K. These findings indicate that a transcribed pseudogene can exert tumor suppressor activity independent of its parental gene by generation of esiRNAs that regulate human cell growth. To our knowledge, this is the first investigation of an esiRNA-mediated role of human pseudogenes in HCC as well as verification of computational prediction. This study provides further information for elucidating the human TPG function, regulation and the relationships between protein-coding genes.