由反轉錄跳躍Alu序列所導引的核醣核酸干擾現象

Abstract

RNA干擾現象是真核細胞體內抑制特定基因的一種現象。它是當細胞內導入與內源性mRNA編碼區相配合之雙股RNA後，使mRNA發生降解導致基因表達默化的一種現象。在自然界中，其扮演著病毒防禦、抑制跳躍基因以及基因調控等功能。RNAi具有高特異性及高效性，因此已經成為研究基因功能，以及發展成基因治療的有用工具。Alu序列屬於反轉錄跳躍子中SINE的一族，它在人類基因體中佔了約15﹪。完整的Alu序列長約250∼300bp，多存在於插入子（intron）、3’-UTR及基因間。Alu來自於7SL RNA基因，由於演化歷史久遠，因為突變而可分為數個家族。我們利用人類H1 promoter當啟動子，TTTTT當終結子以建構siRNA表現載體。經由插入一段已知可用來產生siGFP的序列後，發現轉染細胞時，會經由降低mRNA及蛋白表現量以抑制GFP基因的表現，證明我們建構的載體是有功能性的。我們從資料庫中搜尋出帶有Alu序列的基因，依據Alu序列的長度及相似度進行分析，從中挑選出基因HSD17B12、OACT1、LOC134218、LOC146909和SMG1以做研究。這幾個基因的特點是它們都位於不同的染色體上，而所帶的Alu序列相似高。我們將HSD17B12的Alu序列調出，以進一步建構成siAlu的表現載體。經過二次插入形成重複顛倒序列，我們建構了pH1-siAlu95的表現載體。經由共同轉染3’端帶有Alu序列的GFP基因載體到細胞後，可以達到抑制GFP基因表現量的影響，證明Alu序列也可以作為RNAi的作用標的。經由RT-PCR篩選有表現5個所要研究的基因的細胞，發現HEK293、H1299、SC-M1及K562都會表現這些基因，因此我們選用HEK293以做研究。經過轉染pH1-siAlu95後，我們發現到HSD17B12及OACT1的mRNA表現量明顯的降低，而其他三個基因並不受影響。實驗結果證明，Alu序列可作為RNAi的標的，透過它可同時抑制多個基因的表現。

RNA interference, a phenomenom where dsRNA specifically blocks the expression of its homologous gene, is discovered by Fire and Mell in 1998. It is clear that RNAi occurs in all eukaryotic cells and plays a role in viral defense and transposon silencing mechanisms. RNAi becomes an important method for analyzing gene functions and development of therapeutic gene silencing. Alu sequence is primate-specific member of the SINE（short interspersed element） retrotransposon family，and comprises about 15﹪ of human genome. Full –length Alu sequence is about 250∼300bp long and is commonly found in intron，3’ untranslated regions of genes and intergenic regions. Alu sequence was ancestrally derived from the 7SL RNA gene. Mutations accumulated in the Alu sequence caused it classified of several distinct subfamily. We used H1 promoter amplified from human genomic DNA and synthetic TTTTT as terminator to construct our siRNA expression vector. It can reduce GFP expression after constructed as siGFP expression vector. This means that the constructed siRNA expression vector is functional. After searching the Alu sequence containing genes from the database, we chose gene HSD17B12、OACT1、LOC134218、LOC146909 and SMG1 to research. After amplifying the trancated Alu sequence from the gene HSD17B12, we insert it into the vector as a inverted repeat to construct siAlu expression vector,named pH1-siAlu95. After transfecting into cells, it resulted in reducing expression of GFP gene containing Alu sequence in 3’ end, showed that Alu sequence can serve as RNAi target. After examination, HEK293, H1299, SC-M1 and K562 cells have expressed the five Alu containing genes and we chose HEK293 for continuous research. Our results showed that the expression of gene HSD17B12 and OACT1 are repressed after transfection of pH1-siAlu. It demonstrated that Alu sequence can serve as RNAi target. To use Alu sequence for RNAi target can simultaneously downregulate several genes by redused the expression level of mRNA and protein﹒