核糖核酸編輯對於在肝癌失調之微型核糖核酸的影響

Abstract

近年來許多研究指出，微型核糖核酸在癌症中扮演著重要角色，可能扮演腫瘤抑制基因及致癌基因等，而在肝細胞癌(Hepatocellular carcinoma,HCC)中，發現有許多的微型核糖核酸表現量有不正常的上升或下降。事實上目前已知有許多機制可調控微型核醣核酸的表現量，在最近研究發現，A-to-I核糖核酸編輯發生在微型核糖核酸上，成為其中一種調控微型核醣核酸表現量的機制，導致微型核糖核酸生合成步驟受到影響，進而造成表現量改變。有鑑於此，本研究計劃希望探討在HCC表現量改變的微型核糖核酸中，是否有A-to-I核糖核酸編輯參與在肝癌生成機制中，使得微型核糖核酸表現量改變。 實驗以Huh7肝癌細胞株為材料，以質體方式外送在此細胞株中表現負責A-to-I核糖核酸編輯的三種ADAR蛋白質，後續利用High resolution melting (HRM) curve analysis做篩選，發現了miR-214可能有被核糖核酸編輯，經由定序發現，絕大多數是T-to-C核甘酸改變，少部分是A-to-I，其中某些特定位置的核甘酸改變在臨床檢體中也同樣被發現。針對miR-214，先前研究指出miR-214存在一個對股RNA，我們提出一個可能性T-to-C鹼基改變可能來自於對股RNA A-to-I編輯。為了證明這個假設，我們藉由於細胞中大量表現特定股的RNA與ADAR蛋白質，來釐清送入哪一股的RNA會有哪些編輯發生，在這個實驗設計下，原本的A-to-I編輯只有在表現正股 pri-miRNA與ADAR蛋白質組別發現，而T-to-C則只有在表現與正股完全互補的對股 RNA與ADAR蛋白質組別發現，證實了T-to-C鹼基改變來自於對股RNA編輯。 接下來研究的議題放在與正股完全互補的對股 RNA的功能與編輯對於對股 RNA的功能的影響。為了研究miR-214的對股是否會產生微型核糖核酸，並且藉由核糖核酸編輯影響其功能與表現量的可能性，我們建立一個靈敏度較高的北方墨點法的分析方法，雖然可以偵測到miR-214陽性對照組，但是目前尚未發現miR-214對股有微型核糖核酸的證據。因此 miR-214的對股是否會產生微型核糖核酸仍有待後續的研究。總括來說，目前結果初步指出ADAR2為主要負責編輯miR-214對股的蛋白質，尚待討論的問題為對股編輯對於正股miR-214有何功能上的影響，核糖核酸編輯是否會影響對股RNA或是假想可能由對股所產生微型核糖核酸之功能。

Increasing evidence suggested the involvement of microRNAs (miRNAs) in hepatocarcinogenesis, and a panel of miRNAs were identified aberrantly expressed in hepatocellular carcinoma (HCC). However, the mechanism underlying the deregulation of specific microRNAs has not been extensively. Recently, RNA editing by ADARs has been identified in several miRNA precursor, which either affects the processing of miRNA or changes the cognate target genes. Since a subgroup of miRNAs with amount changes in HCC occurs at the step of pri- to pre-miRNA processing, a step RNA editing affects, we thus proposed to test the possibility if such miRNA changes could be attributed by the RNA editing during hepatocarcinogenesis. We approached this hypothesis by overexpressing the individual ADAR enzymes in Huh7 HCC cell line and then evaluated their effects on the miRNAs with amount changes in HCC. Screened by the very sensitive mutant detection assay of high resolution melting (HRM) analysis, miR-214 was found possibly edited by specific ARARs. The editing events at specific nucleotides have been verified by TA cloning and subsequent sequencing analysis. Intriguingly, the editing at several specific nucleotides induced by overexpressed ADAR2 was also identified in the clinical specimens, mainly in HCC but not in the adjacent non-tumorous liver tissues. But there remains one major puzzle, showing that most of the nucleotide changes belong to T-to-C rather than the A-to-I changes, which ADARs cause. Since an anti-sense transcript complementary to the pri-miR-214 was documented, we thus proposed a hypothesis that the T-to-C changes caused by ADAR2 overexpression could be due to the A-to-I RNA editing at the anti-sense RNA. To test this hypothesis, we ectopically express either the sense or the antisense RNA transcript covering pre-miR-214 with the individual ARARs into Huh-7 cells, which can help evaluate the editing of specific sense of RNA transcript. Only the anti-sense RNA showed A-to-I editing by overexpressed ADAR2, implicating that the ADAR2 induced T-to-C changes miR-214 could be resulted by A-to-I RNA editing at the anti-sense RNA. The next issue is to study the functional effect of the specific editing on either the sense miR-214 or on the antisense transcript. Interestingly, one recent report reported that an antisense RNA transcript complementary to the sense pri-miRNA could encode a novel miRNA. We tried to explore if the anti-sense RNA transcript of miR-214 also encodes a miRNA. A modified LED Northern blot analysis has been established to examine the existence of any miRNA generated by the anti-sense transcript. Although the detection sensitivity achieves 5 foml, no signal for the anti-sense miRNA has been identified yet. In conclusion, study identified a specific RNA editing by ADAR2 on the anti-sense transcript complementary to miR-214. The functional effect of such RNA editing on either sense strand or antisense RNA transcript awaits further investigation.