MicroRNA在肝癌形成過程中表現量的改變及相關致癌機轉之研究

The Change of MicroRNA Expression Patterns and A Related Carcinogenic Mechanism in Hepatocarcinogenesis

盧卓群(Cho-Chun Lu)

指導教授 ： 葉秀慧

國立臺灣大學/醫學院/微生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02711

摘要

MicroRNAs 是具有19至25個核苷酸的小片段RNA，它主要會辨認到 mRNA 的3端未轉譯區，形成不完全的配對而抑制基因的蛋白質表現。它在細胞裡扮演著很重要的功能，例如發育的調控，還有細胞的脂肪代謝、增生、分化及細胞的死亡和抗病毒機制等。另外，miRNA 的不正常表現可能和疾病的發生有相關，已有文獻指出 miRNA 在腫瘤細胞中可能扮演著致癌基因或是腫瘤抑制基因的角色，因此分析在腫瘤的形成過程中，有哪些的miRNA 有不正常的表現情形，可以幫助我們了解腫瘤的致病機轉。
我們根據 (1) 之前在文獻中利用microarray分析所指出在HCC中有顯著差異的miRNAs 或是可能有性別表現差異的 miRNAs；(2)經由網站預測出與肝癌形成可能相關基因之miRNAs；(3) 在肝臟細胞內有高度表現量的 miRNA；(4) 和位在第四對染色體上於肝癌好發易斷裂區的miRNAs，經由此種方式，我們共選擇了29 個miRNAs來進行本論文針對miRNA於HCC癌化過程中是否產生變化之分析。為探討miRNA之變化是否和病毒感染因子以及男女性別差異之特性相關，我們分析之臨床檢體主要包含了不同病毒感染因子相關之男女配對之tumor 及其non-tumor肝臟組織，並以配對 FNH (Focal nodular hyperplasia，肝臟結節性增生)肝臟組織當作對照組。我們使用定量real-time PCR 的方式去偵測肝組織內的 miRNA 的表現量，以t-test 分析並把p-value 小於0.05視為有顯著的差異。
在我們的研究中，我們發現有好幾個 miRNAs 在 HCC 內的表現量有明顯的改變。另外，我們也發現有些 miRNAs 在依病毒或是性別分群上有表現量的差異，但仍需要進一步證明。為了指出 miRNAs 在癌化過程中的作用機制，我們選擇了2個 miRNAs : mir-130a 和 mir-493-5p 做為進一步實驗。 因為我們發現mir-130a 和 mir-493-5p 在 HCC 的表現量是有顯著下降，且mir-130a 和 mir-493-5p是被預測會去調控 AR 的miRNAs，因此我們進行實驗想去探討mir-130a 或mir-493-5p 是否能辨認到 AR 並調控其表現。
我們所使用的方法是把具有AR的3端未轉譯區接在 pGL3-promoter 之後，轉染入人類肝癌細胞株，再使用感染 lentivirus 的方式去大量表現 mir-130a 或是 mir-493-5p。為了證實這辨識的特異性，我們把 mir-130a 和 mir-493-5p 所辨識的 seed site 做了4個核苷酸的突變。雖然在初步的實驗中，可以發現這兩個 miRNA 可以造成具有AR-3端未轉譯區的reporter 活性下降，但是這兩個 miRNAs 對於內生性 AR 的蛋白質表現量抑制的程度並不明顯。
結論，在我們的研究中指出有幾個miRNAs 在HCC 的表現量有顯著的差異，但它們在癌化過程中所扮演的角色，則需要更進一步的實驗去證明。

關鍵字

肝癌 ； 男性荷爾蒙受體

並列摘要

MiRNAs are 19- to 25-nucleotide-long RNAs which can inhibit the protein synthesis by binding to the complementary sequences at the 3’-untranslated regions (3’-UTR) of target mRNAs. They play regulatory roles in various biological functions, including development, cell fate determination, proliferation, differentiation, cell death, and antiviral mechanisms etc. Intriguingly, an increasing list of miRNAs have been reported to function as putative oncogenes or tumor suppressor genes and showed aberrant expression patterns during carcinogenic process. Therefore, it is assumed that analysis of the miRNA expression patterns of caner cells can help researcher to understand the carcinogeneic mechanisms of specific tumors.
Aiming to study the functional involvement of miRNA in liver cancers, I started a pilot study by examining the expression patterns of numerous specific miRNAs in paired HCC and corresponding non-tumorous tissues from HBV-, HCV-, and nonB/nonC related HCCs collected both from the male and female patients. Such an approach can help to elucidate the correlation of specific miRNA expression pattern with the viral etiology and gender factor. The selected miRNAs in the current study were based on the criteria listed as followed. (1) The ones showing significant difference or possible gender difference revealed by the previous studies approached by microarray analysis; (2) the predicted miRNAs target genes which are associated with hepatocarcinogenesis; (3) the ones with high expression level in the liver tissues; and (4) the ones locating in the chromosome 4q region with frequent allelic loss. The assay used to determine the amount of miRNA in liver tissues is the quantitative real-time PCR analysis, which is assumed to be a much more reliable method for such analysis.
The results from current analysis indeed pointed out several miRNAs showing significant different expression patterns between tumorous and non-tumorous tissues (p<0.05, t-test). Moreover, I have identified a few miRNAs showing viral etiological or gender difference, which still awaits further verification. To illustrate the functional mechanism of specific miRNA in carcinogenesis, I have chosen two miRNA, the mir-130a and mir-493-5, which were predicted to target to the androgen receptor (AR) gene, as an example in the subsequent functional analysis. Since these two miRNAs showed significant down-regulated expression in HCCs, I tried to test if these two miRNAs indeed target to AR gene and regulate its expression negatively. The strategy is to deliver the mi-RNA expression constructs into hepatoma cells with the luciferase reporter construct fused with the 3’–UTR of AR gene, which contains the putative recognition sites of both miRNAs. To verify the specificity of the recognition, I also included a reporter construct with mutations at the target recognition sequence as a control. Although the preliminary results showed down-regulation of reporter activity by both miRNAs, their effect on the AR protein expression is not evident. In conclusion, current study pointed out several miRNAs with significant changes in HCCs. Yet, their functional role in carcinogenic process warrants further clarification.

並列關鍵字

microRNA ； HCC ； Hepatocarcinogenesis ； androgen receptor

參考文獻

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