在膀胱 癌中 c-myc 擴增 的現象經由 miR-34a ceRNA 機制導致 CD44的過度表現

Amplification of c-myc Leads to Overexpression of CD44 Through a miR-34a-mediated ceRNA Mechanism in Bladder Cancer

黃雯宇(Huang Wen-Yu)

指導教授 ： 陳永恩

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2014年)

摘要

膀胱癌位居世界常見癌症排名第六，在台灣特別是在西南沿海一帶有比較高的發生率。其中一種癌症形成的原因是癌症幹細胞理論，在癌症幹細胞的表面會表達CD44抗原。Micro RNA(miRNAs)是一種內源性非編碼RNA，具有抑制他標的mRNA的功能。然而，miRNAs在膀胱癌幹細胞中到底扮演著怎樣的調控角色，尚未完全被研究透徹。我們主要是想研究在膀胱癌中miRNA 是怎樣去調控癌症幹細胞的標記蛋白CD44。生物資訊預測miR-34a 會調控CD44的表現量。miR-34a的表現量在膀胱癌細胞中只有在UMUC3細胞有表現量降低的情形。在這株細胞中過度表達miR-34a會使CD44的表現量降低，所以我們認為CD44是miR-34a的目標mRNA。經過組合重亞硫酸鹽限制分析法(combined bisulfite restriction analysis, COBRA)、重亞硫酸鹽焦磷酸定序(bisulphite pyrosequencing) 和表基因研究常用藥物，結果表明UMUC3細胞的miR-34a表現量降低是透過啟動子CpG島的高度甲基化。但是只有其中一株膀胱癌細胞經由啟動子CpG島甲基化而造成表基因沉默，所以我們研判是還有其他的機制會調控CD44的表現。最近的文獻提出了一個假說¬---競爭性內源RNA (competitive endogenous RNA ,ceRNA)認為mRNA也會去調控miRNA的功能。為了瞭解是不是有ceRNA再調控CD44，我們利用生物資訊預測，發現c-myc也是miR-34a的目標mRNA。先前研究發現，在膀胱癌中常有8q24異常擴增的現象，而c-myc就位在其上。我們認為過度表現c-myc會經由ceRNA理論也使CD44表現量增加。非常有趣的，在TCCSUP和TSGH8301兩株膀胱癌細胞中過度表達c-myc 3’UTR都會導致CD44的表現量增加。臨床上，在公開的微陣列(expression array ,GDS1479)和膀胱癌病人檢體中c-myc和CD44的表現量也是呈現正相關性的。重要的是，我們發現， c-myc表現量低但CD44表現量高的病人在miR-34a的啟動子CpG島甲基化而造成表基因沉默。 最後，過度表現miR-34a會抑制膀胱癌細胞的生長以及入侵鄰近組織能力，並且會增加對抗癌藥物的敏感度。 總結以上結果，異常的啟動子CpG島甲基化和ceRNA理論都會影響miR-34a在膀胱癌中的功能。miR-34a 抑制腫瘤的功能會如何調控癌症幹細胞的表現型是值得被更深入研究的。

關鍵字

膀胱癌

並列摘要

Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. One of the hypotheses suggests that bladder cancers arise from cancer stem-like cell (CSC) with surface antigen CD44. MicroRNAs (miRNAs) are endogenous, non-protein-coding RNAs that can regulate the expression of their target mRNAs.However, the role of miRNAs in the regulation of CSC in bladder cancer is not fully explored. We aim to investigate the role of miRNAs in the regulation of CSC marker, CD44 in bladder cancer. Bioinformatics prediction identified that miR-34a regulates the expression of CD44. Expression analysis showed that miR-34a was only down-regulated in UMUC3 bladder cancer cells. Over-expression of miR-34a resulted in a down-regulation of CD44 in this cell thus suggesting that CD44 is a target of miR-34a. Further analysis by COBRA, bisulphite pyrosequencing and epigenetic drug treatment demonstrated that down-regulation of miR-34a in UMUC3 cells is due to promoter hypermethylation. As miR-34a is only epigenetically silenced in a bladder cancer cell line, we suspect that additional mechanism may play a role in the regulation of CD44. Recently, competitive endogenous RNA (ceRNA) mechanism has been demonstrated to control the function of miRNAs. To test if ceRNA play a role in the regulation of CD44, we performed additional bioinformatic analysis and found that c-myc is also a target of miR-34a. Since amplification of chromosome 8q24 where c-myc resides is frequently observed in bladder cancer, we suspect that overexpression of c-myc may also up-regulate the expression of CD44 through ceRNA mechanism. Interestingly, overexpression of c-myc 3’UTR in TCCSUP and TSGH8301 bladder cancer cells which expressed miR-34a resulted in an up-regulation of CD44. Clinical study also demonstrated a positive correlation between the expression of c-myc and CD44 in a publicly available expression array data set (GDS1479) and a cohort of bladder cancer patient samples. Importantly, patients with low expression of c-myc but high expression of CD44 demonstrated a promoter methylation of miR-34a. Finally, overexpression of miR-34a resulted in a suppression of cell growth, invasion, and enhanced chemosensitivity in UMUC3 cells. In conclusion, aberrant promoter methylation and ceRNA mechanism may attenuate the function of miR-34a in bladder cancer. The tumor suppressive role of miR-34a in controlling CSC phenotype in bladder cancer deserves further investigation.

並列關鍵字

bladder cancer

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