MicroRNA與大腸直腸癌的相關性研究

Investigation of microRNAs associated with colorectal cancer

潘冠頤(Kuan-Yi Pan)

指導教授 ： 陳韻如

共同指導教授 ： 林尊湄(Tsun-Mei Lin)

義守大學/醫學院/生物科技學系/碩士(2021年)

摘要

大腸直腸癌在台灣與世界上是最常見的癌症之一，所以發展能偵測與預測大腸直腸癌進程的特異性生物標記是很重要的。MicroRNAs (miRNAs)是種能調節基因表現的短鏈且非編碼的RNA序列，就像潛在的致癌基因和腫瘤抑制基因。這些分子表現的改變與許多癌症疾病的發生有關，因此它們被認為是非侵入性癌症診斷和預後的分子工具。這研究一開始主要目的是評估大腸直腸癌病人血漿中各種miRNAs表現量，透過次世代定序技術，分析分別來自於第一、二期大腸直腸癌病人、第三、四期大腸直腸癌病人及正常健康人的等體積混合血漿，發現有9種miRNAs在早期和晚期大腸直腸癌病人與正常健康人相較表現量是共同上升的，如miR-4532、miR-21-3p、miR-204-5p、miR-4326、miR-22-5p、miR-6721-5p、miR-30b-5p、miR-1-3p與miR-320e，而miR-323a-3p與miR-1268a在早期和晚期大腸直腸癌病人與正常健康人相較表現量共同下降的。這些分析出來的miRNAs種類後續以qRT-PCR方式進一步驗證其它個別檢體和腫瘤組織，結果發現miR-4326、miR-320e和miR-323a表現量在早期與晚期大腸直腸癌病人血漿中有顯著差異(p值<0.05)。然而，若比較週遭正常組織，腫瘤組織的miR-4326與miR-320e表現量則會隨著癌症分期而上升。考量miR-4326在大腸直腸癌中是種新興的miRNA且功能尚未被探索研究，因此我們以大腸直腸癌細胞株的增殖、移動和侵犯能力實驗來驗證miR-4326造成的影響。當miR-4326的過度表現會抑制大腸直腸癌細胞株移動、侵犯和細胞聚落形成的能力，反之移除miR-4326則會加強癌細胞的上述能力。然而，無論miR-4326過度表現或去除，對於以XTT測試的大腸直腸癌細胞株增殖能力，結果則無任何影響。最後透過公開生物資訊工具預測miR-4326假定標靶基因，顯示miR-4326有可能與structural maintenance of chromosomes (SMC) 1A這種促進大腸直腸癌生長及轉移的致癌基因作用。未來需要進一步研究來評估MiR-4326的功能，以解開大腸直腸癌腫瘤開始與癌症進展的分子機制，並期望miR-4326能被用作大腸直腸癌早期偵測的生物標記，並且能成為有潛力的治療標的物。

關鍵字

大腸直腸癌 ； 生物標記

並列摘要

Colorectal cancer (CRC) is one of the most common cancers in Taiwan and the world. Developing specific biomarkers for detection and prediction of CRC progression is important. MicroRNAs (miRNAs) are short non-coding RNA sequences to regulate gene expression. MiRNAs can function as potential oncogenes or tumor suppressors. Altered expression of these molecules was correlated with the occurrence of many cancer diseases and therefore they are considered as a molecular tool for non-invasive cancer diagnosis and prognosis. The primary aim of this study was to evaluate miRNAs expression profiles in the plasma of CRC patients. Three pooled samples from CRC patients of stage I&II, stage III&IV and matched healthy controls were analyzed by next generation sequencing. There are 9 miRNAs to be co-upregulated, including miR-4532, miR-21-3p, miR-204-5p, miR-4326, miR-22-5p, miR-6721-5p, miR-30b-5p, miR-1-3p and miR-320e and miR-323a-3p and miR-1268a were co-downregulated in CRC patients at both early- and late-stage cancers as compared with those of healthy controls. Differentially expressed miRNAs are further validated in individual sample and tumor tissues using quantitative reverse-transcription polymerase chain rection. The expressions of miR-4326, miR-320e and miR-323a were demonstrated to be significantly different between early- and late-stage CRC patients (p<0.05). In addition, compared to the normal adjacent tissues, miR-4326 was found to be up-regulated in accordance to the stages. Since miR-4326 is a novel miRNA and its function has not been explored, the effects of miR-4326 on proliferation, migration, and invasion in CRC cell lines were examined. Overexpression of miR-4326 inhibited CRC cell migration, invasion and colony formation. In contrast, miR-4326 knockdown enhanced cancer cell migration, invasion and colony formation. However, overexpression and knockdown of miR-4326 had no effect on CRC cell proliferation as analyzed by XTT assay. The assumed miR-4326 target genes predicted by public bioinformatics tools revealed that miR-4326 might target structural maintenance of chromosomes (SMC) 1A, which could promote CRC growth and metastasis. Further investigation is needed to assess the functions of miR-4326 to uncover the molecular mechanisms of tumor initiation and progression in CRC. Therefore, miR-4326 may be used as an early detection biomarker and potential therapeutic target in CRC patients in the future.

並列關鍵字

CRC ； Biomarkers ； MicroRNA ； MiR-4326 ； SMC1A

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