利用電腦分析方法預測及實驗驗證在胃癌中微小核醣核酸目標基因

Abstract

胃癌（GC）是最常見的惡性腫瘤之一，在癌症中為第二最常見癌症相關死亡的原因。由於缺乏早期診斷，大多數患者確診時為晚期胃癌。儘管在手術和化療策略進步，但整體的預後還是沒有很好。因此找尋新型的分子生物標記物或治療靶標為胃癌的治療的重要關鍵。近期許多研究都集中在MicroRNA（miRNA）與在胃癌中的作用。miRNA是一類21-23個鹼基對的單鏈RNA分子，並透過miRNA與mRNA的非編碼區之間的特異性鹼基配對相互作用調控標靶基因表達。miRNA在癌症功能可能為致癌基因或腫瘤抑制基因，並且可以潛在地用在胃癌診斷，預後和治療。本研究的總體目的是提供與胃癌相關的完整miRNA和其標靶基因的關係，並可找出潛在的miRNA生物標記以利早期診斷來成功治療胃癌。第一，利用與miRNA表現和胃癌的文獻回顧分析中，發現miR-21 和 miR-375分別為促進調控和抑制調控的miRNA。第二，生物資訊分析實驗驗證的miRNA標靶基因在胃癌中的關係，得到共543個miRNA標靶基因交互作用，其中有200個miRNA和323個標靶基因。透過基因富集分析，發現多數基因調控細胞凋亡，細胞死亡，細胞週期，細胞運動，粘著連接，p53信號通路。最後，利用系統化整合分析方法，結合 GEO 和 TCGA 胃癌的 miRNA 和 基因表現資料，挑選表現量負相關的miRNA 和 基因，發現分別共有79和1042個差異表現的miRNA 和 基因。利用miRNA-標靶基因預測工具，miRanda, PITA 和 RNAhybrid預測，發現共有79個miRNA和774個標靶基因，並建立關係網路。此外，miR-204的異位表達抑制細胞增生和胃癌細胞的轉移。利用qRT-PCR分析五個目標基因（CD55, CKS1B, CXCL1, GPRC5A 和 TNS4）和miR-204關係。表明這些靶基因受miR-204抑制。利用冷光報導基因分析四個基因（CD55, CKS1B, CXCL1和GPRC5A在 miR-204）大量表現時，發現螢光素酶顯著減少從而指示miR-204調空了這四個靶基因的功能。因此，這些研究提供了CD55，CKS1B，CXCL1，GPRC5A和TNS4受miR-204調控的一個證據和作為潛在的腫瘤抑制基因，同時可能為胃物標記。

Gastric cancer (GC) is one of the most common malignancies and the second most frequent cause of cancer-related death worldwide. Most of the patients are late diagnosed with advanced gastric cancer due to lack of early diagnosis. Despite the advances in operative and chemotherapy strategies, the overall prognosis is not good for gastric cancer. Therefore identifying the factors that contribute to the advancement of this cancer may serve as the novel molecular biomarkers or therapeutic targets for the treatment of gastric cancer. Recently, many studies have focused in the role of microRNA (miRNA) in gastric cancer. Micro-RNAs are a class of single stranded RNA molecules of 21-23 base pairs and regulate target genes expression through specific base-pairing interactions between miRNA and untranslated regions of mRNAs. miRNAs function as oncogenes or tumor suppressors and could be potentially used as diagnostic, prognostic and therapeutic targets in gastric cancer. The overall intention of carrying out this study is to provide comprehensive information of miRNAs and their targets that are associated with the gastric cancer and to find out the potential miRNA and its targets that can function as therapeutical biomarkers in treatment of gastric cancer. So at first, the systematic review of published miRNA profiling studies was carried out in this study that compared the miRNA expression profiles between GC tissues and paired noncancerous gastric tissues and revealed that miR-21 and miR-375 were the most consistently reported up-regulated and down-regulated miRNAs in GC. Secondly, the bioinformatics analysis of the experimentally validated miRNA targets in gastric cancer yielded 543 interactions between 200 miRNAs and 323 target genes with enriched gene ontology terms of the target genes like regulation of apoptosis, cell death, cell cycle, cell motion and enriched pathway like cell cycle, adherens junction, p53 signaling pathway. And lastly, the multistep approach in combination with integrated analysis and anti-correlation ship to identify the differentially expressed miRNAs and mRNAs from the expression profiling studies collected from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) revealed 79 and 1042 differentially expressed miRNAs and mRNAs respectively from gastric cancer samples and their corresponding normal epithelial tissues. A total of 3206 miRNA-mRNA pairs combined by 79 dysregulated miRNAs and their 774 target mRNAs predicted by three prediction tools miRanda, PITA and RNAhybrid were shown in miRNA and mRNA network. Moreover, ectopic expression of miR-204 suppressed cell proliferation and migration of gastric cancer cells. The quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) of five targets; CD55, CKS1B, CXCL1, GPRC5A and TNS4, of miR-204 showed that these target genes are suppressed by miR-204 and are thus regulated by miR-204. When the construct containing 3’UTR of four targets; CD55, CKS1B, CXCL1 and GPRC5A, of miR-204 were transfected along with miR-204 mimics, in gastric cancer cells there was the significant reduction in the luciferase reporter assay thus indicating the functional role of miR-204 in regulating these four target genes expression via their 3’UTR. Thus these studies provide the first evidence of regulation of the CD55, CKS1B, CXCL1, GPRC5A and TNS4 by miR-204 and it acts as potential tumor suppressor and therefore is useful as a promising therapeutic biomarker for gastric cancer.