臺灣博碩士論文加值系統

在許多癌症中，已發現microRNA的失衡和癌症的進展和轉移有關。 CpG island 中的DNA甲基化在抑癌基因的表現量下降中扮演重要的角色，這其中也包含miRNA。 miR-22的已被證明在某些癌症中失衡表現，包括乳癌和多發性骨髓瘤。採用real-time RT-PCR分析，我們也發現了miR-22的表現量在多數口腔癌細胞株和70% 臨床病人檢體中皆是下降。進一步，我們利用CpG island 預測軟體，在pre-miR-22 上游序列發現三個CpG island，1 - 3。 我們選擇兩株miR-22 表現量比較低的口腔癌細胞，處理1 ~ 5 μM的甲基化抑制劑5’-azaC ，5天後萃取出其RNA，並利用 real-time PCR分析其miR-22的表達量。實驗結果顯示口腔癌細胞在處理不同劑量的5’-azaC之後, 皆會誘導miR-22表現量的增加。這結果似乎意味著口腔癌細胞中miR-22 的表現量下降是受到表觀遺傳修飾調節。首先，我們利用bisulfite sequencing PCR (BSP) 分析 CpG island中的CpG sites的甲基化情形，並檢視與miR-22的表現量下降有關的重要區域。我們將最長的CpG island 3（約1.1 kb的長度）分為1 - 5, 5個區域，我們的初步結果顯示CpG island 3中3 - 5區域在某些口腔癌細胞中有DNA甲基化修飾的情形，並且其DNA甲基化的程度可受到5’-azaC處理而下降。接下來，我們利用in vitro methylation 和luciferase assays 探討CpG island 的缺失和DNA甲基化是否影響相對的luciferase活性。啟動子從-558到+2440（轉錄起始點為+1）和其不同截斷區段的序列分別克隆到pGL3-basic載體。我們發現CpG island 3的消失，其啟動子活性顯著下降。 但CpG island 3 的存在與否皆不會影響DNA甲基化抑制啟動子的效力，這顯示non-CpG sites 也具有調節作用。更進一步，發現轉錄因子p53和retinoid acid receptors也參與調節的miR-22啟動子活性。雖然需要更多的研究證實，但這項研究說明了DNA甲基化和轉錄調控參與調控miR-22在口腔癌細胞中失調表現

The deregulation of certain microRNAs has been associated with the progression and metastasis of various cancer types. DNA methylation in the CpG islands plays a crucial role in the down-regulation of tumor suppressor genes, including those encoding miRNAs. MiR-22 has been shown to be deregulated in several cancer types, including breast cancer and multiple myeloma. Using real-time RT-PCR analysis, we also found that miR-22 was down-regulated in the majority of oral cancer cell lines and 70% of the tested clinical specimens when compared with their normal counterparts. Consistent with the notion that DNA methylation might play a role in the gene silencing, we did detect three putative CpG islands, 1 - 3, located upstream from the first nucleotide of pre-miR-22 using CpG island prediction softwares. Two oral cancer lines with low miR-22 expression were treated with a methylation inhibitor, 5’-azaC at 1 ~ 5 M, for 5 days. Total RNA was isolated from the treated cells for real-time quantitative PCR analysis of miR-22 expression. The expression of miR-22 was induced by the inhibitor, suggesting the involvement of epigenetic regulation in the decreased expression of miR-22 in oral cancer cells. First, bisulfate specific PCR coupled with DNA sequencing was used to analyze the methylation status of CpG sites in the putative CpG islands, and pinpoint the critical region related with the silencing of miR-22 expression. Following dividing the longest CpG island 3 (~1.1 kb in length) into 5 regions, 1 - 5, our preliminary data show that only regions 3 to 5 in the putative CpG island are subjected to DNA methylation in certain oral cancer lines, which can be attenuated by 5’-azaC treatment. We next used in vitro methylation and promoter-driven luciferase assays to investigate whether CpG island deletion and DNA methylation affected the relative luciferase activity. A putative promoter segment from -558 to +2440 (transcription start as +1) and its serial truncations were cloned into pGL3-basic vectors. We found a significant reduction of the promoter activity driven by CpG island 3 deletion. DNA methylation suppressed the promoter activity regardless of the presence of CpG island 3, suggesting the involvement of non-island CpG sites in the regulation. Futthermore, the transcription factors including p53 and retinoid acid receptors also participate in the regulation of miR-22 promoter activity. Although more studies are needed, this study implicates that both DNA methylation and transcriptional regulation are involved in the deregulation of miR-22 in oral cancer cells.

Abstract in Chinese 1
Abstract in English 3
Acknowledgement 5
Content 6
List of Figures 8
Abbreviations 9
I. Introduction
Oral cacner 10
Genetic and epigenetic changes in oral cancer 10
TP53 alternations in oral cancer 11
MicroRNA dysregulation in oral cancer 12
DNA Methylation 12
Hypermethylation of tumor suppressor microRNAs 13
MiR-22 dysregulation in human cancer 14
Previous lab studies on miR-22 14
II. Specific aims 16
III. Materials and methods
Cell lines 17
Genomic DNA extraction and bisulfite sequencing PCR 17
Plasmid contruct 18
In vitro methylation 18
Transient transfection and luciferase promoter assays 18
Site-directed mutagenesis 19
Cisplatin treatment and Western blot analysis 19
Primer list 19
IV. Results
DNA methylation in the putative miR-22 promoter in oral cancer cell lines 21
CpG island 3 and its adjacent region are crucial for the miR-22 promoter activity 22
DNA methylation plays a direct role in regulating miR-22 promoter activity 23
Tumor suppressor p53 and retinoid acid metabolites participate in the regulation of miR-22 promoter activity 23
p53 mutants, R175H and R248Q, attenuates p53-mediated inhibition of miR-22 promoter activity 24
Retinoid acid, a differentiation reagent, and its metabolite induces miR-22 promoter activity 25
V. Discussion 26
VI. References 28
VII. Appendix 50

VI.References

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