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研究生：	劉家仁 Liu, Chia-jen
論文名稱：	miR-320與miR-185在口腔癌發展過程中所扮演的角色 The role of miR-320 and miR-185 in oral cancer progression
指導教授：	陳玉玲 Chen, Yuh-ling
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2008
畢業學年度：	96
語文別：	英文
論文頁數：	44
中文關鍵詞：	反轉錄病毒系統、口腔鱗狀上皮細胞癌
外文關鍵詞：	miR-185, Retrovirus system, miR-320, Oral squamous cell carcinoma (OSCC)
相關次數：	點閱：135 下載：1
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MicroRNAs (miRNAs)是屬於一群小片段且不轉譯出蛋白質的核醣核酸，目前發現的功能為結合mRNA並且抑制其蛋白質轉譯或使其降解。最近的研究指出，在許多人類癌症中發現miRNAs突變或是表現量改變，同時也發現miRNAs扮演著腫瘤抑制基因或是致癌基因的角色。在先前研究藉由微矩陣方式，發現miRNAs在口腔癌細胞株(OC2)中表現與正常口腔角化細胞(NHOK)不同。所以在本實驗中，想進一步研究miRNA在口腔臨床檢體的表現量是否與細胞株實驗結果一致，並研究這些miRNA在口腔癌病程發展中可能造成的影響。首先利用即時定量PCR方式，檢測27組口腔鱗狀上皮細胞癌臨床病人的腫瘤組織與非腫瘤組織的配對檢體，發現miR-185和miR-320在口腔癌組織中的表現量比正常口腔黏膜組織明顯減少，miR-185和miR-320分別在55.6% (15/27)及70% (19/27)的口腔癌檢體有表現量降低的情形。接著利用外送miR-320 先驅物質來觀察口腔癌細胞株(OC2、HSC3)增生功能及細胞週期的改變，其結果並沒有顯著差異。在細胞移行實驗當中，發現外送miR-320 先驅物質能使OC2移行能力增加，但卻使HSC3移行能力下降，miR-320對不同口腔癌細胞株的移行活性有不同的調控影響此機轉還有待進一步釐清。為了研究miRNAs在活體動物的功能及有效率且大量的表現miR-320，本研究也建立反轉錄病毒系統來製作miRNAs病毒，將miR-320先驅序列基因構築到反轉錄病毒載體中，讓miR-320在細胞中持續表現。選用不同長度的miR-320基因片段，分別為102、182、332個鹼基對，其中都包含miR-320的先驅序列基因。實驗結果發現，在長度為102以及182個鹼基對載體感染的前列腺癌細胞株(PC3)中，雖然利用RT-PCR可偵測到外送的整段基因序列表現，但利用quantitative real time polymerase chain reaction (qPCR)並沒有偵測到成熟 miR-320序列在感染反轉錄病毒的細胞中增加。因此未來會朝microRNAs processing機轉準則探討，首先加長構築序列的長度，使其能過度表現成熟miR-320，以利於將來miR-320生物活性及調控機轉之研究。

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Recent evidence has shown that miRNAs mutations or mis-expression were correlated with various human cancers and indicated that miRNAs can function as tumor suppressors and oncogenes. In the previous studies, we found that several miRNA genes that were differently expressed between oral cancer cells (OC2) and normal human oral keratinocytes (NHOK) by microarray analyses. In this study, we want to analyze whether the expression of these miRNAs was correlated with oral cancers and to characterize their possible effects on oral cancer progression. We analyzed the RNA levels of miR-320 and miR-185 in 27 OSCC tissues and matched-pair non-cancer tissues by Quantitative RT-PCR and found that both miRNAs were significantly down-regulated in oral cancer specimens. There is no significant effect on proliferation and cell cycle progression in oral cancer cell lines by transporting exogenous miR-320 precursor. In migration assay, miR-320 overexpreesion increases migratory ability of OC2 but has opposing effects on HSC3. The mechanism should be studied in the future. We further constructed various primary miR-320 genes (pri-miR-320) with different length of flanking sequence (10, 50, 125-bp) in a retroviral plasmid to produce pri-miR-320-retrovirus. The data showed that mature miR-320 wasn’t detected in pri-miR-320-retrovirus (10, 50-bp) transduced cells. We will figure out the problems of miRNAs experiments in the future according to the rules of miRNAs processing, for example, elongating flanking sequence up to 125-bp. Besides, the overexpresion of miR-320 in regulating cell functions will be studied.

CONTENTS

摘要	I
ABSTRACT	III
誌謝	V
CONTENTS	VI
TABLE CONTENTS	VIII
FIGURE CONTENTS	IX
ABBREVIATIONS	X
INTRODUCTION	1
 Oral cancer	1
 MicroRNAs	1
 MicroRNAs biogenesis	2
 MicroRNAs and cancer	2
 MicroRNAs database	3
 MicroRNAs regulate migration	4
 Purposes of research	4
MATERIALS AND METHODS	6
 Tissue samples	6
 Whole tissue RNA extraction	6
 Reverse transcription (RT)	7
 Polymerase chain reaction (PCR)	7
 MicroRNA qRT-PCR	8
 Cell culture	9
 Cell proliferation assay (MTS assay)	9
 Plasmid construction	10
 Virus production	10
 Apoptosis analysis by propidium iodide (PI) Assay	11
 Cell migration assay	12
 Western blot analysis	12
 Statistical analysis	14
RESULTS	15
 Expression of mature miR-185 and miR-320 in clinical OSCC specimens	15
 Clinicopathological characteristics	15
 Expression of mature miR-320 in oral cells	16
 miR-320 does not affect cell viability and proliferation in oral cancer cell lines	16
 No significant in cell cycle analysis of both cell lines	17
 miR-320 exerts opposite effects on the migration of two oral cancer cell lines	18
 The retroviral construct for miR-320 expression	18
DISSCUSSION	20
CONCLUSION	25 
REFERENCE	26
自述	44


TABLE CONTENTS 

Table 1. The correlation between miR-185 expression and the clinicopathological parameters in 27 OSCC patients were analyzed by Chi-square test.	29
Table 2. The correlation between miR-320 expression and the clinicopathological parameters in 27 OSCC patients were analyzed by Chi-square test.	30


FIGURE CONTENTS

Figure 1. Expression of mature miR-185 and miR-320 in OSCC.	31
Figure 2. miR-320 expression in oral cancer cell lines.	32
Figure 3. miR-320 Genomic DNA sequence of cell lines	33
Figure 4. miR-320 doesn’t significantly affect OC2 cell morphology and proliferation.	34
Figure 5. Cell cycle analysis showed no significant differences in cell cycle distributions between OC2 transfected with control miR and miR-320.	35
Figure 6. miR-320 doesn’t significantly affect HSC3 cell morphology and proliferation.	36
Figure 7. Cell cycle analysis showed no significant differences in cell cycle distributions between HSC3 transfected with control miR and miR-320.	37
Figure 8. Overexpression of miR-320 in OC2 cells can enhance migration.	38
Figure 9. Overexpression of miR-320 in HSC3 cells can effectively inhibit migration	39
Figure 10. The retroviral constructs for miR-320 expression.	40
Figure 11. The precursor of miR-320 (102) can be overexpressed in transfected PC3 cells, but its mature form was not successfully produced..	41
Figure 12. The precusor of miR-320 (182) can be overexpressed in transfected PC3 cells, but its mature form was not successfully produced.	42
Figure 13. Summary of miR-320 expression and maturation from vectors containing various lengths of miRNA genomic fragments	43
                                    

Bartel DP (2004). MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116: 281-97.

Bova GS, Carter BS, Bussemakers MJ, Emi M, Fujiwara Y, Kyprianou N et al (1993). Homozygous deletion and frequent allelic loss of chromosome 8p22 loci in human prostate cancer. Cancer Res 53: 3869-73.

Brown BD, Gentner B, Cantore A, Colleoni S, Amendola M, Zingale A et al (2007). Endogenous microRNA can be broadly exploited to regulate transgene expression according to tissue, lineage and differentiation state. Nat Biotechnol 25: 1457-67.

Calin GA, Dumitru CD, Shimizu M, Bichi R, Zupo S, Noch E et al (2002). Frequent deletions and down-regulation of micro- RNA genes miR15 and miR16 at 13q14 in chronic lymphocytic leukemia. Proc Natl Acad Sci U S A 99: 15524-9.

Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD et al (2004). MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A 101: 11755-60.

Chan JA, Krichevsky AM, Kosik KS (2005). MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Cancer Res 65: 6029-33.

Chen CZ, Li L, Lodish HF, Bartel DP (2004). MicroRNAs modulate hematopoietic lineage differentiation. Science 303: 83-6.

Chen SY, Wang Y, Telen MJ, Chi JT (2008). The genomic analysis of erythrocyte microRNA expression in sickle cell diseases. PLoS ONE 3: e2360.

Fasanaro P, D'Alessandra Y, Di Stefano V, Melchionna R, Romani S, Pompilio G et al (2008). MicroRNA-210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin-A3. J Biol Chem 283: 15878-83.

Garzon R, Fabbri M, Cimmino A, Calin GA, Croce CM (2006). MicroRNA expression and function in cancer. Trends Mol Med 12: 580-7.

Hsieh IS (2007). MicroRNA profiling in oral squamous cell carcinoma. Masteral thesis. Institute of Oral Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.

Ikuta M, Podyma KA, Maruyama K, Enomoto S, Yanagishita M (2001). Expression of heparanase in oral cancer cell lines and oral cancer tissues. Oral Oncol 37: 177-84.

Kozaki K, Imoto I, Mogi S, Omura K, Inazawa J (2008). Exploration of tumor-suppressive microRNAs silenced by DNA hypermethylation in oral cancer. Cancer Res 68: 2094-105.

Kusenda B, Mraz M, Mayer J, Pospisilova S (2006). MicroRNA biogenesis, functionality and cancer relevance. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 150: 205-15.

Lin SC, Liu CJ, Chiu CP, Chang SM, Lu SY, Chen YJ (2004). Establishment of OC3 oral carcinoma cell line and identification of NF-kappa B activation responses to areca nut extract. J Oral Pathol Med 33: 79-86.

Matsuyama H, Pan Y, Yoshihiro S, Kudren D, Naito K, Bergerheim US et al (2003). Clinical significance of chromosome 8p, 10q, and 16q deletions in prostate cancer. Prostate 54: 103-11.

Maziere P, Enright AJ (2007). Prediction of microRNA targets. Drug Discov Today 12: 452-8.

Meng F, Henson R, Lang M, Wehbe H, Maheshwari S, Mendell JT et al (2006). Involvement of human micro-RNA in growth and response to chemotherapy in human cholangiocarcinoma cell lines. Gastroenterology 130: 2113-29.

Nilsen TW (2007). Mechanisms of microRNA-mediated gene regulation in animal cells. Trends Genet 23: 243-9.

Smalheiser NR (2008). Regulation of mammalian microRNA processing and function by cellular signaling and subcellular localization. Biochim Biophys Acta.

Voorhoeve PM, le Sage C, Schrier M, Gillis AJ, Stoop H, Nagel R et al (2006). A genetic screen implicates miRNA-372 and miRNA-373 as oncogenes in testicular germ cell tumors. Cell 124: 1169-81.

Ye H, Pungpravat N, Huang BL, Muzio LL, Mariggio MA, Chen Z et al (2007). Genomic assessments of the frequent loss of heterozygosity region on 8p21.3-p22 in head and neck squamous cell carcinoma. Cancer Genet Cytogenet 176: 100-6.

Zhang HH, Wang XJ, Li GX, Yang E, Yang NM (2007). Detection of let-7a microRNA by real-time PCR in gastric carcinoma. World J Gastroenterol 13: 2883-8.

Zhang L, Huang J, Yang N, Greshock J, Megraw MS, Giannakakis A et al (2006). microRNAs exhibit high frequency genomic alterations in human cancer. Proc Natl Acad Sci U S A 103: 9136-41.

Zhao Y, Srivastava D (2007). A developmental view of microRNA function. Trends Biochem Sci 32: 189-97.

Zhu S, Wu H, Wu F, Nie D, Sheng S, Mo YY (2008). MicroRNA-21 targets tumor suppressor genes in invasion and metastasis. Cell Res 18: 350-9.

校內：2013-08-28公開
校外：2013-08-28公開

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