抑制口腔癌和乳癌細胞轉移的微型核醣核酸｜國立中央大學博碩士論文系統

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研究生：	黃暐捷 Wei-Chieh Huang
論文名稱：	抑制口腔癌和乳癌細胞轉移的微型核醣核酸 MicroRNAs acting as potent metastasis suppressors in oral cancer and breast cancer
指導教授：	王陸海 Lu-Hai Wang 陳盛良 Shen-Liang Chen
口試委員:
學位類別：	博士 Doctor
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	163
中文關鍵詞：	口腔癌、乳癌、轉移、微型核醣核酸、局部黏著斑激酶、口腔鱗狀上皮細胞癌
外文關鍵詞：	oral cancer, breast cancer, metastasis, microRNA, focal adhesion kinase, OSCC
相關次數：	點閱：15 下載：0
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轉移是癌症患者治療失敗的主要臨床因數。微RNA分子在癌症進展和轉移扮演一個重要的角色。此項研究是深入探討微RNA分子與高侵入性的口腔癌及乳癌細胞之間的關係。我們分別使用了體外和體內的篩選方式建立了高度侵入性的口腔鱗狀細胞癌（OSCC）及乳癌細胞，並且藉由微小RNA微陣列技術分析這些高度轉移細胞株與其低侵入性母代細胞株的轉錄圖譜。我們以基因表現差異兩倍為門檻，並且使用gene ontology軟體分析找出與侵入相關的微RNA分子以及其下游標的基因。研究顯示所篩選出的全部高侵入口腔癌細胞株及乳癌細胞株其miR-491-5p或miR-149分別表現顯著減少。同時研究也顯示當miR-491-5p或miR-149分別大量表達在高侵入性口腔癌或乳癌細胞時會抑制其爬動和侵入能力，在老鼠模式中也發現大量表達這些微RNA分子會抑制癌細胞轉移的能力。藉由3'UTR報告基因分析證實G protein-coupled receptor kinase-interacting protiein 1（GIT1）同時是miR-491-5p及miR-149的目標基因。另一方面在口腔癌細胞中大量表達GIT1可以回復miR-491-5p及miR-149所抑制的爬動、侵入及轉移的能力。若抑制GIT1的表現則會抑制口腔癌細胞的爬動、侵入和肺轉移的能力。這個研究也證實miR-491-5p藉由抑制GIT1的表現進而減少口腔癌細胞的focal adhesion，同時導致paxillin蛋白的降解及減少paxillin、FAK及EGF/EGFR調控ERK1/2的活性，也抑制了MMP2/9的表現量與活性。在乳癌方面，miR-149同樣藉由抑制GIT1的表現進而促使paxillin及α5β1 integrin蛋白分別走向proteasome及lysosome的分解路徑。此外，我們發現miR-491-5p和FOCAD同時位於染色體9p21.3，我們的實驗證明miR-491-5p是一個intronic微RNA分子，其座落在FOCAD的intron 4。實驗證明當FOCAD的表現被抑制時也會導致口腔癌細胞癌轉移及侵入的能力減少。進一步我們也證實FOCAD與miR-491-5p會一同被表現出來，並且藉由抑制GIT1調控路徑來減少口腔癌細胞的爬動、侵入和轉移的能力。因此，我們的結論是miR-491-5p及miR-149可以同時藉由GIT1的調控路徑有效的抑制口腔癌及乳癌細胞的轉移能力，這些發現暗示著本研究探討的調控機制可能成為臨床診斷和治療的標的。

Metastasis is an important clinical parameter for patient prognosis and the major cause of treatment failure for cancer. MicroRNAs are molecules that could play an abstrusive role in cancer progression and metastasis. The study is to identify relevant microRNAs associated with invasive phenotype of oral and breast cancers. We have established isogenic highly invasive oral squamous cell carcinoma (OSCC) lines and breast cancer lines from their respective low invasive parental lines via in vitro and in vivo selection protocols. MicroRNA array analysis was used for transcriptome profiling between each pair of the parental and the highly invasive subline. Using threshold of 2-fold change of gene expression, we analyzed the microarray data by gene ontology enrichment of Partek and identify genes as well as microRNAs revealed to be significantly associated with invasive phenotype. We found that miR-491-5p and miR-149 level were significantly decreased in the selected OSCC invasive lines and highly invasive breast cancer lines respectively. Overexpression of miR-491-5p or miR-149 in those highly invasive cells suppressed their migration/invasion in vitro and metastatic ability in a xenograft mouse model. The G protein-coupled receptor kinase-interacting protiein 1 (GIT1) is a direct target of miR-491-5p and miR-149 as revealed by 3'UTR reporter assays. The miR-491-5p- and miR-149-mediated inhibition of migration/invasion and lung metastasis could be rescued by overexpression of GIT1. Depletion of GIT1 inhibited migration/invasion and lung metastasis of OSCC and breast cancer cells. MiR-491-5p-mediated GIT1 repression reduced focal adhesion and concurrently decreased steady state levels of paxillin, phospho-paxillin,phospho-FAK, EGF/EGFR-mediated ERK1/2 activation as well as decreased MMP2/9 levels and activities in OSCC cells. Overexpression of miR-149 or depletion of GIT1 led to enhanced protein degradation of paxillin and α5β1 integrin via proteasome and lysosome pathways respectively. In addition, we found that miR-491-5p and focadhesin (KIAA1797/FOCAD) gene are located on chromosome 9p21.3 together. Our data suggest that miR-491-5p is an intronic miRNA processed form FOCAD intron 4 rather than being transcribed as a separate RNA. Depletion of FOCAD promoted cell migration/invasion abilities in human oral cancer cells. Furthermore, FOCAD/miR-491-5p were co-expressed in OSCC cells and they suppresses OSCC cell migration/invasion and metastasis, suggesting potential application of the miR-491-5p/GIT1 pathways in OSCC prognosis and therapy. Therefore, we conclude that miR-491-5p and miR-149 suppresses migration/invasion and metastasis of OSCC and breast cancer cells, respectively, by targeting GIT1, suggesting potential application of the miR-491-5p/GIT1 and miR-149/GIT1 pathways in clinical diagnosis and therapeutics.

Table of contents
Declaration    I
Publications arising from this thesis    II
(A). Referred papers:    II
(B). Abstracts presented in meetings:    II
中文摘要    IV
Abstract    VI
Acknowledgments    VIII
Table of contents    XI
Abbreviation    XVII
Chapter I: Introduction    1
Epidemiology of oral cancer and breast cancer    1
Neck lymph node metastasis of oral squamous cell carcinoma    2
Breast cancer metastasis    2
microRNA biogenesis and nomenclature    3
The role of microRNAs in oral cancer and breast cancer invasion and metastasis    5
Regulation of focal adhesion complexs in cancer cell migration, invasion and metastasis    6
Figures    11
Figure I-1. The microRNA processing pathways and their regulation    11
Figure I-2. GW (glycine-tryptophan repeats) proteins are recruited to mRNA via direct interaction with the slicer activity (miRNA–AGO complex) (49).    13
Figure I-3. Schematic of a miRNA precursor    14
Figure I-4. Focal adhesion components    15
Figure I-5. GIT1 domain structure    16
Figure I-6. Summary of the GIT1 protein interaction and signaling pathways    17
Figure I-7. Focal adhesion kinase (FAK)-SRC-ERK-paxillin signaling regulating cell migration and focal adhesion formation    18
Figure I-8. The model showing that FOCAD is a novel component of the focal adhesion complex.    20
Chapter II: Materials and Methods    21
Tissue samples acquisition.    21
Cell culture, DNA and RNA transfections and stable cell line generation.    21
Vectors, antibodies and reagents.    23
3'UTR reporter assays.    24
Reverse transcriptase (RT)-PCR and qRT-PCR.    25
Cell chemotatic migration and invasion assay.    25
In vivo metastasis assays.    26
Focal adhesion assay and Immunofluorescence microscopy.    27
Western blotting    27
Paxillin degradation assays    28
Immunohistochemistry (IHC) and Fluorescence in situ hybridization (FISH).    28
Gelatin zymography    29
Transfection efficiency of miRNAs    29
Statistical analysis.    30
Figure    31
Figure II. The predicted highly conserved miR-491-5p and miR-149 target sequences located in the 3′UTR of GIT1 mRNA.    31
Chapter III: Functional role and clinical evidence of microRNA-491-5p in oral cancer metastasis    32
Rationale    32
Results    34
Establishment of isogenic pairs of high and low invasive OSCC lines.    34
Down-regulation of miR-491-5p was found in the highly invasive OSCC cells and was correlated with poor survival of OSCC patients.    35
The miR-491-5p inhibits migration, invasion and lung metastasis of OSCC cells.    36
GIT1 is a direct target of miR-491-5p.    37
Suppression of GIT1 by its siRNA inhibits OSCC cell migration, invasion and metastasis.    38
Re-expression of GIT1 significantly reverses miR-491-5p-mediated suppression of invasion and metastasis of C9-lung-IV2 cells.    39
Inhibtion of GIT1 by miR-491-5p enhances degradation of paxillin and impaires focal adhesion signaling in OSCC cells.    39
Dominant-negative form of FAK (DN-FAK) partially inhibits OSCC cells invasiveness which could be rescued by GIT1.    41
miR-491-5p targets GIT1 to regulate expression level and activity of Matrix metalloproteinases2/9 (MMP2/9) via EGFR/ERK1/2 signaling pathway .    41
Correlation of miR-491-5p and GIT1 expression with grades of OSCC and inverse correlation between miR-491-5p and GIT1 .    43
Discussion:    44
Figures    50
Figure III-1. Selection of highly invasive oral cancer cells.    50
Figure III-2. The miR-491-5p expression is downregulated in highly invasive OSCC lines and oral cancers.    52
Figure III-3. The miR-491-5p inhibits migration, invasion and metastasis of OSCC cells.    54
Figure III-4. Overexpressing miR-491-5p inhibits lung metastasis in SCID mice.    56
Figure III-5. Identification of GIT1 as the direct target of miR-491-5p.    57
Figure III-6. The GIT1 mRNA level is upregulated in highly invasive OSCC lines.    59
Figure III-7. GIT1 is overexpressed in the highly invasive oral cancer lines.    60
Figure III-8. GIT1 expression correlates with lymph node metastasis and knockdown of GIT1 inhibits OSCC cell migration, invasion and metastasis.    61
Figure III-9. GIT1 depletion enhances degradation of paxillin, reduces FAK phosphorylation and decreases focal adhesion formation.    63
Figure III-10. Knockdown of GIT1 by siRNA-3 has no effect on paxillin mRNA expression.    65
Figure III-11. DN-FAK suppresses GIT1-induced OSCC cell migration and invasion.    66
Figure III-12. Effect of GIT1 on activation and expression of MMP2/9 and on EGF-induced ERK1/2 phosphorylation.    67
Figure III-13. Effect of ERK1/2 on MMP2/9 mRNA expression in OSCC cells.    69
Figure III-14. Overexpression of miR-491-5p has no effect on proliferation of OSCC cells measured up to 72h.    71
Figure III-15. Effect of the DN-MEK or PD98059 on GIT1 induced MMP2/9 mRNA expression in OSCC cells.    73
Figure III-16. miR-491-5p and GIT1 expression levels exhibits an inverse relationship in OSCC specimens.    74
Figure III-17. Depletion of FAK and GIT1 decreases the paxillin and FAK phosphorylation.    76
Figure III-18. CGHNC9 cells transfected with Fluorescein amidite (FAM)-labeled miR-491-5p.    77
Tables    78
Table III-1: Clinicopathologic features of OSCC patients    78
Table III-2: The list of primers used in oral cancer study    79
Table III-3: Venn digram showing miR-491-5p targets derived from the TargetScan and DIANA-microT    80
Chapter IV: The role of microRNA-149 in migration, invasion and metastasis of breast cancer    81
Rationale    81
Results    83
MiRNA microarray profiling identified miRNAs differentially expressed in the in vivo-selected metastatic breast cancer cells versus their parental cells.    83
miR-149 suppresses breast cancer cell migration and invasion in vitro    84
miR-149 functions as a metastasis suppressor in vivo    84
GIT1 is a direct target of miR-149    85
Low miR-149 and high GIT1 level correlates with the invasive phenotype of breast cancer cell lines    86
Knockdown of the endogenous GIT1 expression by siRNAs impairs cell migration and invasion    87
Re-expression of GIT1 partially reverses miR-149-mediated suppression of migration and invasion    88
Re-expression of GIT1 partially rescues miR-149-suppressed lung metastasis    88
Inhibition of GIT1 expression by miR-149 or GIT1 siRNAs impairs focal adhesions and focal adhesion signaling, which is significantly rescued by re-expression of GIT1    89
Depletion of GIT1 leads to enhanced proteasome-mediated degradation of paxillin    90
Depletion of GIT1 leads to enhanced lysosome-mediated protein degradation of α5β1 integrin    91
Depletion of GIT1 impairs α5β1-mediated cell adhesion to fibronectin and collagen    93
miR-149 and GIT1 expression significantly correlates with clinical stages and lymph node metastasis of breast cancer    93
Discussion    95
Figures    99
Figure IV-1. Schematics for the establishment of highly metastatic breast cancer cells using a mouse model.    99
Figure IV-2. Metastatic IV2 sublines exhibit increased invasive ability as compared to the parental 231 cells.    100
Figure IV-3. Whole organ photographs and histological analysis of lung metastases generated by parental 231 and IV2-1 cells.    101
Figure IV-4. Hierarchical clustering of miRNA expression among the parental 231 cells and three metastatic IV2 sublines, IV2-1, IV2-2 and IV2-3, reveals a set of differentially expressed miRNAs.    102
Figure IV-5. Taqman qRT-PCR validation of selected candidate miRNAs.    103
Figure IV-6. miR-149 is down-regulated in highly metastatic IV2 sublines and suppresses breast cancer cell migration, invasion and metastasis.    104
Figure IV-7. Overexpression of pre-miR-149 has no effect on proliferation of the IV2-1 cells.    106
Figure IV-8. Overexpression of pre-miR-149 has no effect on proliferation of the Hs578T breast cancer cells.    107
Figure IV-9. miR-149 reduces lung targeting ability of metastatic IV2-1 cells.    108
Figure IV-10. GIT1 is a direct target of miR-149 in breast cancer cells.    109
Figure IV-11. miR-149 suppresses GIT1 expression at the mRNA level in IV2-1 cells.    111
Figure IV-12. miR-149 sponge suppresses miR-149 expression in parental 231 cells.    112
Figure IV-13. miR-149 and GIT1 expression are associated with the invasive phenotype of breast cancer cell lines.    113
Figure IV-14. miR-149-dependent inhibitory effect on cell migration/invasion and metastasis can be partially reversed by re-expression of GIT1 in vitro and in vivo.    114
Figure IV-15. Knockdown of GIT1 using siRNA transfection has no effect on GIT2 mRNA expression.    116
Figure IV-16. Depletion of GIT1 has no effect on proliferation of the IV2-1 cells.    117
Figure IV-17. Inhibition of GIT1 in the invasive Hs578T cells reduces their migration and invasion.    118
Figure IV-18. ArfGAP activity of GIT1 is not required for GIT1-mediated cell migration and invasion of IV2 cells.    119
Figure IV-19. Inhibition of GIT1 expression by miR-149 or GIT1 siRNAs suppresses phosphorylation of FAK and paxillin and focal adhesion formation of metastatic IV2 cells.    121
Figure IV-20. Depletion of GIT1 leads to enhanced protein degradation of paxillin and α5β1 integrin in metastatic IV2 cells.    123
Figure IV-21. GIT1 depletion greatly reduces α5β1integrin-mediated cell adhesion to fibronectin in metastatic IV2 cells.    125
Figure IV-22. Effect of depleting GIT1 on paxillin and α5β1 integrin level under different matrix-coating conditions.    126
Figure IV-23. Expression level of miR-149 and GIT1 in 90 breast tumor specimens and 50 adjacent normal tissues as determined by qRT-PCR.    127
Figure IV-24. Low level of miR-149 and high level of GIT1 correlate with lymph node metastasis.    128
Figure IV-25. Model of miR-149-GIT1 pathways in the regulation of breast cancer metastasis.    130
Figure IV-26. Evaluation of the transfection efficiency of pre-miR-149 using FAM-labeled pre-miR-149.    131
Tables    132
Table IV-1. Metastasis ability between parental 231 cells and IV2-1 cells.    132
Table IV-2. Candidate miRNAs down-regulated in IV2 lines.    133
Table IV-3. The list of primers and oligomers used in this study.    134
Table IV-3 continued. The list of primers and oligomers used in this study.    136
Table IV-4. Information of antibodies and reagents.    138
Chapter V: General discussion and future work    139
The Role of miRNA-491-5p and miR-149 in tumor progression    139
miRNAs as therapeutic targets and tools for cancer growth and metastasis    140
Role of GIT1, a target of miR-491-5p, as a therapeutic targets and tools for cancer progression    142
MiR-491-5p as an intronic microRNA is likely co-expressed with its host gene, FOCAD    144
Figures    148
Figure V-1. MiR-491-5p coding pre-sequence is located in intron 4 of the FOCAD gene    148
Figure V-2. Expression of intronic miR-491-5p and FOCAD    149
Figure V-3. miR-491-5p and FOCAD expression are down-regulated in the highly invasive oral cancer lines.    151
Figure V-4. Expression of targets of miR-491-5p are downregulated in OSCC cells overexpressing FOCAD intron 4 filament.    152
Figure V-5. Transfection of CGHNC9 cells with three set of FOCAD siRNAs effectively suppressed FOCAD expression.    154
Figure V-6. Inhibition of FOCAD expression promotes migration and invasion of human OSCC cells    155
References:    156


                                

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