The EGFR-induced microRNA miR-7 via Ras/ERK/c-Myc pathway modulates lung cancer oncogenesis by downregulation of ets2-repressor factor, ERF

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研究生：	林華珩 Lin, Hua-Heng
論文名稱：	The EGFR-induced microRNA miR-7 via Ras/ERK/c-Myc pathway modulates lung cancer oncogenesis by downregulation of ets2-repressor factor, ERF 表皮生長因子接受器(EGFR)誘導微核糖核酸7(miR-7)經Ras/ERK/c-Myc路徑微調肺癌癌化藉由ets2抑制因子(ERF)
指導教授：	吳成文 Wu, Cheng-Wen
口試委員:	張文祥 Chang, Wun-Shaing Wayne 謝義興 Shieh, Yi-Shing 吳成文 Wu, Cheng-Wen
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物科技研究所 Biotechnology
論文出版年：	2011
畢業學年度：	99
語文別：	英文
論文頁數：	51
中文關鍵詞：	肺癌、微核醣核酸、表皮生長因子接受器
外文關鍵詞：	lung cancer, miRNA, EGFR
相關次數：	點閱：4 下載：0
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MicroRNAs (miRNAs), a novel class of gene regulators, have been associated with tumorigenesis. Aberrant expression and function of epidermal growth factor receptor (EGFR) are reported in most lung cancer cases and are correlated with poor prognosis. Through a miRNA microarray screen for miRNAs regulated by EGFR signaling in lung cancer, we discovered that EGFR induces miR-7 expression. Overexpression of the active EGFR mutant (L858R) enhanced the miR-7 expression. Once transfected in lung cancer cells, Ras and c-Myc promoted miR-7 level; whereas blocking the ERK pathway with inhibitors decreased the miR-7 level, suggesting that the EGFR induces miR-7 expression through the Ras/ERK/c-Myc pathway. Ectopic expression of miR-7 in lung cancer cells promoted cell growth and tumor formation, and significantly increased the mortality of nude mice, indicating miR-7 is an oncomiR. Using a SILAC (stable isotope labeling with amino acids in cell culture) analysis, we found that miR-7 decreases the levels of ERF, suggesting the downstream target of miR-7 is ERF. Furthermore, a miR-7 complementary sequence was identified in the coding sequence of ERF. miR-7 inhibited ectopic expression of the wild-type but not the mutated ERF coding sequence, supporting that ERF is the direct target of miR-7 in lung cancer. Overexpression of miR-7 inhibited ERF expression and rescued ERF-mediated cell growth arrest. Our finding that miR-7 is overexpressed in primary lung cancer and correlated positively with EGFR mutation indicates an important role for miR-7 in the modulation of EGFR-mediated oncogenesis and can serves as a novel biomarker for cancer prognosis.

中文摘要    8
Abstract    9
   Introduction    10
1.    Lung cancer and EGFR    10
2.    MicroRNA    10
3.    ERF tumor suppressor    11
4.    EGFR-induced miRNA    11
   Materials and Methods    13
1.    Materials    13
2.    Cell culture    13
3.    Construct generation    13
4.    Virus generation and infection    14
5.    Oligonucleotide transfection    14
6.    Human miRNA oligo microarray analysis    15
7.    Quantitative real-time PCR (qRT-PCR)    15
8.    Cell proliferation assay    15
9.    Soft agar anchorage-independent assay    16
10.    Immunoblotting    16
11.    Luciferase reporter assay    17
12.    SILAC labeling and cell culture    17
13.    In-gel digestion    18
14.    In-solution digestion    18
15.    Peptide isoelectric focusing    19
16.    Mass spectrometric analysis    19
17.    Proteins identification and quantification    20
18.    Xenograft tumorigenicity assay and orthotopic implantation    21
19.    Patients and tissue samples    21
20.    Statistical Analysis    22
   Results    23
1.    EGFR induces miR-7 expression    23
2.    Ras/ERK/c-Myc pathway regulates miR-7 expression    23
3.    Ectopic expression of miR-7 enhances oncogenic properties of lung cancer    24
4.    miR-7 targets ERF for suppression    26
5.    Elevated miR-7 expression in human lung cancer    27
   Discussion    29
   Figure and figure legends    33
Figure 1. miR-7 expression is regulated by EGFR signaling    33
Figure 2. Silencing of EGFR promoted cell death in CL1-5 cells    34
Figure 3. Ras/ERK/c-Myc signaling controls miR-7 expression    35
Figure 4. EGFR regulates pre-miR-7-1 expression    36
Figure 5. Ectopic expression of miR-7 was detected in lung cancer    37
Figure 6. The effect of miR-7 on tumor cell growth    39
Figure 7. Overexpression of miR-7 increased the mortality of nude mice    40
Figure 8. miR-7 regulates ERF expression    41
Figure 9. miR-7 targets the coding sequence of ERF for inhibition    43
Figure 10. miR-7 is up-regulated in primary lung cancer and negatively correlated with disease-free survival    45
Figure 11. overexpression of miR-7 attenuates EGFR expression    45
Table 1 miRNAs induced by EGFR in CL1-5 lung cancer cells    46
   Reference    47
   Appendix    51
1.    Clinicopathologic features and EGFR mutation patterns of the 5 patients with non-small cell lung cancer    52
2.    Pearson Product Moment Correlation for miR-7 and EGFR mRNA expressions in 10 pairs of squamous cell carcinoma versus adjacent normal tissue    52
3.    Primers designed for real-time PCR    52
4.    Primer designed for plasmid construction    53

                                

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