肺腺癌差异表达基因文库的建立以及ERGIC3在肺癌中的病理生理功能
文献类型：学位论文
| 作者 | 吴明松 |
| 学位类别 | 博士 |
| 答辩日期 | 2013-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 曹毅 |
| 关键词 | 肺癌 抑制性消减杂交 ERGIC3 Erv46 基因文库 基因筛选 |
| 学位专业 | Construction of Lung Adenocarcinoma-related Gene Libraries and Pathophysiological Function of ERGIC3 in Lung Cancer |
| 中文摘要 | 肺癌居肿瘤相关死亡的首位，其5年生存率仅为10%到15%。按照组织学类型分类，肺癌分为小细胞肺癌和非小细胞肺癌，非小细胞肺癌由腺癌、鳞癌和大细胞癌组成，约占肺癌的80%。近年来，肺腺癌发病率急剧上升，已成为肺癌最多的亚类。众所周知，肿瘤是在多种因素作用下，导致细胞内基因突变或表达改变，积累至一定量后产生的。因此，差异表达基因成为了肿瘤研究的中心环节。目的：研究肺癌差异基因表达模式，寻找新的肺癌相关基因，新的肺癌标志物；研究差异表达基因的表达及功能。方法：用抑制性消减杂交技术建立了肺腺癌的差异表达基因文库，然后用生物信息学对文库进行深入分析，并在文库中选取16个基因，用定量PCR法在肺癌组织和其配对的癌旁组织中进行筛选，然后用定量PCR、免疫组化、免疫荧光、免疫印迹、RNA干扰、基因过表达和细胞功能实验（迁移、增殖）等方法研究差异表达基因在肺癌中的表达和生物学功能。结果：在正向杂交文库 (FSL)中，获得177个基因和44条未知的ESTs片段，在反向杂交文库 (RSL)中，获得59个基因和10条未知的ESTs片段。在这些基因中，正向杂交文库中的152个基因和反向杂交文库中的54个基因在过去的肺癌消减杂交文库中未报道过。进一步生物信息学分析显示，这些差异表达基因具有重要的病理生理功能，参与了癌症的发生、分子运输、细胞信号转导、细胞周期、细胞增殖、细胞凋亡、细胞运动等功能。用定量PCR法对16个基因分析结果显示，在肺癌组织中，来自于正向杂交文库的ERGIC3，DDR1，HSP90B1，SDC1，RPSA，LPCAT1的mRNA明显上调 (P<0.05)，来自于反向杂交文库的GPX3, TIMP3则明显下调 (P<0.05)。此外，来自于正向杂交文库的DDX58, CCNDBP, TMSB4X, CXCL17, FOXA2, C4BPA, SCGB3A1水平上调，但没有统计学差异 (P>0.05)；而来自于正向杂交文库和反向杂交文库所共有的基因CD9水平上调，也没有统计学差异 (P>0.05)。对ERGIC3深入研究，发现ERGIC3的mRNA水平和蛋白水平在肺癌组织和多个肺癌细胞系中表达异常升高。免疫组化结果表明ERGIC3在肺癌组织的细胞质中呈强阳性染色，在支气管上皮组织和正常肺组织的细胞中染色呈阴性。统计分析结果显示，染色的阳性率与肺癌的组织学类型和分化程度有明显相关性 (P<0.05)，在腺癌中的阳性率高于鳞癌，高分化肺癌染色阳性率高于低分化肺癌；但是，ERGIC3阳性率与肺癌患者的年龄、性别、吸烟和临床TNM分期没有明显相关性。免疫荧光染色结果显示，在体外培养的肺癌细胞中，ERGIC3主要定位于高尔基体和内质网。在肺癌细胞EPLC-32M1，801D和NCI-H446中，ERGIC3主要分布于细胞核一侧，形成“新月状”结构；而在肺癌细胞SPCA-1，GLC-82 和A549中，ERGIC3分布于细胞核周围，形成“圆环状”结构。ERGIC3与另外两个主要定位于高尔基体的蛋白MUC1、ST6Gal I共定位。细胞功能实验结果显示，在内源性ERGIC3表达很高的GLC-82中，抑制ERGIC3的表达，细胞的增殖能力和迁移能力受到抑制；而在内源性ERGIC3表达很低的永生化人支气管上皮细胞BEAS-2B中，增加ERGIC3的表达，细胞的增殖能力和迁移能力明显加强。为进一步研究ERGIC3在肺癌中的功能，我们用A549细胞建立稳定过表达ERGIC3细胞株，划痕实验结果表明，稳定过表达ERGIC3后，A549细胞的迁移速度也明显加快 (P<0.05)。但是，培养液中分泌蛋白MUC2的浓度没有明显改变，细胞的形态学也没有明显变化。结论：我们建立两个肺腺癌差异表达基因文库，发现一批新的肺癌相关基因，这些差异表达基因为进一步研究非小细胞肺癌的生物学机制，寻找肺癌诊断、治疗靶标提供基础；ERGIC3在肺癌中异常高表达，在正常肺组织和支气管上皮组织中不表达，可能是一个新的潜在的肺癌标志物；ERGIC3促进细胞增殖和迁移，ERGIC3可能是一个新的肺癌相关基因。 |
| 英文摘要 | Lung cancer is the leading cause of cancer-related death and the global 5-survival rate is only 10% to 15%. Lung cancer is also more variable in its biological behavior and can be divided into two histological groups: small-cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). NSCLC accounts for approximately 80% of all lung cancers, including adenocarcinoma (AC), squamous cell carcinoma (SCC) and large-cell carcinoma. The incidence of adenocarcinoma appears to be increasing globally. To understand the carcinogenesis caused by accumulated genetic and epigenetic alterations and identify novel biomarkers for various cancers, studying differentially expressed genes between cancerous and normal tissues is crucial. Here we constructed two cDNA libraries of differentially expressed genes using lung adenocarcinoma tissue and adjacent nonmalignant lung tissue by suppression subtractive hybridization: the forward-subtracted library (FSL) contained 177 genes and 44 unknown expressed sequence tags (ESTs), as well as the reverse-subtracted library (RSL) 59 genes and 10 unknown ESTs. Bioinformatic analysis demonstrated these genes were involved in a wide range of cellular functions: molecular transport, cell signaling and interaction, cell cycle, cellular growth and proliferation, apoptosis, and cellular movement, and so on. The vast majority of these genes were newly identified to be linked to lung cancer. In the first stage of the screening for 16 genes, we compared lung cancer tissues with their adjacent nonmalignant tissues at the mRNA level by quantitative real-time polymerase chain reaction, and found six genes (ERGIC3, DDR1, HSP90B1, SDC1, RPSA, and LPCAT1) from the FSL were significantly up-regulated while two genes (GPX3 and TIMP3) from the RSL were significantly down-regulated (P<0.05). Moreover, DDX58, CCNDBP, TMSB4X, CXCL17, FOXA2, C4BPA, SCGB3A1 from FSL, CD9 from FSL and RSL were up-regulated, but they were not statistically significant (P>0.05). Then the gene ERGIC3 was studied further. The ERGIC3 protein was also over-expressed in lung cancer tissues and cultured cells; likewise its expression was correlated with the differentiated degree and histological type of lung cancer. However, it was not correlated with the patients’ age, gender as well as smoking and TNM stage. Immunofluorescence analysis showed ERGIC3 was mainly located at the Golgi apparatus and endoplasmic reticulum in the lung cancer cell lines. Interestingly, ERGIC3 was distributed at the side of nucleus in EPLC-32M1, 801D, and NCI-H446 cells, but uniformly present around nucleus in SPCA-1, GLC-82 and A549 cells. We also found that ERGIC3 was co-localized with the epithelia mucin MUC1 and β-galactoside α2,6 sialyltransferase which were principally located at the ER and Golgi apparatus in the cultured cells. Furthermore, the up-regulation of ERGIC3 in BEAS-2B cells could promote cellular migration and proliferation while down-regulation of ERGIC3 in GLC-82 cells could demote cellular migration and proliferation in vitro. In stable over-expression of ERGIC3 in A549 cells, over-expression of ERGIC3 significantly promoted the cellular migration (P<0.05), but it did not change cell morphology as well as the secretion of MUC2. In summary, Our data suggests that these two libraries of differentially expressed genes may provide the basis for new insights into finding novel lung cancer-related genes. ERGIC3 was strongly expressed in lung cancers and was a new potential biomarker. ERGIC3 promoted the cellular proliferation and migration and may be a novel lung cancer-related gene. |
| 语种 | 中文 |
| 源URL | [http://159.226.149.26:8080/handle/152453/10158]  |
| 专题 | 昆明动物研究所_分子病理学 |
| 推荐引用方式 GB/T 7714 | 吴明松. 肺腺癌差异表达基因文库的建立以及ERGIC3在肺癌中的病理生理功能[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：昆明动物研究所
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