【作者】 方程；

【导师】 龙行华；

【作者基本信息】 武汉大学 ， 临床检验诊断学， 2017， 博士

【摘要】 目的:建立人乳腺癌他莫昔芬(Tamoxifen,TAM)耐药细胞株,描述其生物学特性,寻找耐药性产生的潜在分子机制,为开展肿瘤耐药机制的研究提供科研模型;应用Hiseq2500测序技术筛选人乳腺癌他莫昔芬耐药细胞株与敏感细胞之间的miRNA差异表达谱,并对差异表达miRNA的靶基因进行功能注释,为进一步研究miRNA在肿瘤耐药细胞中的调控作用奠定基础;在上述研究工作的基础上,采用Meta分析方法,全面系统地评价miRNA-21与乳腺癌患者临床病理学参数的相关性,为临床诊治乳腺癌提供参考。方法:1.以人乳腺癌细胞MCF-7为亲代细胞,以他莫昔芬为筛选药物,在体外采用低浓度逐步加量诱导法和高浓度逐步加时诱导法分别建立MCF-7C和MCF-7T耐药细胞株;倒置显微镜观察并记录细胞的形态学变化;采用CCK-8法绘制敏感及耐药细胞株的生长曲线;采用MTT法测定敏感及耐药细胞株对不同浓度Tamoxifen的敏感程度;采用Western blot检测敏感及耐药细胞株表面标志雌激素受体(ERα)的表达变化。2.采用HiSeq 2500测序技术和生物信息学相结合的方法,筛选乳腺癌他莫昔芬耐药细胞株与敏感细胞之间的miRNA差异表达谱,并对差异表达miRNA的靶基因进行功能注释。3.在研究工作的基础上,制定严格的检索策略和纳入标准,采用Meta分析方法,全面系统地评价miR-21与乳腺癌患者临床病理学参数(包括雌激素受体、孕激素受体、表皮生长因子受体、TNM分期及淋巴结转移)的相关性。结果:1.建立了人乳腺癌他莫昔芬耐药细胞株(MCF-7C和MCF-7T),且能在含10μM他莫昔芬药物浓度的培养液中稳定生长。倒置显微镜下观察MCF-7敏感细胞和耐药细胞存在明显的形态学差异;CCK-8结果显示,MCF-7C和MCF-7T细胞在体外的增殖速度显著高于MCF-7细胞;MTT结果显示,在相同浓度的TAM药物干预下,MCF-7细胞的存活率低于MCF-7C和MCF-7T细胞;Western blot结果显示,ERα蛋白在他莫昔芬耐药细胞中表达显著增高。2.测序结果显示,所有样品共预测出1,646个miRNA,其中已知miRNA有1,376个,新预测的miRNA有270个。MCF-7C耐药株与MCF-7敏感株相比较,162种差异表达miRNA具有统计学意义;MCF-7T耐药株与MCF-7敏感株相比较,62种差异表达miRNA具有统计学意义;MCF-7C耐药株与MCF-7T耐药株相比,175种差异表达miRNA具有统计学意义。三组间差异表达miRNAs靶基因的GO功能分类、COG功能分类以及KEGG代谢信号通路分析的结果基本一致,说明耐药肿瘤细胞的生长与细胞的代谢以及相关功能蛋白的调节密切相关。3.本次研究共纳入11篇相关文献,总的Meta分析结果显示,miR-21过表达与乳腺癌患者的HER-2阳性表达显著正相关;晚期乳腺癌患者miR-21高表达的发生率显著高于早期乳腺癌患者,淋巴转移乳腺癌患者中miR-21高表达的发生率显著高于无淋巴转移的患者。亚组分析结果显示,miR-21表达与乳腺癌患者的激素受体表达有着显著相关性,且在中国和其他国家人群中存在差异关联。结论:1.利用两种不同的方法建立了乳腺癌他莫昔芬耐药的MCF-7C和MCF-7T细胞株,可用于TAM耐药机制的研究;2.他莫昔芬耐药细胞株与敏感细胞株之间存在显著miRNAs差异表达谱,提示miRNA在乳腺癌TAM耐药形成过程具有重要调控作用;3.miR-21过表达与乳腺癌患者的激素受体、HER-2、TNM分期和淋巴转移情况均具有相关性,提示,miR-21可作为乳腺癌恶性程度的标记物和内分泌治疗的潜在作用靶点。更多还原

【Abstract】 Objective To establish human breast cancer tamoxifen resistant cell lines,describe their biological features and explore the potential molecular mechanisms of drug resistance,providing models for the research of tumor resistant mechanism.To screen the differentially expressed miRNAs among tamoxifen resistant and sensitive breast cancer cell lines.Then functional expression of the target genes of those differentially expresses miRNAs was investigated,so as to lay a foundation for further study of the regulatory role of miRNA in drug-resistance.On the basis of the research work,a meta-analysis was performed to comprehensively investigate the relationships between miRNA-21 and clinicopathological features of breast cancer patients.Methods1.The two drug resistant cell lines MCF-7C and MCF-7T were established from the parent MCF-7 cell line by using tamoxifen as a selective drug.MCF-7C cell line was established based on dose stepwise increment exposure to tamoxifen,while MCF-7T was established based on a high concentration of tamoxifen with time stepwise increment exposure.The morphological changes of the cells were observed by microscope.The cell growth curves were plotted using the CCK8 assay and MTT assay was performed to determine the sensitivity of different cell lines to different concentrations of tamoxifen.Moreover,the estrogen receptor(ERa)protein expression was detected by Western blot.2.The HiSeq 2500 sequencing technology and bioinformatics were used to detect the differentially expressed miRNAs in MCF-7 sensitive cell line and tamoxifen resistant cell lines.Then functional expression of the target genes of those differentially expresses miRNAs was investigated.3.On the basis of the research work,a meta-analysis was performed to investigate the relationships between miRNA-21 and clinicoathological features,including estrogen receptor,progesterone receptor,human epidermal growth factor 2,TNM stage and lymph node status of breast cancer patients.Results1.The human breast cancer tamoxifen-resistant cell lines(MCF-7C and MCF-7T)were established and can grow steadily in a culture medium containing 10 μM tamoxifen.The different morphological changes of the cell lines were observed by microscope.The results of CCK-8 assay showed that MCF-7C and MCF-7T cell lines exhibited significantly greater proliferation than MCF-7 cell lines in vitro.MTT results showed that,at the same concentration effect of TAM,tamoxifen-resistant cell lines(MCF-7C and MCF-7T)exhibited greater cell variability than MCF-7 cell.Western blot assay showed that the expression of ERa protein was increased in tamoxifen-resistant cell lines(MCF-7C and MCF-7T).2.A total of 1,646 miRNAs were predicted,with 1,376 known miRNAs and 270 newly predicted miRNAs.162 miRNAs were differentially expressed between MCF-7C and MCF-7 cell lines,62 miRNAs were differentially expressed between MCF-7T and MCF-7 cell lines,and 175 miRNAs were differentially expressed between MCF-7C cell and MCF-7T cell lines.The function classification of GO,and COG and KEGG metabolic signal pathway analysis between differentially expressed miRNAs were almost consistent,indicating that the growth of drug-resistant tumor cells was closely related to the cell metabolism and the regulation of related functional proteins.3.A total of 11 relevant articles were included in this meta-analysis.The overall results showed that miR-21 overexpression was associated with positive HER-2 expression and advanced TNM stage as well as lymph node metastasis.Subgroup analysis revealed significant associations between miR-21 expression and hormone receptor status,and the associations varied in Chinese and other populations.Conclusion1.The establishment of the tamoxifen resistant breast cancer cell lines(MCF-7C and MCF-7T)can be used to further investigate the mechanisms of tamoxifen resistance.2.miRNAs were differentially expressed between tamoxifen-resistant cell lines and sensitive cell lines,suggesting that miRNAs could play an important role in the formation of TAM resistance in breast cancer.3.Overexpression of miR-21 was associated with hormone receptor,HER-2,TNM stage and lymphy node metastasis of breast cancer patients,suggesting that miR-21 could serve as a promising biomarker of an aggressive breast cancer phenotype and a novel tumor therapeutic target of breast cancer.更多还原