【作者】 夏琳；

【导师】 樊代明； 张德新；

【作者基本信息】 第四军医大学 ， 内科学， 2008， 博士

【摘要】 【背景】胃癌细胞发生多药耐药(multidrug resistance,MDR)是导致胃癌患者化疗失败的最主要原因。经过几十年的研究,目前已明确胃癌MDR是一个多因素参与的复杂过程。近年来随着基因组学和蛋白质组学研究手段的飞速发展,新的胃癌耐药相关分子不断得以发现,然而胃癌MDR的分子机制仍未完全阐明。值得思考的是,目前的研究大多热衷于挖掘耐药与亲本/药敏细胞之间差异表达的基因,进而探究其介导耐药的下游分子通路,而忽略了导致诸多基因差异表达的幕后操纵者,即这些耐药相关基因的上游调控网络是研究中的盲点。如果逆向探寻上游的调节分子,找到处于耐药分子网络中的“始作俑者”,将其作为靶点来逆转胃癌MDR,将为胃癌化疗开辟新的道路。microRNA (miRNA)是近年来得以发现的一类广泛存在于动植物体内的非编码小RNA,在转录后水平负性调控基因的表达。据预测,每个miRNA可以调控大约200个基因的表达,存在于人体内的大约1000个miRNAs可调控超过1/3的蛋白编码基因的表达,影响着几乎所有的信号通路。miRNA已被证实广泛参与多种生理病理过程,包括肿瘤的发生和发展。由此,我们推测:miRNA可能是我们要寻找的一类处于胃癌耐药分子网络上游的调节分子,调控众多耐药相关基因的表达,进而影响胃癌细胞的耐药表型。目前为止,miRNA在胃癌MDR中的作用及机制尚未见报道。【目的】探讨miRNA在胃癌MDR中的调节作用及其分子机制,以期更全面的阐明胃癌MDR机制,并为寻找新的耐药逆转策略提供理论依据。【方法】1.通过miRNA芯片检测胃癌多药耐药细胞SGC7901/VCR和其亲本细胞SGC7901的miRNA表达谱,找出差异表达的miRNA;2.利用real-time RT-PCR法对miRNA芯片结果进行验证;3.通过细胞转染,利用miR-15b/miR-16/let-7a特异的前体寡核苷酸分别上调SGC7901/VCR细胞中miR-15b/miR-16/let-7a的表达,miR-15b/miR-16/let-7a特异的反义寡核苷酸抑制物分别下调SGC7901细胞中miR-15b/miR-16/let-7a的表达;4.通过MTT试验检测miR-15b/miR-16/let-7a对胃癌细胞(SGC7901、SGC7901/VCR)体外药物敏感性的影响;5.通过小鼠肾包膜下移植法(SRCA)检测miR-15b/miR-16/let-7a对胃癌细胞(SGC7901/VCR)体内药物敏感性的影响;6.利用生物信息学网站或软件预测miR-15b/miR-16/let-7a可能调控的靶基因;7.利用Western blot、RT-PCR以及荧光素酶报告基因实验验证miR-15b/miR-16/let-7a调控的靶基因;8.通过AnnexinV/PI染色流式细胞术和caspase-3/7活性试验分析miR-15b/miR-16对胃癌多药耐药细胞SGC7901/VCR的凋亡敏感性的影响;9.借助宿主细胞再活化试验分析let-7a对胃癌多药耐药细胞SGC7901/VCR的DNA损伤修复能力的影响。【结果】1. 12个miRNAs在胃癌多药耐药细胞SGC7901/VCR和其亲本细胞SGC7901中差异表达(>2倍),在SGC7901/VCR细胞中表达升高的miRNAs为miR-302b和miR-492,表达降低的miRNAs为let-7a、miR-15b、miR-16、miR-17-5p、miR-20a、miR-23b、miR-106a、miR-106b、miR-196a和miR-320。其中一些miRNAs已报道与肿瘤相关,即‘oncomir’,而与胃癌多药耐药的关系未见报道;2. real-time RT-PCR结果显示:miR-15b、miR-16和let-7a的表达在SGC7901/VCR细胞较SGC7901细胞中显著降低,与芯片结果一致;3.体外药物敏感性实验表明,上调miR-15b/miR-16/let-7a的表达,SGC7901/VCR细胞对不同化疗药物的敏感性显著增加,而下调miR-15b/miR-16/let-7a的表达可显著降低SGC7901细胞对不同化疗药物的敏感性;4. SRCA体内药物敏感性实验证实,上调miR-15b/miR-16/let-7a的表达可显著增强SGC7901/VCR细胞体内对顺铂的敏感性;5. miR-15b和miR-16属于同一miRNA家族,具有结构上的同源性,生物信息学预测的众多miR-15b和miR-16的靶基因中包括已明确的耐药相关分子Bcl-2,而let-7a的预测靶基因中包括已明确的耐药相关分子Ras;6.与对照细胞相比,转染miR-15b/miR-16特异前体的SGC7901/VCR细胞的Bcl-2蛋白水平明显降低,而mRNA水平则无显著差异。miR-15b/miR-16通过结合克隆于荧光素酶基因编码区下游的Bcl-2 3’UTR的靶位点,抑制荧光素酶表达,破坏该靶位点可以解除miR-15b/miR-16对荧光素酶表达的抑制效应;7.与对照细胞相比,转染let-7a反义抑制物的SGC7901细胞的H-Ras蛋白水平明显升高,而mRNA水平则无显著差异。let-7a通过结合克隆于荧光素酶基因编码区下游的H-Ras 3’UTR的靶位点,抑制荧光素酶表达。8. AnnexinV/PI染色流式细胞术、caspase-3/7活性试验的结果显示:与对照细胞相比,转染miR-15b/miR-16特异前体的SGC7901/VCR细胞对化疗药物诱导的凋亡的敏感性显著增加;9.宿主细胞再活化试验结果表明:与对照细胞相比,转染let-7a特异前体的SGC7901/VCR细胞的DNA损伤修复能力显著降低。【结论】1. miRNA在胃癌多药耐药细胞和其亲本细胞中差异表达,可能参与胃癌多药耐药的发生;2. miR-15b,miR-16参与调节胃癌细胞多药耐药表型,其可能的分子机制是:miR-15b、miR-16负性调控靶基因Bcl-2的表达,从而调节胃癌细胞对于化疗药物诱导的凋亡的敏感性;3. let-7a参与调节胃癌细胞多药耐药表型,其可能的分子机制是:let-7a负性调控靶基因Ras的表达,从而影响胃癌细胞的DNA损伤修复能力。更多还原

【Abstract】 【Background】The development of multidrug resistance (MDR) in cancer cells is the primary cause for failed chemotherapy in gastric cancer patients. It is now well acknowledged that mechanisms responsible for MDR in gastric cancer are likely to be multifaceted and extremely intricate. New MDR-related molecules keep emerging owing to the great advances in genomics and proteomics approaches, but the understanding to gastric cancer MDR still remains incomplete. It is worth noticing that while great efforts have been made in identifying differentially expressed genes between drug-resistant and parental/ drug-sensitive cells, and then exploring its downstream signal pathway, much less insight has been gained into the regulatory networks that establish such altered gene expression states. If the‘crime boss’in the MDR molecule network could be identified by inversely exploring upstream regulators, strategies targeting them may pave a new road for chemotherapy of gastric cancer.microRNAs (miRNAs) are a newly discovered class of small noncoding RNAs encoded by the genomes of a wide range of multicellular organisms and negatively regulate gene expression at posttranscriptional level. It is predicted that each miRNA could regulate about 200 genes and the ~1000 miRNAs in the human body may regulate the expression of up to 1/3 of human protein-coding genes, which implies the potential influence of miRNAs on almost every genetic pathway. Indeed, miRNAs have been shown to play crucial roles in many physiological and pathological processes, including cancer development and progression. Thus, we postulated that miRNA could be such class of modulators located in the upstream of MDR molecule network, which regulate the expression of numerous MDR-related genes, hence the MDR phenotype of gastric cancer cells. Until now, there has been no report on the potential role of miRNAs in MDR of gastric cancer.【Aims】To investigate the regulatory role of miRNAs in MDR of gastric cancer cells and the underlying mechanisms, with the aim of better elucidating the mechanisms of gastric cancer MDR and laying a foundation for formulating novel MDR reversing strategies.【Methods】1. The differentially expressed miRNAs between multidrug-resistant gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901 were identified by miRNA profiling of these two cell lines using miRNA microarray; 2. The results obtained by microarray profiling were validated using real-time RT-PCR analysis; 3. The specific miRNA precursor for miR-15b/miR-16/let-7a was respectively transfected into SGC7901/VCR cells to up-regulate its expression and the specific antisense inhibitor for miR-15b/miR-16/let-7a was transfected into SGC7901 cells to down-regulate its expression; 4. the effect of miR-15b/miR-16/let-7a on in vitro drug sensitivity of gastric cancer cells (SGC7901、SGC7901/VCR) was determined by MTT assay; 5. The effect of miR-15b/miR-16/let-7a on in vivo drug sensitivity of gastric cancer cells (SGC7901/VCR) was determined by subrenal capsule xenograft assay (SRCA); 6. The putative target genes of miR-15b/miR-16/let-7a were predicted using bioinformatics website or software; 7. The putative target genes of miR-15b/miR-16/let-7a were validated by Western blot, RT-PCR and luciferase reporter assay; 8. The effect of enforced miR-15b or miR-16 expression on drug-induced apoptosis in SGC7901/VCR cells was evaluated using AnnexinV/PI staining flow cytometry and caspase-3/7 activity assay; 9. The effect of let-7a on DNA repair capacity in SGC7901/VCR cells was assessed by host cell reactivation assay.【Results】1. There are 12 miRNAs differentially expressed (>2 fold) in multidrug-resistant gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901. The up-regulated miRNAs in SGC7901/VCR cell line were miR-302b and miR-492 and the down-regulated miRNAs were let-7a、miR-15b、miR-16、miR-17-5p、miR-20a、miR-23b、miR-106a、miR-106b、miR-196a and miR-320. Among them, some have been implicated in cancer, or known as‘oncomir’, but their involvement in MDR of gastric cancer has yet been reported; 2. In accordance with the microarray data, real-time RT-PCR showed decreased miR-15b, miR-16 and let-7a levels in SGC7901/VCR cells compared with its counterpart; 3. In vitro drug sensitivity assay demonstrated that overexpression of miR-15b/miR-16/let-7a sensitized SGC7901/VCR cells to different anticancer drugs whereas inhibition of them conferred SGC7901 cells multidrug resistance; 4. SRCA in vivo drug sensitivity assay demonstrated that overexpression of miR-15b/miR-16/let-7a sensitized SGC7901/VCR cells to CDDP in vivo; 5. miR-15b and miR-16 belong to miR-15/16 family and exhibit structural homology, thus they potentially target same genes by in silico analysis. Among the numerous putative target genes for miR-15b, miR-16 and let-7a, some are well-defined MDR-related molecules, e.g. Bcl-2 for miR-15b and miR-16, and Ras for let-7a; 6. Compared with precursor control-transfected cells, Bcl-2 protein level in miR-15b or miR-16 precursor-transfected cells were decreased significantly, whereas no visible difference was observed in mRNA level. miR-15b and miR-16 caused a decrease in relative luciferase activity by binding to the target site in Bcl-2 3’UTR which was cloned immediately downstream of luciferase gene coding region. As expected, this suppression was abolished by disrupting the target site; 7. Compared with inhibitor control-transfected cells, H-Ras protein level in let-7a antisense inhibitor-transfected cells were increased significantly, whereas no visible difference was observed in mRNA level. let-7a caused a decrease in relative luciferase activity by binding to the target site in H-Ras 3’UTR which was cloned immediately downstream of luciferase gene coding region; 8. AnnexinV/PI staining flow cytometry and caspase-3/7 activity assay showed that miR-15b or miR-16 precursor-transfected SGC7901/VCR cells exhibited much higher sensitivity to VCR-induced apoptosis compared to precursor control-transfected ones; 9. Host cell reactivation assay revealed that DNA repair capacity in let-7a precursor-transfected SGC7901/VCR cells were greatly depressed compared with precursor control-transfected ones.【Conclusions】1. miRNAs are differentially expressed in multidrug-resistant gastric cancer cell line and its parental cell line, which suggests that miRNAs may play a role in the development of MDR in gastric cancer cells; 2. miR-15 and miR-16 were demonstrated to modulate MDR phenotype in gastric cancer cells and the underlying mechanism is that miR-15 and miR-16 regulate target gene Bcl-2, thereby modulating the sensitivity of gastric cancer cells to drug-induced apoptosis; 3. let-7a was demonstrated to modulate MDR phenotype in gastric cancer cells and the underlying mechanism is that let-7a regulates target gene H-Ras, thereby modulating DNA repair capacity of gastric cancer cells.更多还原