【作者】 张志栋；

【导师】 李勇； 赵群；

【作者基本信息】 河北医科大学 ， 外科学， 2014， 博士

【摘要】 在全球范围内，胃癌是癌症相关的死亡率第二位的癌症。它在亚洲的发病率高于其他地区。在我国，胃癌在恶性肿瘤中死亡率第二，发病率占第三。由于胃癌通常在癌症晚期或因转移性胃癌被发现，所以预后一般较差，5年生存率小于30%。外科手术是目前最有效的治疗策略，但大约50-70%进展期胃癌病人术后出现复发。因此，化疗已被广泛应用于手术后的患者及晚期胃癌姑息性治疗，以提高患者的生活质量和延长生存期。尽管胃癌被认为是对化疗不敏感的癌症之一，但铂类及氟尿嘧啶类药物仍为胃癌化疗一线方案，其中几种重要的化疗药物如顺铂（cisplatin, DDP）和5-氟尿嘧啶（cisplatin,5-FU）的治疗胃癌还是作为一线药物有一定效果，但是生存时间一直不理想。多种证据表明，由于多药耐药（multidrug resistance, MDR）的发生导致了长期化疗不能消除所有癌细胞，进而造成癌症复发。膜联蛋白家族属于Ca(2+)调节的磷脂和膜结合蛋白，家族成员膜联蛋白A2(ANXA2)参与了细胞骨架重排。在肿瘤的形成、增殖、凋亡和信号转导形成过程中都扮演重要角色。然而，ANXA2在不同肿瘤中的表达模式不同，提示ANXA2在不同肿瘤发生发展中可能起不同作用。如在肝脏和乳腺组织中表达上调，而在前列腺和食管癌组织中表达下调。ANXA2可以作为肿瘤治疗的靶标以及肿瘤的形成、增殖、凋亡和信号转导及判断患者预后的生物标志。然而，ANXA2在胃癌中的表达和功能仍不十分清楚。目的：本研究以临床切除的胃癌组织及胃癌细胞系为研究对象，观察ANXA2在胃癌中的表达及其在胃癌耐药细胞中的作用机制。以阐明ANXA2在胃癌发生发展过程中的重要作用，为胃癌的靶向治疗提供理论依据。方法：通过实时定量RT-PCR、免疫印迹等方法观察ANXA2在胃癌和癌旁组织及不同分化程度的胃癌及胃癌细胞耐药株中的表达，通过RNA干扰封闭ANXA2表达，观察ANXA2对胃癌耐药细胞SGC7901/DDP细胞药物敏感性、耐药基因表达及信号通路激活的影响，结果：本课题五部分的具体结果内容如下：第一部分胃癌组织中ANXA2的表达研究第一部分实验主要利用荧光定量RT-PCR和Western blot方法观察ANXA2在临床切除的胃癌标本和癌旁组织中的表达情况。结果如下：1ANXA2在胃癌组织和癌旁组织中转录水平的差异应用荧光定量RT-PCR方法检测了所有临床切除的胃癌样本及癌旁对照组织中ANXA2的mRNA表达。结果显示，与癌旁组织相比，胃癌组织中ANXA2的mRNA表达水平明显升高。结果表明，ANXA2在胃癌发生过程中转录水平发生改变，ANXA2在胃癌发生过程中可能发挥重要作用。2ANXA2在胃癌组织和癌旁组织中翻译水平的差异为了明确ANXA2与胃癌发生的关系，我们进一步通过Western blot观察了胃癌组织和癌旁组织中ANXA2蛋白水平，结果显示，与癌旁组织相比，胃癌组织中ANXA2的蛋白表达水平明显升高。结果表明，ANXA2在胃癌发生过程中翻译水平发生改变，ANXA2在胃癌发生过程中可能发挥重要作用。第二部分不同分化程度胃癌的ANXA2表达的研究第二部分实验主要利用荧光定量RT-PCR和Western blot方法观察ANXA2在临床切除的不同分化程度胃癌标本和不同分化的胃癌细胞株中的表达情况，以初步探讨ANXA2胃癌癌分化中的意义。结果如下：1ANXA2基因在不同分化胃癌组织中的表达为了明确ANXA2与胃癌发生的关系，我们进一步观察了ANXA2基因表达与胃癌分化程度的相关性。我们通过荧光定量PCR和western blot对高、中和低分化胃癌组织中ANXA2进行检测，结果显示，胃癌组织分化程度越高ANXA2的mRNA和蛋白表达越低。结果表明，Annexin A3基因表达与胃癌细胞株分化程度呈负相关。2ANXA2基因在不同分化胃癌细胞株中的表达为了进行体外机制研究，我们接着检测了不同分化程度的胃癌细胞株中ANXA2的表达。我们通过荧光定量PCR和western blot对不同程度胃癌细胞株中ANXA2的表达进行检测，结果显示，ANXA2的表达由低到高为：高分化腺癌细胞株MKN-28、中分化腺癌细胞株SGC-7901、低分化腺癌细胞株BGC-823。结果表明，Annexin A3基因表达与胃癌细胞株分化程度呈负相关。第三部分ANXA2-siRNA转染对胃癌细胞株耐药性的影响第三部分实验主要利用荧光定量RT-PCR和Western blot方法观察ANXA2在胃癌细胞及胃癌耐药细胞中的表达情况，及封闭ANXA2（小干扰RNA技术）表达后胃癌细胞耐药性的改变，以阐ANXA2在胃癌细胞耐药中的作用机制。结果如下：1胃癌耐药细胞中ANXA2mRNA和蛋白的表达情况我们首先检测了DDP耐药细胞SGC7901/DDP及其对照细胞SGC790的IC50值，MTT结果显示，SGC7901/DDP细胞对DDP的IC50值为5.36±0.73μg/ml，而对照细胞SGC7901为0.26±0.03μg/ml。我们进一步检测了ANXA2mRNA在胃癌耐药细胞中的表达，荧光定量RT-PCR分析结果显示：DDP耐药细胞SGC7901/DDP中ANXA2的mRNA表达水平明显高于对照细胞SGC7901中ANXA2的表达。我们同样检测了ANXA2蛋白在胃癌耐药SGC7901/DDP细胞中的表达，Western blot分析结果显示：DDP耐药细胞SGC7901/DDP中ANXA2的蛋白表达水平明显高于对照细胞SGC7901中ANXA2的表达。2ANXA2-siRNAs对SGC7901/DDP细胞ANXA2表达的影响为了进一步阐明ANXA2表达对胃癌细胞耐药的影响，我们采用ANXA2-siRNAs转染高表达ANXA2因子的SGC7901/DDP胃癌耐药细胞，同时以转染control-siRNA细胞作为阴性对照，观察ANXA2-siRNA对SGC7901/DDP细胞ANXA2表达的影响。荧光定量RT-PCR和Western blot结果显示，不同浓度（20、40和80nM）的ANXA2-siRNAs转染SGC7901/DDP细胞48h后，ANXA2的mRNA和蛋白表达均有不同程度下降，其中80nM浓度ANXA2-siRNAs对ANXA2的敲低作用达到了90%以上。而细胞转染control-siRNA后ANXA2表达没有明显改变。我们进一步检测了不同时间（24,48或72h）ANXA2-siRNAs转染SGC7901/DDP细胞对ANXA2mRNA和蛋白表达的影响。荧光定量RT-PCR结果和Western blot结果显示，80nM的ANXA2-siRNAs转染SGC7901/DDP细胞24,48或72h后，ANXA2的mRNA和蛋白表达逐渐降低，相比于转染control-siRNA对照，其中48h及72h时ANXA2mRNA和蛋白表达均下降了90%以上。以上结果表明，实验所用ANXA2-siRNAs的能高效、特异、持久抑制内源性ANXA2表达。3ANXA2-siRNAs转染对SGC7901/DDP细胞耐药的影响我们进一步研究了ANXA2-siRNAs转染对SGC7901/DDP细胞耐药的影响。MTT实验结果显示，与转染control-siRNA对照相比，80nM浓度ANXA2-siRNAs转染48h后，SGC7901/DDP细胞对doxorubicin,5-fluorouracil及cisplatin IC50值明显降低，结果表明，ANXA2-siRNAs可提高SGC7901/DDP细胞对化疗药物的敏感性。第四部分ANXA2-siRNA转染对胃癌细胞耐药相关基因表达的影响第四部分实验主要利用小干扰RNA技术，通过荧光定量RT-PCR和Western blot方法观察抑制内源性ANXA2后，观察胃癌耐药SGC7901/DDP细胞多药耐药因子表达的影响。结果如下：1ANXA2-siRNAs转染对耐药相关基因mRNA表达的影响为进一步阐明ANXA2在胃癌多药耐药中的作用，我们采用荧光定量RT-PCR的方法检测了耐药相关基因mRNA的表达情况，结果显示：与对照组相比，80nM的ANXA2-siRNAs转染48h后，SGC7901/DDP细胞中经典耐药途径中耐药相关基因P-gp、MRP1和Bcl-2的mRNA表达水平均明显降低，而Bax则显著升高。但是其他的几个非经典耐药途径中耐药相关因子GST-π、TOPO-I和TOPO-II的mRNA表达并没有明显的变化。2ANXA2-siRNAs转染对耐药相关基因蛋白表达的影响我们同时检测了ANXA2-siRNAs转染后耐药相关基因蛋白的表达变化。Western-blot检测结果显示：与对照组相比，80nM的ANXA2-siRNAs转染48h后，SGC7901/DDP细胞中经典耐药途径中耐药相关基因P-gp、MRP1和Bcl-2的蛋白表达水平均明显降低，而Bax则显著升高。但非经典耐药途径中耐药相关因子GST-π、TOPO-I和TOPO-II的蛋白表达并没有明显的变化。这与mRNA的检测结果一致。第五部分ANXA2-siRNA转染对胃癌耐药细胞MAPK及PI3K/Akt信号通路蛋白表达的影响第五部分实验进一步应用RNA干扰技术特异性抑制内源性ANXA2表达，应用Western blot观察SGC7901/DDP细胞中MAPK信号转导通路和PI3K/AKT通路活化情况，以阐明ANXA2在胃癌细胞耐药形成中的作用机制。结果显示，80nM的ANXA2-siRNAs转染SGC7901/DDP细胞48h后，MAPK信号通路分子AKT、P38和JNK蛋白表达均无明显变化，但AKT和P38的磷酸化水平明显下降。结论：综合上述五部分内容，本研究得出如下结论：1ANXA2在胃癌组织中高表达，且与肿瘤分化程度关系密切，提示ANXA2在胃癌形成过程中可能发挥了重要作用。2ANXA2的表达与胃癌细胞耐药有关，抑制内源性ANXA2表达后胃癌耐药细胞株的药敏性增强，这与ANXA2调节P-gp、MRP1和Bcl-2等多药耐药相关基因有关，3抑制内源性ANXA2表达后胃癌耐药细胞株AKT和p38的磷酸化水平明显下降，表明AKT和p38的磷酸化过程可能参与ANXA2对胃癌细胞耐药的形成。更多还原

【Abstract】 Globally, gastric cancer (GC) is the second mostly frequent cause of cancer-related mortality and the incidence is much higher at Asia than other geographic areas. In china, gastric cancer is the second leading cause of cancer-related mortality and has the third highest incidence. Because gastric cancer is usually diagnosed in the advanced or metastatic stage, the prognosis of gastric cancer is generally rather poor, with a5-year survival rate of less than30%. Surgical operation remains the standard therapy strategy at present, but recurrence appears in approximately50-70%of patients with advanced disease. Therefore, chemotherapy has been widely accepted as palliative treatment for both resectable and advanced gastric cancer, leading to improvements in quality of life for patients and prolonged survival. Although gastric cancer is considered as relatively insensitive to chemotherapeutics and several important chemotherapeutic agents such as cisplatin (DDP) and5-fluorouracil (5-FU) are effective to date, the survival time has been unsatisfactory so far. Multiple lines of evidence suggest that long-term chemotherapy often fails to eliminate all cancer cells due to the development of multidrug resistance (MDR), which can further caused the cancer recurrence.AnxA2(ANXA2), a calcium dependent phospholipid binding protein, is involved in maintaining the plasticity and rearrangement of the actin cytoskeleton. There is overwhelming evidence that ANXA2is a multifunctional protein which is involved in tumor progression, proliferation, apoptosis, and signaling transduction. ANXA2may act as tumor suppressors or tumor promoters depending on the types of tumor cells and tissues. For example, increased ANXA2tissue levels have been observed in malignancies of the liver and breast, however, decreased ANXA2tissue levels have been observed in malignancies of the esophageal and prostate. Therefore, ANXA2may serve as an ideal target for cancer therapy and a biomarker for studying tumor progression, proliferation, apoptosis, and tumor patients’ prognosis. However, the role of ANXA2in gastric cancer is not clear.Objectives To investigate the ANXA2expression in gastric cancer as well as its impact and mechanisms on drug resistance. To clarify the role of ANXA2in the development of gastric cancer occur, as to provide theoretical basis for targeted therapy of gastric cancer.Methods ANXA2expression in both gastric cancer tissues and cell lines were detected by quantitative real-time PCR (RT-qPCR) and Western blotting. The cell proliferation was measured by SRB assay. The pool of siRNA against ANXA2were designed and synthesized and then transfected into resistant gastric cancer SGC7901/DDP cells. ANXA2expression was detected by RT-qPCR and Western blotting. Drug sensitivity of SGC7901/DDP cells to one P-gp-related drug (doxorubicin) and on P-gp-non-related drugs (5-FU and cisplatin) were measured by the SRB assay. In addition, the effect of ANXA2siRNA on the expression of MDR-related genes and activation of some molecular in signaling pathway were also observed.Results are as follows:Part one: The study of ANXA2expression in gastric cancer tissuesIn the first part, we investigated the expression of ANXA2in gastric cancer tissue by quantitative real-time RT-PCR and Western blot. The results are as follows:1Expression differences of ANXA2mRNA between gastric cancer tissue and adjacent normal tissuesThe mRNA level of ANXA2in the clinical samples of gastric cancer and adjacent normal tissues were detected by RT-PCR. The results showed that ANXA2mRNA expression was significantly higher in cancer tissues compared to that in adjacent normal tissues, suggesting that Annexin A3plays an important role in gastric carcinogenesis.2Expression differences of ANXA2protein between gastric cancer tissue and adjacent normal tissuesThe protein level of ANXA2in the clinical samples of gastric cancer and adjacent normal tissues were detected by western blot. The results showed that ANXA2protein expression was significantly higher in cancer tissues compared to that in adjacent normal tissues, suggesting that Annexin A3plays an important role in gastric carcinogenesis.Part two: The study of ANXA2expression in gastric cancer differentiationIn the second part, to investigate the significance of ANXA2in gastric carcinoma differentiation, we detected the expression of Annexin A3in gastric cancer tissue and gastric cancer cell lines with the various differentiat ion by quantitative real-time RT-PCR, and Western blot.1Correlation of Annexin A3expression with gastric cancer tissues with various differentiation gradesGenerally, the poorer GC differentiation, the worse the prognosis was. To assess whether ANXA2were related with GC differentiation, the expression of ANXA2in GC tissues from stage I-Ⅲ GC patients was further detected by RT-QPCR, and western blotting analysis. And the results showed that the higher differentiation stage, the lower expression level of ANXA2was, indicating that the expression level of ANXA2was negative correlated with the differentiation stage of gastric tumor.2Correlation of Annexin A3expression with cultured gastric cancer cell lines with various differentiation gradesTo further verify the correlation of ANXA2expression with GC differentiation and the feasibility of following study for mechanism, ANXA2expression in GC cell lines (MKN28, SGC7901and BGC823) with various differentiation stage was also detected. quantitative real-time RT-QPCR, western blotting analysis, and immunocytochemistry staining. The results of RT-QPCR and western blotting analysis showed that ANXA2expression were MKN-28, SGC-7901and BGC-823in increasing order, which is consistent with ANXA2expression in differentiation stage of gastric cancer tissues. Part three: Effect of ANXA2-siRNA transfection to chemosensitivity of gastric cancerIn the third part, to investigate the significance ANXA2expression of in a gastric cancer cell line SGC7901and a DDP resistant gastric cancer cell line, and gastric cancer cell resistance change after ANXA2-siRNA transfection, to make clear ANXA2mechanism of drug resistance in gastric cancer cell by quantitative real-time RT-PCR and Western blot.1Expression of ANXA2in MDR gastric cancer cellsTo address the role of ANXA2in the multidrug resistance of GC, the IC50values of DDP in the SGC7901/DDP and parent SGC7901cells were determined to establish the multidrug resistance cell line firstly. The results of SRB showed that the IC50values of DDP is (6.08±1.52) μg/ml and (0.26±0.02) μg/ml in the SGC7901/DDP and SGC7901cells, respectively, which indicated that the SGC7901/DDP cells were23.4times more resistant to DDP than SGC7901cells.Further, we compared the expression of ANXA2in DDP-resistant SGC7901/DDP cells and parent SGC7901cells. quantitative real-time RT-QPCR and western blotting analysis results revealed that the expression of ANXA2was higher in SGC7901/DDP cells than that in parent SGC7901cells at both mRNA and protein levels. These data suggest that ANXA2is associated with the development of MDR in gastric cancer cells.2Effect of siRNA on ANXA2expression in SGC7901/DDP cellsTo further investigate the effects of ANXA2in the multidrug resistance of GC, siRNA techniques was employed in this study. We first investigated the ability of an ANXA2siRNA to inhibit the expression of ANXA2in SGC7901/DDP cells. The results of quantitative real-time RT-QPCR and western blotting showed that20,40, and80nM ANXA2siRNA downregulated the endogenous ANXA2mRNA and protein expression efficiently, and that its optimal concentration is80nM, with an inhibition of approximately90%compared to controls, suggesting that ANXA2siRNAs efficiently inhibited the transcription and translation of ANXA2in SGC7901/DDP cells.We also detected ANXA2expression in SGC7901/DDP cells after80nM ANXA2siRNA transfection for24,48, and72h, the results of quantitative real-time RT-QPCR and western blotting showed that endogenous ANXA2mRNA and protein expression were decreased in a time-dependent manner. After80nM ANXA2siRNA transfection for24,48, and72h, more than90%of the ANXA2expression in SGC7901/DDP cells was inhibited compared to controls, suggesting that ANXA2siRNAs efficiently inhibited the transcription and translation of ANXA2in SGC7901/DDP cells.3Effect of ANXA2-siRNA on MDR of SGC7901/DDP cellsAlthough SGC7901/DDP cells were selected with the chemotherapeutic drug cisplatin, they also displayed multiple resistances to other chemotherapeutic drugs. Therefore, we futher investigate the effects of ANXA2siRNA on the drug sensitivity of SGC7901/DDP to one P-gp-related drug (doxorubicin) and on P-gp-non-related drugs (5-FU and cisplatin). The IC50values for doxorubicin,5-fluorouracil, and cisplatin were all higher in SGC7901/DDP cells than that in parent SGC7901cells. The results showed that drug resistant drug resistant SGC7901cells displayed cross resistance to these chemotherapeutic drugs. In addition, after transfection of80nM ANXA2siRNA, the IC50values for doxorubicin,5-fluorouracil, and cisplatin was decreased significantly compared with non-targeted siRNA and Lipofectamine2000TM group, indicating that ANXA2siRNA could partially reverse resistance to DDP in multi-drug resistant SGC7901cells.Part four: Effect of ANXA2on the MDR-related genes expression in SGC7901/DDP cells after ANXA2-siRNA transfectionIn the fourth part, the expression of MDR-related genes including P-gp, MRP1, Bcl-2, and Bax were detected in SGC7901/DDP cells after ANXA2-siRNA transfection by quantitative real-time RT-QPCR and western blotting.1Effect of ANXA2on the mRNA levels of MDR-related genes in SGC7901/DDP cells To further explore the potential mechanism of ANXA2on development of MDR, the MDR-related genes including P-gp, MRP1, Bcl-2, and Bax were detected by quantitative real-time RT-QPCR. We observed that the expression of P-gp, MRP1, Bcl-2was significantly downregulated, while the expression of Bax was markedly upregulated by80nM ANXA2siRNA in SGC7901/DDP cells. However, the expression of GST-π,TOPO-I, and TOPO-II were not changed.2Effect of ANXA2on the protein levels of MDR-related genes in SGC7901/DDP cellsThe protein level of the MDR-related genes were also detected by western blotting. The results showed that the protein expression of P-gp, MRP1, Bcl-2was significantly downregulated, while the expression of Bax was markedly upregulated by80nM ANXA2siRNA in SGC7901/DDP cells. However, the expression of GST-π,TOPO-I, and TOPO-II were not changed.Part five: Effect of ANXA2on the phosphorylation of MAPKs and PI3K/Akt signaling pathway in SGC7901/DDP Cells after ANXA2-siRNA transfectionIn the last part, the phosphorylation of MAPKs and PI3K/Akt signaling molecular were detected in SGC7901/DDP cells after ANXA2-siRNA transfection by western blotting.Western blotting analysis of MAP kinases and AKT revealed that phosphorylation of p38-MAPK and AKT, but not ERK1/2and JNKs was specifically decreased in SGC7901/DDP after transfection of80nM ANXA2siRNA.Conclusions:1Overexpression of ANXA2was found in gastric cancer tissues, and significant relationship between expression of ANXA2and degree of tumor differentiation was also found in present research. All results suggested that ANXA2may play an important role in carcinogenesis and progression of gastric cancer.2ANXA2was nvolved in multidrug resistance of gastric cancer. After endogenous ANXA2was inhibited by siRNA, the chemosensitivity in vitro of SGC7901/DDP became more sensitive, which was related to the ability of ANXA2to regulate multidrug resistance genes such as P-gp, MRP1, Bcl-2, etc.3Activation of p38-MAPK and AKT signaling pathway was inhibited by the ANXA2siRNA transfection in resistant gastric cancer cells. Our results suggested that drug resistance in SGC7901/DDP cells is attributed at least partially to the activation of p38-MAPK and AKT signal pathway and inhibition of these pathways could increase the sensitivity of SGC7901/DDP to P-gp-unrelated drugs as well as P-gp-related drug.更多还原