【作者】 朱亮；

【导师】 赵秋；

【作者基本信息】 华中科技大学 ， 内科学， 2012， 博士

【摘要】 目的胰腺癌是一种恶性程度高、易早期转移、预后极差的消化系统肿瘤。深入研究胰腺癌的浸润和转移机制,对于控制其进展,改善预后具有重要意义。研究表明,转化生长因子-β(transforming growth factor-B, TGF-B)诱导胰腺癌发生上皮间质转化(epithelial-mesenchymal transition, EMT)是促进胰腺癌侵袭和转移的重要因素之一,但其具体机制尚未阐明。补体反应基因-32(response gene to complement-32, RGC-32)是一种新发现的补体激活基因,在人类多种组织和器官中广泛表达,参与细胞增殖和分化调控。RGC-32在多种肿瘤中异常表达,但其表达丰度和作用不尽相同,而其在胰腺癌中的作用尚无报道。生理细胞的研究表明,RGC-32参与TGF-B诱导的EMT过程,但其在胰腺癌EMT中的作用还不得而知。本研究拟通过组织水平和细胞水平的研究,阐明RGC-32在胰腺癌中的作用,探讨其对胰腺癌细胞的EMT过程是否具有调控作用,机制如何,从而进一步完善胰腺癌EMT的调控机制,为临床探索胰腺癌基因治疗的分子靶点提供新的思路。方法应用免疫组织化学法分别检测RGC-32和E-cadherin在胰腺癌、慢性胰腺炎以及正常胰腺组织中的表达,分析不同组织中RGC-32阳性表达率、表达强度的差异以及E-cadherin异常表达率的差异,并分别分析RGC-32阳性表达率及E-cadherin异常表达率与胰腺癌病理学特征之间的关系,进一步探讨RGC-32阳性表达率与E-cadherin异常表达率之间是否具有相关性；以Smad4纯合子缺失(Smad4 homozygous deleted)型胰腺癌细胞株BxPC-3作为研究对象,给予TGF-β1刺激,并应用基因转染过表达和RNA干扰技术处理细胞,分别采用实时定量RT-PCR及Western blot法从mRNA及蛋白质水平检测RGC-32及EMT相关分子的表达,探讨RGC-32是否受TGF-β调控并参与后者诱导的EMT过程；利用Smad非依赖性信号通路的化学抑制剂阻断相应通路,进一步筛选RGC-32介导TGF-β诱导的EMT过程的主要通路；应用Transwell细胞迁移实验探讨RGC-32是否介导TGF-β诱导的细胞迁移；分别采用细胞增殖试剂盒CCK-8 (cell counting kit-8)及流式细胞术分析RGC-32对BxPC-3细胞增殖和凋亡的影响。结果免疫组织化学结果显示,胰腺癌组织中RGC-32的阳性表达率较慢性胰腺炎和正常胰腺组织显著升高(P值均<0.05),并与肿瘤的淋巴结转移和TNM分期有关(P值均<0.05),胰腺癌组织中E-cadherin的异常表达率较慢性胰腺炎和正常胰腺组织显著升高(P值均<0.05),并与肿瘤分化、淋巴结转移和TNM分期有关(P值均<0.05)。进一步分析显示,RGC-32的阳性表达率与E-cadherin的异常表达率呈正相关(P<0.01,R=0.458)。细胞试验表明,持续TGF-β刺激可诱导胰腺癌BxPC-3细胞发生EMT,并上调RGC-32的表达水平。基因转染过表达和RNA干扰试验证实,RGC-32不仅可介导TGF-β诱导的EMT过程,而且可独立诱导EMT的发生,并且这一过程可不依赖于Smad信号通路。信号通路抑制试验证实,RGC-32至少可通过Erk-MAPK和p38-MAPK两条信号通路介导TGF-β诱导的EMT过程。Transwell细胞迁移实验证实,RGC-32同样介导TGF-β诱导的细胞迁移过程。细胞增殖和凋亡实验表明RGC-32可抑制BxPC-3细胞的增殖,而对细胞凋亡无显著影响。结论RGC-32可能是一种新的胰腺癌转移促进因子,其可通过介导TGF-β诱导的EMT过程增强胰腺癌细胞的转移表型。更多还原

【Abstract】 Objective Pancreatic cancer is one of digestive system carcinomas with high malignancy, early metastasis and extremely poor prognosis. The investigation of invasion and metastasis mechanisms of pancreatic cancer is of great importance for controlling progression and improving prognosis of this deadly disease. It has been shown that transforming growth factor-β(TGF-β)-induced epithelial-mesenchymal transition (EMT) is one of the important factors for promoting invasion and metastasis of pancreatic cancer, although the underlying mechanisms are still unclear. Response gene to complement-32 (RGC-32) is a newly discovered gene activated by complement. RGC-32 is extensively expressed in many kinds of human tissues and organs and is involved in cell proliferation and differentiation. RGC-32 is aberrantly expressed in different kinds of tumors, with diverse abundance and effects. However, the role of RGC-32 in pancreatic cancer is still unknown. In the researches of normal cells, RGC-32 was found to mediate TGF-β-induced EMT. However, nothing is known about its role in the EMT of pancreatic cancer. In this study, in vitro, we try to clarify the effect of RGC-32 in pancreatic cancer and explore its possible role in regulating the EMT of pancreatic cancer cells and the underlying mechanisms, so as to further improve the controlling mechanisms of EMT and provide new insight into molecular targets for gene therapy of pancreatic cancer.Methods Immunohistochemical staining of RGC-32 and E-cadherin was performed on pancreatic cancer, chronic pancreatitis and normal pancreatic tissues. The positive expression rate and staining intensity of RGC-32 and the abnormal expression rate of E-cadherin were compared respectively among different kinds of tissues. The relationship between positive expression rate of RGC-32 and pathological features of pancreatic cancer and relationship between abnormal expression rate of E-cadherin and pathological features of pancreatic cancer were analyzed respectively. Furthermore, the correlation between positive expression rate of RGC-32 and abnormal expression rate of E-cadherin was determined. To explore whether RGC-32 is controlled by TGF-βand its possible role in TGF-β-induced EMT, human pancreatic cancer cell line BxPC-3 (Smad4 homozygous deleted) was treated with TGF-β1, and RGC-32 siRNA silencing and gene transfection were performed as well. The expression of RGC-32 and EMT-related markers at mRNA and protein levels was determined by real-time quantitative RT-PCR and western blot respectively. In order to screen the signal pathways by which RGC-32 mediated TGF-β-induced EMT, chemical inhibitors of Smad-independent pathways were used. Transwell cell migration assay was employed to determine whether RGC-32 mediated TGF-β-induced cell migration. Finally, cell counting kit-8 (CCK-8) and flow cytometry were used to determine the influence of RGC-32 on cell proliferation and apoptosis of BxPC-3 cells respectively.Results Immunhistochemical staining and analysis showed that the positive expression rate of RGC-32 in pancreatic cancer tissues was higher than that in chronic pancreatitis and normal pancreatic tissues (P<0.05) and was correlated with lymph node metastasis and TNM staging (F<0.05); the abnormal expression rate of E-cadherin in pancreatic cancer tissues was higher than that in chronic pancreatitis and normal pancreatic tissues (P<0.05) and was correlated with tumor differentiation, lymph node metastasis and TNM staging(P<0.05). Further analysis demonstrated that there was a significantly positive correlation between positive expression of RGC-32 and abnormal expression of E-cadherin (P<0.01, P=0.458).In vitro, we found sustained TGF-βstimuli induced EMT and up-regulated RGC-32 expression in BxPC-3 cells. By means of gene over-expression and RNA interference, we further demonstrated that RGC-32 not only mediated TGF-β-induced EMT, but also induced EMT independently in BxPC-3 cells, which was independent of Smad signal pathways. Signal pathway inhibiting study indicated that RGC-32 could mediate TGF-β-induced EMT at least via Erk-MAPK and p38-MAPK pathways. Finally, transwell migration assays demonstrated that RGC-32 mediated TGF-β-induced cell migration as well. Cell proliferation and apoptosis detection demonstrated that RGC-32 inhibited the proliferation of BxPC-3 cells, but had no obvious effect on cell apoptosis.Conclusion RGC-32 might be a new metastasis promoting factor for pancreatic cancer and it can enhance metastatic phenotype of pancreatic cancer cells by mediating TGF-β-induced EMT.更多还原