胰腺癌上调因子（PAUF）在胰腺癌SP亚群细胞中的表达及其与吉西他滨耐药机制关系的研究

Pancreatic cancer is highly lethal and is resistant to current therapies, including chemotherapy, radiotherapy and targeted agents. Many of the key genetic changes involved in pancreatic cancer progression have been identified through a variety of approaches and validated in mouse models of the disease; however these have yet to translate into effective therapies. Thus, novel approaches are needed to treat this deadly cancer. Gemcitabine is the only FDA-approved chemotherapeutic drug for the treatment of advanced and metastatic pancreatic cancer.However, it has not proven very effective clinically, and improvement in patient's survival undergoing gemcitabine therapy is only minimal.The identification of novel tumor-specific antigens and signal pathways could improve prognosis and help overcome obstacles such as late diagnoses,aggressive tumors, and resistance to existing therapeuticregimens. Recently,Kim etl identified a novel secretory protein, pancreatic adenocarcinoma up-regulated factor (PAUF), which potentiates metastasis of pancreatic cancer cells via upregulation CXCL12-CXCR4 signalling;enhancing the expression of β-catenin ,leading to a rapid proliferation of pancreatic cells; promoteing adhesiveness of pancreatic cancer cells by modulating focal adhesion kinase.Therefore, PAUF plays an important role in cancer progression and may have the potential to become a novel diagnostic and therapeutic target in pancreatic cancer. in our previous reports, PAUF was higher in SP compared with non-sp of PANC-1 pancreatic carcinoma cells.We also demonstrated that SP cells had high drug-resistance.we aimed to evaluate whether pauf may maintain drug resistance in human pancreatic cancer cells, and therapeutic strategies to inhibit pauf and reverse drug resistance should be evaluated. Overall, the proposed work will not only enhance our understanding how pauf is linked to pancreatic oncogenesis in the molecular level, but also will identify novel drugs that potentially can be used in the clinic for treatment of pancreatic cancer.

吉西他滨为治疗进展期及转移胰腺癌的主要化疗药物，但现有的耐药机制学说尚不能解释其耐药问题。胰腺癌干细胞（CSC）是对化疗不敏感的主要原因。PAUF是一新发现的蛋白因子，在胰腺癌高表达，与胰腺癌迁徙、转移等密切相关，但具体机制尚未阐明，尤其与吉西他滨耐药关系尚未研究。而前期我们发现具有干细胞样SP亚群胰腺癌细胞与吉西他滨耐药相关，且PAUF在SP亚群高表达，推测其在耐药机制中发挥重要作用。我们拟在组织水平上，研究PAUF表达与胰腺癌患者预后及其与吉西他滨化疗耐药的关系；继而研究吉西他滨干预对胰腺癌CSC耐药的影响以及PAUF 表达改变；通过外源性表达以及基因沉默研究PAUF 与胰腺癌CSC吉西他滨耐药关系；将PAUF耐药机制抑制，观察对吉西他滨耐药的影响；从而探讨PAUF 作为逆转吉西他滨耐药靶点的可行性。本研究将为阐明吉西他滨化疗耐药机制建立良好的基础；有利于提高化疗效果，改善患者预后。

背景 胰腺癌细胞对吉西他滨耐药的具体机制以及发现新型及有效的化疗策略已经成为这一领域的重要挑战。胰腺癌上调因子（Pancreatic adenocarcinoma up-regulated factor ,PAUF）作为一种新型的癌基因，能够激活CXCR4，β-catenin等信号通路，同时这些信号通路在胰腺癌吉西他滨耐药中也发挥关键作用。因此我们推测PAUF也可能在胰腺癌吉西他滨耐药中具有一定作用。目前国内外尚没有关于PAUF与胰腺癌吉西他滨耐药关系及其相关机制的研究。.目的 明确PAUF与胰腺癌吉西他滨耐药的关系；探讨沉默PAUF增加胰腺癌细胞对吉西他滨敏感性的机制。.方法 应用慢病毒转染建立PAUF低表达的实验组BxPC-3_shPAUF细胞株以及PAUF高表达的对照组BxPC-3_shCtrl细胞株；有限稀释法挑选PAUF表达差异最明显的实验组和对照组细胞；不同浓度吉西他滨作用后，应用MTT检测细胞增殖率及流式细胞术检测细胞凋亡率来评估两组细胞对吉西他滨的敏感性；建立人类胰腺癌裸鼠移植瘤模型，通过观察吉西他滨作用后两组肿瘤生长速度以及TUNEL检测两组肿瘤的凋亡率来评估在体内PAUF对胰腺癌细胞吉西他滨敏感性的影响；应用基因芯片检测BxPC-3_shPAUF及BxPC-3_shCtrl细胞中表达不同的基因，挑选出与耐药可能相关的基因并验证；应用免疫组化检测两组人类胰腺癌裸鼠移植瘤组织标本中耐药可能相关基因的蛋白表达。.结果 不同浓度吉西他滨作用后，实验组增殖抑制率，细胞凋亡率均明显高于对照组；吉西他滨作用于人类胰腺癌裸鼠移植瘤模型后，实验组肿瘤生长速度明显低于对照组，细胞凋亡率明显高于对照组；基因芯片检测及Real-time PCR和Western blot验证后找出与PAUF诱导胰腺癌细胞吉西他滨耐药可能相关的基因包括MRP2，MRP3，MDR1，PIK3R1，NFkB2，TGFβ2及Notch3等;免疫组化检测人胰腺癌裸鼠移植瘤中以上7种基因的表达发现MRP2，MRP3，MDR1，PIK3R1，NFkB2在两组间有显著差异。.结论 1.体内外实验中，沉默PAUF能够增加BxPC-3细胞对吉西他滨的敏感性； 2.沉默PAUF增加BxPC-3细胞对吉西他滨的敏感性可能与下调MRP2，MRP3，MDR1，PIK3R1，NFkB2等基因有关。