原癌基因PAGE4调控胃癌发生及影响藏红花素联合顺铂的药敏性机制研究

Base on our previous work, we identified that the sensitivity of cisplatin on gastric cancer were enhanced by combined with crocin. By using the gene chip and other expression strategies we found that proto oncogene prostate related gene 4 (PAGE4) was significantly decreased by treating with crocin and cisplatin. Further we found that PAGE4 was over expressed in gastric carcinoma tissue. It indicated that PAGE4 is a potential target molecule for combined chemotherapy. PAGE4 is a member of the cancer-testicular antigen family, whether it involved in the development of gastric cancer and the regulation of chemotherapy sensitivity was ambiguous. In this study, qPCR was used to identify the recombinant lentivirus-mediated PAGE4 interference strategy, MTS assay, flow cytometry, qPCR and western blot was used to verify the role and mechanism of PAGE4 in regulating the proliferation, metastasis and apoptosis of gastric cancer and the sensitivity of the combination of crocin and cisplatin both in vitro and in vivo. PAGE4 overexpression tumor bearing mice model was used to investigate the effect of PAGE4 on the gastric cancer growth inhibition of treated with the combination of crocin and cisplatin. To use the clinical gastric cancer specimens and case analysis were utilized to determine the clinical relevance of PAGE4 and the development of gastric cancer. This topic will reveal the new mechanism of PAGE4 on the gastric cancer development and progression regulation, and provide a potential therapeutic target for the clinical treatment of gastric cancer, and offer a theoretical basis for the combination of crocin and cisplatin.

前期研究发现藏红花素与顺铂联用增加抑制肿瘤增殖作用。我们利用全基因表达谱芯片等表达策略检测到前列腺相关基因（PAGE4）在藏红花素联合顺铂处理的胃癌细胞表达下调，在胃癌组织中呈现高表达。提示PAGE4是联合化疗潜在的重要靶分子。PAGE4 是癌-睾丸抗原家族成员之一，但未有其参与胃癌发生发展和化疗敏感性调控的报道。本课题将利用重组慢病毒介导的PAGE4干涉策略，在细胞模型验证PAGE4调控胃癌细胞增殖、转移、凋亡等生物学特性的作用和机制；探讨PAGE4在胃癌细胞藏红花素联合顺铂敏感性中作用及相关性；利用胃癌荷瘤模型，验证过表达 PAGE4对藏红花素联合顺铂抑制胃癌生长的作用；结合临床胃癌组织标本及病例分析，确定PAGE4与胃癌发生和发展的临床相关性。该课题将揭示PAGE4调控胃癌发生和发展的分子新机制，为临床治疗胃癌提供潜在治疗靶点，并为藏红花素和顺铂联用提供理论基础。

胃癌是异质性很强的肿瘤，寻找新的胃癌治疗靶标及治疗方法以提高治疗疗效仍是目前研究的热点。本项目前一部分研究结果表明，藏红花素与顺铂联合不能协同抑制胃癌细胞增殖；藏红花素对胃癌细胞耐药株、敏感株作用不一致，其对胃癌耐药细胞中具有促进增殖作用；通过基因芯片筛选出PAGE4基因在藏红花素联合顺铂处理后的胃癌细胞中明显下调，但高通量筛选发现PAGE4基因是胃癌细胞增殖的阴性基因。由此，本课题对研究内容进行调整。经过基因芯片及高通量筛选、细胞功能学试验发现TPM4基因在藏红花素处理后的胃癌细胞中表达并明显下调，且藏红花素通过下调TPM4基因发挥抑制胃癌细胞增殖、凋亡、侵袭、转移的作用。为进一步阐明藏红花素通过下调TPM4基因发挥抗肿瘤作用的分子机制，采用基因组学方法分析出TPM4基因与EGFR基因表达有一定的相关性,且TPM4/EGFR信号通路可能是藏红花素抑制胃癌增殖、凋亡的重要分子通路；采用蛋白质组学分析出TPM4基因可能在Cdc42-WASP-Arp2/3通路中通过抑制下游APRC3蛋白表达进而促进胃癌侵袭、转移；采用qRT-PCR法、Western Blot法、免疫组化验证TPM4基因在多株胃癌细胞及胃癌组织表达；利用生物信息学分析胃癌组织中TPM4基因的临床病理特征并在组织样本中验证。综合上述研究结果，本研究为TPM4基因成为藏红花素抑制胃癌生长及胃癌治疗关键靶标提供理论和临床依据，为藏区特有药物资源在胃癌治疗中的应用提供论文支持。