苦瓜核糖体失活蛋白MAP30调控未成熟同源蛋白增强基因抑制膀胱尿路上皮癌增殖和下游药靶基因机制研究

One of the most problem in treatment of bladder cancer was its high recurrence, and postoperative bladder infusion to prevent recurrence was not so effectively. Momordica Charantia MAP30 protein can inhibit the proliferation of cancer cells, no toxic to normal cells, hardly get through the cell membrane, is a potential intravesical instillation drug; ERH, which was an oncogene first found by us highly expressed in bladder cancer cell lines, may have an effect on cancer proliferation and apoptosis. We screened MAP30 gene from Momordica charantia and obtained purified recombinant MAP30 protein. MAP30 protein will be used to inhibit the bladder cancer cells’ proliferation, apoptosis, invasion and metastasis and the change of ERH in vitro. Down-regulate ERH expression to find the effect on cancer cells’ development. Establish a bladder cancer model in vivo, then investigate the effect of MAP30 on the invasion and metastasis, the effect of angiogenesis, changes of surface molecules and changes of ERH gene expression in bladder cancer cell lines. ChIP-seq method to screen the transcriptional activity directly under the control of ERH downstream pathway gene. WB / qPCR was used to verify the expression level of downstream response gene in the target gene interference group. The functional response experiment determines whether ERH is the target of MAP30. To elucidate if the molecular mechanism of MAP30 to regulate downstream drug target, the ERH gene and explore the specific mechanism of MAP30 of cancer inhibition.

膀胱癌因术后高复发性是临床治疗难题，目前尚无预防复发满意药物。苦瓜MAP30能抑制癌细胞增殖，对正常细胞几无毒性，但具体抗癌机制不明；未成熟同源蛋白增强基因（ERH）是我们首次报道在膀胱癌高表达的基因，与癌增殖相关；选择ERH为MAP30调控下游药靶基因切入点，体外实验研究MAP30抑制膀胱癌细胞增殖、侵袭转移及潜在药靶基因ERH的基因表达量变化，设计阻断ERH基因表达干扰靶点，分析其在癌进展中的作用；建立鼠膀胱癌模型，检测MAP30处理后癌组织侵袭转移、血管生成能力和癌细胞表面分子、ERH基因表达变化，筛选转录活性直接受ERH基因调控的下游通路基因，验证下游应答基因在目的基因干扰后的表达水平，采用功能回复实验确定ERH基因是否为MAP30作用靶点，阐明MAP30抑癌具体机理和调控下游药靶基因的分子机制，证实MAP30可通过调控ERH基因表达而发挥抑癌作用，有助于传统医学同现代医学有效结合

1.获得纯化的具有生物学功能的MAP30蛋白；2.完成了MAP30的脂质体组装；3.对MAP30蛋白进行体外的功能测试：首先确定MAP30蛋白对肿瘤细胞的半数致死量，从而确定最佳作用剂量和时间。然后膀胱癌细胞系进行体外细胞实验，主要包含细胞活性检测，细胞凋亡检测以 及药物对细胞迁移和侵袭的影响；4.MAP30蛋白体内功能实验，即检测其对膀胱癌荷瘤小鼠的治疗效果和机制；5.苦瓜MAP30干预对膀胱尿路上皮癌ERH基因mRNA和蛋白表达变化的影响， 癌细胞的增殖抑制、凋亡和侵袭转移与MAP30干预、ERH基因表达变化之间的相关性；6.MAP30处理的T24人膀胱癌细胞的基因表达谱：通过Affymetrix GeneChip确定MAP30（320μg/ ml）处理的T24细胞的基因表达谱，在层次聚类分析中，所有差异基因根据表达水平聚类以获得树结构。所有结果表明，MAP30处理显着改变了T24癌细胞中的基因表达谱；7.MAP30治疗的T24人膀胱癌细胞中经典途径改变的IPA分析：根据数据集中的基因表达变化，进行了IPA上游分析，以预测重要的上游调节分子，这可能有助于找到实验中涉及的潜在生物学过程。 IPA使用激活Z评分算法来预测上游调节器的激活或抑制。在该项目中，预计TBX2被强烈抑制，并且40个差异表达的基因与其具有一致的表达方向；7.验证MAP30调控的T24人膀胱癌细胞中基因表达：证实了MAP30可以通过调节AKT信号传导途径和EMT途径来抑制T24人膀胱癌。