新基因C8orf84在膀胱癌中的表达、调控和生物学功能研究

Our prior transcriptome analysis revealed that the expression of open reading frame C8orf84 was lower in bladder urothelial cancer tissues compared to that in matched normal bladder tissues, which was confirmed by quantitative real-time PCR (RT-PCR） and immunohistochemical staining (IHC). C8orf84 was low expression levels or no expression in a panel of bladder cancer cell lines, which was in reverse with the methylation status of CpG islands in its promoter regions. Restore expression of C8orf84 can inhibit proliferation of bladder cancer cells. These preliminary data suggested that C8orf84 play an key role in the bladder cancer carcinogenesis. Thus, we hypothesize that C8orf84 functions as a candidate tumor suppressor gene in bladder cancer. But, there is no report on C8orf84 in MEDLINE database. In order to confirm the hypothesize, we will examine C8orf84 expression by RT-PCR, Western blot and IHC, and determinate the association between its expression and clinicopathological parameters in large bladder cancer samples; then we will investigate the epigenetic regulation mechanism of C8orf84 in baldder cancer by bisulfite genomic sequencing and chromatin immunoprecipitation; last we will investigate the biological function and signaling pathway of C8orf84 in bladder cancer by the lentiviral expression system, nude mice model, tandem affinity purification (TAP) combined with liquid chromatography - tandem mass spectrometry (LC-MS/MS). The purpose is to further clarify the roles of C8orf84 in bladder cancer carcinogenesis and its regulation mechanism, and provide new clues to early diagnosis and gene therapy for bladder urothelial carcinoma and other tumors.

课题组前期通过转录组分析，筛选出一个在膀胱癌组织中表达显著低于癌旁正常组织的开放阅读框C8orf84。荧光定量PCR及免疫组化检测证实C8orf84在膀胱癌组织表达下调。C8orf84在膀胱癌细胞株中无表达或低表达，并与其启动子区甲基化程度呈负相关。恢复C8orf84表达显著抑制膀胱癌细胞增殖。这些结果提示C8orf84与膀胱癌的发生发展有关，可能是个新候选抑癌基因，但尚未见该基因相关报道。本课题拟在前期研究基础上，采用RT-PCR、Western blot、免疫组化、亚硫酸氢盐基因组测序、染色质免疫共沉淀、慢病毒载体、串联亲和纯化质谱和裸鼠模型等技术方法分析C8orf84在膀胱癌中的表达及其与患者病情的相关性，分析其表达缺失或下调的表观遗传机制，研究C8orf84的生物学功能及发挥作用的信号通路，明确C8orf84在膀胱癌发生发展中的作用，为膀胱癌及其他肿瘤的早期辅助诊断和治疗提供线索。

膀胱癌是泌尿系统常见的恶性肿瘤。癌症的发生发展是复杂的多因素调控的过程，其涉及到相关癌基因的激活、抑癌基因的表达缺失、DNA错配修复基因异常及其所处的环境中复杂的信号通路，这些信号通路的激活及相互作用介导了肿瘤的发生发展。因此，对膀胱癌分子机制的深入探索，将有助于了解和识别有效的治疗靶标，实现从基础研究到临床应用的转化，具有重大的临床意义和社会意义。.本课题研究发现，C8orf84基因在膀胱癌组织中表达较低，而在人类正常膀胱组织中高表达。免疫组化显示，C8orf84的表达水平与肿瘤分级(p=0.018)和淋巴结转移(p=0.007)具有相关性，与患者的年龄、性别、肿瘤分化程度、T分期无明显相关性。在膀胱癌细胞中过表达C8orf84能抑制癌细胞生长，诱导细胞凋亡和周期阻滞，抑制细胞迁移与侵袭；而沉默C8orf84表达则相反。体内实验显示，用，敲除C8orf84基因的小鼠较对照组的小鼠对BBN诱导膀胱癌更敏感，其膀胱变体积和重量都明显增加；HE染色显示发生明显癌变。.进一步，使用质谱分析和免疫共沉淀实验发现蛋白C8orf84与蛋白HSPA5存在相互作用，免疫荧光发现两者存在共定位。表达C8orf84可以通过抑制HSPA5的活性，抑制PI3K/AKT，MEK/ERK信号通络的激活。可通过抑制EMT抑制膀胱癌的迁移与侵袭。此外，发现C8orf84与HSPA5相互作用可以引发下游未折叠蛋白应答，激活PERK、IRE1α、ATF6和CHOP。.总之，C8orf84是一个潜在的膀胱癌候选抑癌基因，有可能成为膀胱诊断和治疗的一个新的靶标。

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