基于MRPL35功能学分析进行联合自身抗体检测对食管癌早期筛查的研究

Esophageal cancer has a high morbidity in China which is the highest in the world. Of all malignant tumor the death rate of Esophageal cancer ranks sixth. It's significant to explore the pathogenesis of esophageal cancer and establish for early detection of large scale as well as high-risk people's early warning index and method. Mitochondrial ribosome proteins(MRP) is a kind of protein that encoding by nuclear gene involved in protein synthesis in the mitochondria.L35 of the mitochondrial ribosome is proved to be the secondary binding protein, participating in the ribosomal protein transcription of translation.Research of MRPL35 was Focused on the analysis of the structure, there is no research of the function reports in China or abroad. Our initial application KYSE410 esophagus carcinoma cell line ,through the cell experiment and tumorigenicity assay in nude mice, it was found that after muted purpose Gene, The proliferation of cell is significantly suppressed which indicates MRPL35 participate in esophageal cancer cell proliferation. This study proposed to examine the relationship between MRPL35 and cell proliferation by esophageal cancer cell lines TE-1 and ECA109, verifying MRPL35's role in esophageal cancer cell proliferation , validated at the protein level ,using the expression chip at the same time explore its action mechanism to find the key signaling pathways. Also collecting 500 cases of clinical Esophageal cancer tissue samples and 1000 serological samples, using patented autoantibodies detection technologies from the study group, screening the differentially expressed autoantibodies from the serum of newly diagnosed esophageal cancer patients, and compare with previous results to improve the sensitivity and specificity of the detection to achieve the goal of early esophageal cancer diagnosis and provide the basis for diagnosis and treatment of esophageal cancer.

中国食管癌发病率、死亡率逐年升高，探究食管癌发病机制并建立对大范围人群及高危人群早期发现的指标和方法有重要意义。线粒体核糖体L35蛋白被证实参与核糖体中蛋白的转录翻译，目前国内外研究主要集中在其结构的分析，尚无功能学方面的研究报道。我们前期应用食管癌株KYSE410，通过细胞实验和裸鼠成瘤实验发现沉默MRPL35基因后细胞增殖明显受抑制，提示MRPL35参与食管癌细胞的增殖调控，但机制不明。本课题拟通过食管癌株TE-1和ECA109检测MRPL35与细胞增殖的关系，证实MRPL35在增殖中的作用，并通过下游信号的检测和表达谱芯片，探究其作用机制并寻找关键信号通路。同时收集500例临床食管癌组织样本和1000例血液样本，利用课题组多项专利的自身抗体检测技术，筛选初诊食管癌患者血清中差异表达的自身抗体，并联合前期结果提高检测敏感度和特异度，达到早期诊断的目的，以期为食管癌的诊断和治疗提供依据。

中国食管癌发病率、死亡率逐年升高，探究食管癌发病机制并建立对大范围人群的早期筛查指标和方法有重要意义。线粒体核糖体大亚基蛋白35（MRPL35）被证实参与核糖体中蛋白的转录翻译，目前国内外研究主要集中在其结构的分析，尚无功能学方面的研究报道。.项目组利用癌基因芯片数据整合挖掘平台Oncomine和癌症基因组图谱TCGA数据库对MRPL35基因进行数据挖掘分析。同时收集食管癌患者临床样本与正常组织，利用实时定量PCR及Western blotting实验分别从mRNA层面和蛋白层面验证MRPL35在食管癌中存在高表达。以MRPL35 基因为模板设计RNA 干扰靶点序列，制备可实现靶基因敲减的工具病毒载体；利用病毒感染的方式进行基因敲减，筛选并培养MRPL35稳定低表达的食管癌细胞系；通过流式细胞仪等专业设备证明MRPL35 的敲减可以引发细胞增殖、细胞周期和凋亡等细胞生命活动改变。利用高通量测序及转录组分析对MRPL35基因敲减后的全基因组基因表达量变化进行检测，筛选下游差异表达基因后进行基因功能注释与富集分析，并且配合相关实验进行验证，明确敲减MRPL35可通过MAPK家族成员JNK所在通路调控细胞凋亡，关键分子JNK，Bid与细胞色素c可作为MRPL35高表达食管癌的治疗靶点。收集并检测血液样本中SURVIVIN蛋白自身抗体的变化，并联合本课题组前期结果寻找最优自身抗体联合方案提高检测敏感度和特异度，明确自身抗体作为食管癌诊断临床标志物的意义与价值。.本项目旨在探讨MRPL35及其调控通路的关键基因在食管癌发生发展中的作用，并应用课题组专利的自身抗体检测技术检测食管癌患者血清中靶蛋白表达变化，解析食管癌患者的临床表征与检测结果之间的联系，并将关键蛋白作为食管癌相关生物标记物进行流行病学的早期筛查，建立完善的筛查方案，为食管癌的早期诊断与治疗提供了确切的理论依据与数据支持，将进一步降低我国食管癌死亡率。