NIPBL调控PUMA蛋白水平在食管鳞癌顺铂耐药中的作用及机制研究

BACKGROUND: Cisplatin is a well-known chemotherapeutic drug used frequently for treatment of esophageal squamous cell carcinoma (ESCC). Its mode of action has been linked to its ability to crosslink with the purine bases on the DNA, interfering DNA repair, causing DNA damage, and subsequently inducing apoptosis of cancer cells. However, few ESCC cells could escape from apoptosis and lead to drug resistance, recurrence and metastasis. Thus, searching for a new therapeutic target which could increase the sensitivity of ESCC cells to cisplatin will bring new insights to the treatment of ESCC. The cohesin complex is crucial for chromosome segregation during mitosis and has recently also been implicated in transcriptional regulation and chromatin architecture. The NIPBL protein is required for the loading of cohesin onto chromatin. However, the relevance of NIPBL to ESCC development remains unknown. In our previous studies, Kaplan-Meier analysis illustrated that low NIPBL expression predicted poor survival in ESCC patients. Overexpression of NIPBL could distinctly inhibit ESCC cells growth, meanwhile, NIPBL depletion could significantly promote ESCC cells growth, and erase the lethal effect of cisplatin to ESCC cells. PUMA (p53 upregulated modulator of apoptosis), which was a key tumor suppressor gene in mitochondrial apoptotic pathway, was dramatically downregulated by NIPBL depletion. Since NIPBL could recruit HDAC1 and 3 to regulate the expression of its target genes, and HDAC3 could bind to PUMA promoter directly to regulate its expression level, thus, we propose our hypothesis..HEPOTHESIS: NIPBL increase the sensitivity of cisplatin to ESCC cells through the histone deacetylation-dependent upregulation of PUMA..METHODS: More cisplatin resistant and non-resistant samples will be collected from Zhejiang Cancer Hosipital. Double immunofluorescence labeling method will be used to detect the expression of NIPBL and PUMA protein. NIPBL vector and PUMA siRNA or NIPBL siRNA and PUMA vector will be co-transfected into ESCC cells to elucidate whether PUMA could rescue the function of NIPBL in mediating the cisplatin sensitivity to ESCC cells. Chromatin immunoprecipitation and luciferase reporter assay will be used to clarify the transcriptional mechanism between NIPBL and PUMA..RESULTS: Our finding will provide new insights that incombination with cisplatin treatment, targeting NIPBL will be a promising strategy to induce apoptosis of ESCC cells.

顺铂是食管鳞癌化疗的一线用药，抑制肿瘤生长效果显著的同时也极易产生耐药性，因此明确食管鳞癌顺铂耐药的分子机理尤为重要。NIPBL是调控细胞生长和胚胎发育的关键转录因子，我们前期研究发现NIPBL蛋白的表达水平与食管鳞癌患者的预后显著负相关；外源性NIPBL的导入，能够有效地抑制食管鳞癌细胞的增殖，而内源性NIPBL的敲除能够加快细胞周期进程、促进细胞增殖，上调顺铂耐药相关抑癌基因PUMA的表达水平，削弱甚至消除顺铂对肿瘤细胞的杀伤作用。本项目拟在此基础上采用流式细胞术、染色质免疫共沉淀、荧光素酶报告基因系统等技术研究PUMA在NIPBL干预食管鳞癌顺铂敏感性中的作用及其转录调控机制；采用人食管鳞癌细胞皮下移植瘤动物模型和食管鳞癌顺铂耐药与非耐药患者标本来研究NIPBL对食管鳞癌顺铂治疗疗效的影响，探讨NIPBL作为增强食管鳞癌顺铂疗效的靶向分子的可能性，为食管鳞癌的个性化治疗提供新思路。

食管鳞癌是常见的消化道恶性肿瘤之一，我国食管鳞癌发病率和死亡率均居世界首位。顺铂是治疗食管鳞癌的一线用药，但肿瘤细胞在化疗过程中极易出现凋亡逃逸的现象，致使肿瘤细胞产生耐药性，最终导致肿瘤的复发和转移。因此，寻找增强食管鳞癌顺铂敏感性的分子靶点，具有重要的临床应用前景。.NIPBL是新近发现的调控细胞增殖、胚胎发育等重要生物学行为的转录调控因子。研究表明NIPBL基因的异常表达和突变参与了多种恶性肿瘤的发生和发展。目前，尚无深入探讨NIPBL在食管癌中的功能及作用机制的研究结果发表。本项目首次阐明了NIPBL蛋白在食管鳞癌中的“抑癌基因”功能，及其调控食管鳞癌细胞顺铂敏感性的作用机制。NIPBL蛋白的过表达抑制食管鳞癌细胞的生长，相反，NIPBL的表达沉默促进肿瘤细胞的生长。综合分析食管鳞癌细胞的NIPBL表达水平及顺铂敏感性，我们发现NIPBL表达水平越高的食管鳞癌细胞对顺铂越敏感。NIPBL的过表达能够有效增强食管鳞癌细胞的顺铂敏感性，相反，NIPBL的表达沉默能够显著抑制食管鳞癌细胞对顺铂的化疗敏感性。关键的是，NIPBL蛋白的表达水平与PUMA基因的转录活性呈正相关。PUMA的表达沉默能够有效抑制NIPBL的过表达对食管鳞癌细胞顺铂敏感性的增强作用。相反，PUMA的过表达能够逆转NIPBL的缺失对食管鳞癌细胞顺铂敏感性的抑制作用。最后，我们证实了NIPBL蛋白能够通过直接结合于PUMA基因启动子区域来调控其转录活性，且NIPBL、PUMA、HDACs三者的表达水平呈正相关。因此，我们提出了“NIPBL以依赖于组蛋白乙酰化的修饰方式调控PUMA的转录活性，影响食管鳞癌细胞的顺铂敏感性”的科学模型。.本项目首次揭示了NIPBL在调控食管鳞癌细胞顺铂敏感性中的作用及分子机制，阐明食管鳞癌患者顺铂耐药的分子机理，为食管鳞癌的靶向治疗提供潜在的分子靶点，丰富了食管鳞癌个性化治疗的理论基础。