LINC00460在EYA4抑制胰腺癌生长中的作用及机制

We recently reported that EYA4 functioned as a tumor suppressor gene in pancreatic carcinoma in our previous studies, however its mechanisms have not been well-documented. By analyzing the differences in long non-coding RNA (LncRNA) expression profiles between EYA4-overexpressing pancreatic carcinoma cells and its vector control, it was found that the expression of LINC00460 was most markedly upregulated in EYA4-overexpressing pancreatic carcinoma cells. Knocking-down LINC00460 in EYA4-overexpressing pancreatic carcinoma cells could reverse the tumor-suppressing effects of EYA4. These results suggested that LINC00460 might mediate the tumor-suppressing roles of EYA4. Additionally, We found that EYA4 could dephosphorylate the transcriptional factor SP1. Furthermore, it was found that overexpression of LINC00460 could reduce the protein level of ID2, while its mRNA level was not influenced. Based on the results of our previous studies, the present study aims at: (1) Elucidating whether EYA4 up-regulates LINC00460 via dephosphorylating its pivotal transcriptional factor SP1, and its detailed mechanisms. (2) Clarifying whether LINC00460 promotes the ubiquitylation and degradation of ID2 via regulating ASB4 which is an E3 ubiquitin ligase, and its detailed mechanisms. (3) Examining the levels of EYA4,p-SP1,LINC00460 and ID2 in the pancreatic carcinoma tissue samples, and evaluating their correlations and their clinical significances in patients of pancreatic carcinoma. With completion of the present study, the functions and the mechanisms of the novel signal pathway of EYA4 / LINC00460 / ID2 in pancreatic cancer would be clearly elucidated. The results of the present study would provide a new strategy for the diagnosis and management of pancreatic carcinoma.

我们最近报道了EYA4是胰腺癌的抑癌基因，但其抑癌机制尚不清楚。目前通过分析EYA4过表达的胰腺癌细胞长链非编码RNA表达谱，发现LINC00460上调最明显，且敲低LINC00460能逆转EYA4抑制胰腺癌生长的作用，提示其介导了EYA4的抑癌作用。另外，EYA4可去磷酸化转录因子SP1；LINC00460可降低ID2蛋白水平，但不影响其mRNA水平。本项目拟研究（1）EYA4是否通过去磷酸化LINC00460的关键转录因子SP1，从而实现对LINC00460表达的调控；（2）LINC00460是否通过调控E3连接酶ASB4对ID2蛋白的泛素化降解，并明确其机制；（3）临床胰腺癌标本中检测EYA4、p-SP1、LINC00460、ID2的相关性及其临床意义。本项目的完成，将阐明这个新的EYA4/LINC00460/ID2通路在胰腺癌生长中的作用和机制，可能为胰腺癌的诊治提供新策略。

研究背景：胰腺导管腺癌(PDAC)是一种高度致命的恶性肿瘤。吉西他滨耐药是PDAC治疗中的一个主要挑战。.主要研究内容：阐明LINC00460在PDAC耐吉西他滨中的生物学作用及其机制。.主要方法：建立吉西他滨化疗耐药PDAC-PDX模型。用LINC00460/control shRNA转染PDAC细胞株，采用RNA测序和GO分析，研究LINC00460的生物学功能。用FISH法检测LINC00460的细胞定位。RNA pull-down检测可以直接与LINC00460结合的蛋白，LC-MS/MS明确这些蛋白的确切名称，RIP验证其结果。采用IC50法检测胰腺癌细胞对吉西他滨的敏感性。.重要结果：在本研究中，我们发现LINC00460与PDAC患者和PDAC- PDX对吉西他滨的敏感性相关。在PDAC肿瘤细胞中敲降LINC00460后，RNA测序和GO分析结果表明，LINC00460可参与调控生长因子的活性，并介导细胞外机制的重塑。FISH显示LINC00460主要位于细胞质中。RNA pull-down、LC-MS/MS、RIP和免疫印迹结果证实LINC00460可以直接与PDAP1结合。此外，我们发现LINC00460通过PDAP1/PDGFA/PDGFR信号通路介导PDAC肿瘤细胞和CAFs的细胞间相互作用，并调控CAFs的吉西他滨耐药功能，这可以通过PDGFR抑制剂（Crenolanib）逆转。LINC00460表达较低的PDAC-PDX肿瘤对吉西他滨联合Crenolanib治疗有较好的反应。.科学意义：我们的研究结果支持了吉西他滨联合Crenolanib在LINC00460低表达PDAC的精准治疗中的应用。