RIP-SEQ analysis of CGN binding mRNA in PANC-1 cells stably expressing CGN

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies for its difficult early diagnosis and poor prognosis. The molecular determinants dictating pancreatic cancer initiation and progression remain to be defined and are essential to its effective early detection and successful treatment. Little is known on the cingulin (CGN) expression and function in carcinogenesis. In this study, we demonstrate the critical oncogenic role of CGN in PDAC pathogenesis. Increased expression of CGN promoted pancreatic acinar-to-ductal metaplasia (ADM) and CGN is progressively upregulated during pancreatic cancer initiation and progression. CGN was required for pancreatic cancer cell proliferation, gemcitabine resistance, tumorigenesis and invasion and its overexpression predicted a poor patient prognosis. Mechanistically, CGN enhanced EGFR/AXL/ERK signaling pathway through increasing the mRNA stabilization and the protein expression of nuclear-cytoplasmic transport protein, importin-7 (IPO7) and the nuclear translocation of pERK, thus promoting the malignant behaviors of PDAC cells. Therefore, our study has demonstrated that CGN forms a novel oncogenic signaling axis, which could be an effective therapeutic target for PDAC treatment. To investigate the function of CGN in PDAC progression, we established PANC-1-CGN cell lines with CGN overexpressed. Then we analysis of CGN binding mRNA in human PANC-1-CGN cell lines from RIP-SEQ (input, IP)

胰腺导管腺癌（Pancreatic ductal adenocarcinoma, PDAC）是致死率最高的恶性肿瘤之一，因其早期诊断困难且预后极差。调控胰腺癌发生与进展的分子决定因素仍有待明确，而这些因素对于实现胰腺癌的有效早期检测与成功治疗至关重要。目前对于扣带蛋白（cingulin, CGN）在癌变过程中的表达与功能尚不清楚。本研究证实了CGN在PDAC发病过程中发挥关键的致癌作用：CGN表达升高可促进胰腺腺泡-导管化生（pancreatic acinar-to-ductal metaplasia, ADM），且在胰腺癌发生与进展过程中，CGN的表达呈进行性上调；CGN对于胰腺癌细胞的增殖、吉西他滨耐药、成瘤性与侵袭能力均不可或缺，且CGN过表达预示着患者预后不良。从机制上来说，CGN通过增强mRNA稳定性以及核质转运蛋白输入蛋白7（importin-7, IPO7）的蛋白表达，同时促进磷酸化ERK（pERK）的核转位，从而激活EGFR/AXL/ERK信号通路，最终促进PDAC细胞的恶性生物学行为。因此，本研究证实CGN构成了一条全新的致癌信号轴，有望成为PDAC治疗的有效靶点。为探究CGN在PDAC进展中的功能，我们构建了过表达CGN的PANC-1-CGN细胞系；随后我们通过RIP测序（RIP-SEQ，包含input组与IP组），对人源PANC-1-CGN细胞系中与CGN结合的mRNA进行了分析。