DANCR通过调节DANCR-MLL3轴促进胰腺癌进展的机制

Mechanism of DANCR in promoting pancreatic cancer progression via modulation of DANCR-MLL3 axis

摘要

目的：探究长链非编码RNA分化拮抗非蛋白编码RNA（DANCR）及通过调控DANCR-混合谱系白血病3（MLL3）轴影响胰腺癌进展的分子机制。方法：收集泰安市中心医院2019年1月—2022年12月收治的120例胰腺癌患者的肿瘤组织及配对癌旁组织，检测DANCR表达量，分析其与临床病理参数及预后的关联；在胰腺癌细胞系（MIA PaCa-2、PANC-1）中敲低DANCR，通过CCK-8、划痕实验、Transwell实验评估其对细胞增殖、迁移及侵袭能力的影响。采用双荧光素酶报告基因实验验证DANCR对MLL3启动子活性的调控作用。通过蛋白质印迹法检测MLL3、H3K4me3及p57的蛋白表达，并使用MLL3抑制剂MI-503进行回复实验，验证DANCR-MLL3-p57信号轴的功能。结果：胰腺癌组织中DANCR mRNA表达显著高于癌旁组织（P<0.01）；胰腺癌细胞系中DANCR mRNA表达显著高于正常胰腺导管上皮细胞（P<0.01），MLL3 mRNA表达则显著降低（P<0.01），且胰腺癌细胞系中两者表达呈显著负相关（r=-0.808，P<0.01）；DANCR高表达与胰腺癌淋巴结转移、低分化相关（P<0.01），且高表达患者总生存期显著缩短（P<0.01）；敲低DANCR后，MIA PaCa-2、PANC-1细胞增殖活力、划痕迁移率、Transwell迁移及侵袭细胞数均显著降低（P<0.01）；双荧光素酶报告基因实验证实DANCR可抑制MLL3启动子活性（P<0.01）；敲低DANCR可上调MLL3、H3K4me3、p57蛋白表达（P<0.01），而加入MI-503可逆转上述蛋白表达变化及细胞功能抑制效应（P<0.01）。结论：DANCR通过调节DANCR-MLL3轴促进胰腺癌的恶性进展，可能成为胰腺癌潜在的预后生物标志物和治疗靶点。

Abstract

Objective: To investigate the molecular mechanism by which the long non-coding RNA Differentiation Antagonizing Non-protein Coding RNA (DANCR) influences pancreatic cancer progression via regulating the DANCR-Mixed Lineage Leukemia 3 (MLL3) axis. Methods: Tumor tissues and paired adjacent normal tissues were collected from 120 pancreatic cancer patients admitted to Tai'an Central Hospital from January 2019 to December 2022. DANCR expression levels were detected and analyzed for their association with clinicopathological parameters and patient prognosis. DANCR was knocked down in pancreatic cancer cell lines (MIA PaCa-2, PANC-1). The effects on cell proliferation, migration, and invasion were assessed using CCK-8 assay, scratch wound healing assay, and Transwell assay. The regulatory effect of DANCR on MLL3 promoter activity was validated through a dual luciferase reporter gene assay. Protein expression levels of MLL3, H3K4me3, and p57 were detected by Western blotting. Rescue experiments were performed using the MLL3 inhibitor MI-503 to validate the function of the DANCR-MLL3-p57 signaling axis. Results: DANCR expression was significantly higher in pancreatic cancer tissues compared to adjacent normal tissues (P<0.01). Consistently, its expression was upregulated in pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells (P<0.01), while MLL3 expression was significantly reduced (P<0.01), showing a significant negative correlation (r=-0.808, P<0.01). High DANCR expression was associated with lymph node metastasis and poor differentiation in pancreatic cancer patients (P<0.01), and patients with high expression had a significantly shorter overall survival (P<0.01). Knockdown of DANCR significantly reduced proliferation viability, scratch wound healing rate, and the number of migrating and invading cells in both MIA PaCa-2 and PANC-1 cells (P<0.01). Dual-luciferase reporter gene experiments confirmed that DANCR inhibits MLL3 promoter activity (P<0.01). DANCR knockdown upregulated the protein expression levels of MLL3, H3K4me3, and p57 (P<0.01). The addition of MI-503 reversed these protein expression changes and the inhibitory effects on cell function (P<0.01). Conclusion: DANCR promotes the malignant progression of pancreatic cancer by regulating the DANCR-MLL3 axis, suggesting its potential as a prognostic biomarker and therapeutic target for pancreatic cancer.

关键词

胰腺肿瘤 / 分化拮抗非蛋白编码RNA / 混合谱系白血病3 / 增殖 / 侵袭

Key words

Pancreatic neoplasms / Differentiation antagonizing non-protein coding RNA / Mixed lineage leukemia 3 / Proliferation / Invasion

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基金资助

泰安市科技创新发展项目(2023NS271)