Department of Pharmaceutical and Biomedical Sciences, The University of Georgia, Athens, Georgia, USA.
PLoS One. 2013;8(1):e53436. doi: 10.1371/journal.pone.0053436. Epub 2013 Jan 15.
Overexpression of ribonucleotide reductase subunit M2 (RRM2), involved in deoxyribonucleotide synthesis, drives the chemoresistance of pancreatic cancer to nucleoside analogs (e.g., gemcitabine). While silencing RRM2 by synthetic means has shown promise in reducing chemoresistance, targeting endogenous molecules, especially microRNAs (miRNAs), to advance chemotherapeutic outcomes has been poorly explored. Based on computational predictions, we hypothesized that the let-7 tumor suppressor miRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in innately gemcitabine-resistant pancreatic cancer cell lines. Direct binding of let-7 miRNAs to the 3' UTR of RRM2 transcripts identified post-transcriptional regulation of RRM2 influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and chemosensitivity responses in a poorly differentiated pancreatic cancer cell line, MIA PaCa-2. Defective processing of let-7a precursors to mature forms, in part, explained the discrepancies observed with let-7a expressional outcomes. Consistently, the ratios of mature to precursor let-7a were progressively reduced in gemcitabine-sensitive L3.6pl and Capan-1 cell lines induced to acquire gemcitabine resistance. Besides known regulators of let-7 biogenesis (e.g., LIN-28), short hairpin RNA library screening identified several novel RNA binding proteins, including the SET oncoprotein, to differentially impact let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus -resistant pancreatic cancer cells. Further, LIN-28 and SET knockdown in the cells led to profound reductions in cellular proliferation and colony-formation capacities. Finally, defective processing of let-7a precursors with a positive correlation to RRM2 overexpression was identified in patient-derived pancreatic ductal adenocarcinoma (PDAC) tissues. These data demonstrate an intricate post-transcriptional regulation of RRM2 and chemosensitivity by let-7a and that the manipulation of regulatory proteins involved in let-7a transcription/processing may provide a mechanism for improving chemotherapeutic and/or tumor growth control responses in pancreatic cancer.
核苷酸还原酶亚单位 M2（RRM2）的过表达参与脱氧核苷酸的合成，导致胰腺癌对核苷类似物（如吉西他滨）的化学抗性。虽然通过合成手段沉默 RRM2 已经显示出降低化学抗性的潜力，但靶向内源性分子，特别是 microRNAs（miRNAs），以提高化疗结果的研究还很少。基于计算预测，我们假设 let-7 肿瘤抑制 miRNA 将抑制胰腺癌中 RRM2 介导的吉西他滨化学抗性。在天然对吉西他滨耐药的胰腺癌细胞系中，发现大多数 let-7 miRNA 的表达降低，与 RRM2 表达呈负相关。let-7 miRNA 直接结合到 RRM2 转录物的 3'UTR，确定了 RRM2 的转录后调节，影响吉西他滨的化疗敏感性。有趣的是，人前体 let-7 miRNA 的过表达导致在分化不良的胰腺癌细胞系 MIA PaCa-2 中产生不同的 RRM2 表达和化疗敏感性反应。let-7a 前体向成熟形式的加工缺陷，部分解释了观察到的 let-7a 表达结果的差异。一致地，在诱导获得吉西他滨耐药的吉西他滨敏感 L3.6pl 和 Capan-1 细胞系中，成熟到前体 let-7a 的比率逐渐降低。除了 let-7 生物发生的已知调节剂（例如，LIN-28）之外，短发夹 RNA 文库筛选还鉴定了几种新型 RNA 结合蛋白，包括 SET 癌蛋白，它们以不同的方式影响吉西他滨敏感与耐药的胰腺癌细胞中的 let-7 生物发生和化疗敏感性。此外，LIN-28 和 SET 在细胞中的敲低导致细胞增殖和集落形成能力的显著降低。最后，在患者来源的胰腺导管腺癌（PDAC）组织中鉴定到与 RRM2 过表达呈正相关的 let-7a 前体的加工缺陷。这些数据表明，let-7a 对 RRM2 和化疗敏感性的复杂转录后调节，以及涉及 let-7a 转录/加工的调节蛋白的操纵可能为改善胰腺癌的化疗和/或肿瘤生长控制反应提供一种机制。
