p53介导的PVT1 lncRNA表达及加工通路在胰腺癌吉西他滨化疗敏感性调控中的功能研究

Gemcitabine used for pancreatic cancer (PC) therapy represents a major challenge due to drug resistance and the detailed mechanism is not well-established. Our previous data indicate that gemcitabine results in an enhanced stability of PVT1 lncRNA via downregulating the expression of Drosha and DGCR8 in gemcitabine-resistant PC cells. These findings suggest a potential mechanism for gemcitabine chemoresistance in PC treatment. It has been reported that p53 is involved in regulating PVT1 processing into mature microRNAs. Furthermore, abnormal expression of p53 influences pancreatic cancer’s sensitivity to gemcitabine. Whether p53 could regulate pancreatic cancer’s sensitivity to gemcitabine via PVT1 related pathways needs further studies. In our project, we aim to answer these questions. Firstly, the mechanism of p53 in regulating PVT1 processing will be elucidated at transcriptional and proteins interaction levels. Secondly, we hope to explore the correlation between p53 with different genotypes and gemcitabine chemoresistance in PC cell lines and PC-Patient derived xenograft (PC-PDX) mouse models and investigate whether it can be used as a biomarker system for predicting the prognosis of PC chemotherapy and guiding for much more effective therapeutic strategy. Thirdly, we attempt to conduct small molecular drugs for remodifying the function of Drosha/DGCR8 complex and select candidate drugs used for the combined treatment with gemcitabine in PC therapy. Furthermore, the clinical efficacy of gemcitabine in combination with candidate drugs will be evaluated in PC cell lines and PC-PDX mouse models. Altogether, we want to lay the groundwork for the progress of precision medicine for PC prevention.

吉西他滨化疗耐受是胰腺癌治疗面临的一大难题，相关耐药机制至今尚不明确。申请者前期工作显示，吉西他滨在胰腺癌耐药细胞中可诱导Drosha/DGCR8表达下调，促进PVT1稳定性增加。这可能是胰腺癌对吉西他滨耐药的关键机制之一。已有研究表明，p53参与PVT1加工过程的调控；p53的异常表达可影响胰腺癌对吉西他滨治疗的反应。故p53是否可通过PVT1相关通路以调控胰腺癌对吉西他滨化疗的敏感性是我们拟进一步研究的关键问题。本项目拟在基因转录及蛋白质相互作用层面进一步研究p53介导PVT1表达及加工的分子机制，阐明p53突变或缺失等不同基因型与吉西他滨耐药的关系，并研究其做为分子标志物的有效性；同时筛选可以重塑Drosha/DGCR8复合物功能的小分子药物，与吉西他滨联合使用，在细胞水平及人源胰腺癌异种移植瘤模型（PDX）小鼠中系统评估新方案的治疗效果，为胰腺癌精准治疗研究奠定基础。

Pvt1癌基因(非蛋白编码)(PVT1)是肿瘤发生中一种致癌的长链非编码RNA。在目前的研究中，我们证明PVT1的表达与吉西他滨在胰腺癌治疗疗效密切相关。抑制PVT1导致用吉西他滨处理的PC细胞的细胞生长减少。吉西他滨治疗降低PVT1水平并增加其编码的miRNAs，如miR-1207对(miR-1207-5p/3p)。miR-1207对的过度表达增强了细胞对吉西他滨的化学敏感性，而沉默miR-1207-5p/3p以防止吉西他滨治疗诱导导致细胞生长增加。机制研究表明miR-1207-5p和miR-1207-3p分别靶向PC细胞中的SRC原癌基因(非受体酪氨酸激酶)和ras同源家族成员A。特别是吉西他滨诱导Drosha核糖核酸酶III (Drosha)和DGCR8微处理器复合物亚单位(DGCR8)上调，然后触发PVT1加工。Drosha和DGCR8的抑制导致吉西他滨的疗效降低，表明吉西他滨通过促进其加工成miRNAs而降低PVT1的表达，这反过来导致PC细胞中癌基因信号的钝化。此外，我们证明吉西他滨化疗耐药性是AsPC-1细胞和肿瘤细胞移植模型中Drosha和DGCR8表达降低的结果。总的来说，深入了解吉西他滨化疗在胰腺癌中的疗效及新的新的机制。同时我们探讨了PVT1高表达的下游效应，基因集变异分析计算了TCGA胰腺癌组织MsigDb数据库中相应基因集的富集分数。我们发现PVT1的表达水平与增殖相关途径的富集分数密切相关。推测PVT1和MYC的共表达也可能受到表观遗传学的影响。因此，我们分析了胰腺癌的Hi-C和ChIP-seq测序结果，发现PVT1和MYC的启动子在PANC-1和Capan-1中被H3K27ac和H3K4me3修饰，这两种组蛋白修饰可以上调相应基因的表达。结合Hi-C测序结果，我们发现两种胰腺癌细胞中都存在PVT1启动子和MYC启动子的相互作用。由于两者都被激活增强子的特征标记H3K27ac修饰，因此可以通过增强子-启动子相互作用上调彼此的表达水平，这进一步解释了PVT1和MYC共表达修饰的原因。相关的研究仍在继续中。