核糖体新生调控因子PNO1对大肠癌生长的影响及片仔癀的干预作用和机制研究

The morbidity and mortality of colorectal cancer were significantly increased year by year, which is one of the major threaten for public health of human being. The complicated underlying mechanism of carcinogenesis in colorectal cancer remains largely unknown, which is urgent need to be further explored. Our previous study for the first time indicated that the expression of PNO1 in colorectal cancer tissues was significantly increased comparing with non-cancerous colorectal tissues. Survival analysis indicated increase of PNO1 expression in CRC tissues was significantly associated with shorter survival of CRC patients. Moreover, PNO1 knockdown significantly suppressed the tumor growth of CRC cells in vitro and in vivo via activation of p53/p21 signaling pathway. In addition, our series of previous studies with the support from Nation Natural Science Foundation of China indicated that Pien Tze Huang (PZH) treatment significantly suppressed tumor growth, metastasis, drug resistance in CRC cells in vitro and in vivo via modulating multiple signaling pathways. Recently study indicated that PZH treatment significantly down-regulated the expression of PNO1 in CRC cells. These studies suggested that increase of PNO1 expression may play an essential role in tumor growth in CRC cells, and by down-regulating PNO1 expression maybe one of the underlying mechanisms of PZH on anti-CRC. To further confirm our hypothesis, in the present study, we are going to evaluate the effects of PNO1 over-expression and PZH treatment on tumor growth of CRC cells using transgenic mouse model and CRISPR/Cas9 gene editing system, as well as luciferase reporter system. The current study will be helpful for us on understanding the underlying mechanism of CRC carcinogenesis and PZH treatment on anti-CRC. And it also will provide more opportunities for CRC prevention and treatment, as well as provide experimental evidences for clinical use of PZH on anti-CRC.

大肠癌发病率和死亡例逐年呈上升趋势，严重危害人类健康，其发病机制极其复杂，有待进一步深入。课题组系统的研究证实了PNO1在大肠癌组织中的表达显著升高，且与患者预后差密切相关，沉默其表达可通过激活p53/p21通路抑制大肠癌细胞体内外生长；而在多个国家自然基金资助下证实了片仔癀可通过调控多条通路抑制大肠癌的生长、转移、耐药和下调大肠癌细胞中PNO1的表达，提示“通过调节核糖体新生调控因子PNO1表达及其下游p53通路活化”可能是片仔癀发挥抗大肠癌的重要机制。因此，本论文拟通过转基因小鼠、CRISPR/Cas9基因编辑技术和荧光素酶活性检测等技术研究PNO1过表达对大肠癌生长的影响和调控机制及片仔癀的干预作用，以期通过体内外实验一步丰富大肠癌发病机制和片仔癀抗大肠癌作用机制，为临床抗大肠癌治疗提供更多思路、方向，也为片仔癀临床抗癌应用提供实验依据。

背景和目的：片仔癀抗大肠癌疗效确切，但其作用靶点有待深入研究。前期研究发现PNO1对大肠癌细胞生长具有重要调控作用，且片仔癀干预后显著下调，可能是片仔癀发挥抗大肠癌的重要靶点之一。因此，本项目拟深入研究片仔癀干预、PNO1表达对大肠癌体内外生长的影响及对p53/p21通路的调控作用。.方法：构建PNO1过表达稳转细胞株并给予片仔癀干预，检测细胞增殖、凋亡和PNO1/p53/p21通路的活化情况；构建PNO1敲入小鼠和CAC小鼠模型及PNO1敲减移植瘤模型并给予片仔癀干预，评价PNO1敲入、敲减和/或片仔癀干预对瘤体生长的影响和对PNO1/p53/p21通路的影响。.结果：.（1）体外实验：PNO1过表达显著促进大肠癌细胞活力；片仔癀干预显著抑制大肠癌细胞株活力、细胞存活能力和诱导细胞周期从G0/G1期向S期转化和细胞凋亡，而PNO1过表达显著缓解片仔癀干预对细胞活力、细胞存活能力、细胞周期及细胞凋亡的影响；片仔癀干预显著下调PNO1、上调p53和p21在mRNA和蛋白水平的表达，而PNO1过表达显著缓解上述改变。.（2）体内实验：1）成功构建PNO1敲入小鼠，并构建CAC模型。研究发现在CAC模型小鼠中，WT和KI小鼠的体重、DAI指数、瘤体生长均未见显著差异。2）进一步研究发现，片仔癀干预可显著缓解CAC模型小鼠结肠瘤体的大小和数量，降低血清中IL-6、IL-1β和TNF-α等炎性因子的含量，降低肠组织中PCNA的表达和增加凋亡细胞的数量，降低PNO1蛋白表达及升高p21和p53蛋白表达；显著调控Wnt信号通路、PI3K-AKT信号通路及其调控下游多个基因的表达。3）进一步采用PNO1敲减细胞移植瘤模型研究发现，与对照组相比，片仔癀干预和PNO1敲减均可在一定程度上显著降低瘤体体积、重量及GFP荧光强度；而片仔癀干预对PNO1敲减后移植瘤瘤体体积、重量及GFP荧光强度未见显著影响；且片仔癀干预和PNO1敲减均可显著降低PCNA表达，增加细胞凋亡，下调PNO1表达，上调p53和p21表达，而在PNO1敲除基础上给予片仔癀干预，未见显著改变。.结论：片仔癀干预显著抑制CAC瘤体、大肠癌移植瘤及细胞生长，诱导细胞凋亡，且通过调控PNO1及其下游p53/p21通路可能是其重要的机制之一。