Notch3介导Pbx1调控Cyclin D1表达在非小细胞肺癌增殖中的作用

Xuanwei has the highest morbidity and mortality of lung cancer in China due to the unique ingredients of permian period coal and the traditional coal-burning way indoors in Xuanwei. However, the pathogenesis is still poorly understood. Previous studies have demonstrated that Pbx1 is closely related to normal growth and development. Moreover, recent investigations indicated that Pbx1 is involved in carcinogenesis and progression..In this study, we found that cell proliferation was significantly reduced and cell-cycle G1 arrest while Pbx1 was knocked down in XWLC-05 cell line, which was derived from surgical resection tissue of a patient with lung cancer from Xuanwei. These results suggested that Pbx1 is associated with the abnormal proliferation in non-small cell lung cancer..Notably, further studies showed that Pbx1 can be activated by Notch3. Also, bioinformatical analysis showed there exist five potential Pbx1-binding sites (motif) in Cyclin D1 promoter region ..Based on these results, we hypothesize that Notch3 mediated by Pbx1 regulates Cyclin D1 expression leading to abnormal proliferation. In this study, we further use EMSA, CHIP, site-directed mutagenesis and etc. to obtain reliable evidences on the hypothesis so as to identify the key target genes, and to provide a novel scientific proof for targeted therapy on non-small cell lung cancer.

云南宣威肺癌发病居全国首位，独特的二叠纪煤成矿环境和传统的燃煤习惯，使宣威成为特有的肺癌高发现场，然而其发病机制远未阐明。既往研究表明，Pbx1与生长发育相关；新近研究表明，Pbx1参与肿瘤发生发展。我们研究发现：在宣威肺癌细胞系XWLC-05中沉默Pbx1,细胞增殖能力显著降低(结果四,五)，细胞周期G1期出现阻滞(结果六)，提示Pbx1与肺癌恶性增殖相关。新近研究显示：Notch3能够激活Pbx1表达。生物信息学分析提示：Pbx1在Cyclin D1启动子区有五个可能的结合基序。由此，我们提出肺癌细胞增殖可能存在的信号转导机制：Notch3介导Pbx1异常调控Cyclin D1表达，导致细胞恶性增殖。本项目拟进一步用EMSA、CHIP和定点突变技术，旨在获得Notch3激活Pbx1调控Cyclin D1表达的可靠证据；阐明肺癌增殖相关信号转导通路作用机制,为肺癌新靶点治疗提供科学依据。

肺癌起源于支气管黏膜上皮，是全世界癌性死亡的主要原因之一。近年来，虽然在肺癌的预防和治疗方面取得了显著进步，但肺癌的发病率和死亡率仍居高不下。云南宣威肺癌发病居全国首位，独特的二叠纪煤成矿环境和传统的燃煤习惯，使宣威成为特有的肺癌高发现场。组织学上，通常将肺癌分为小细胞肺癌和非小细胞肺癌，其中非小细胞肺癌约占全部肺癌的85%，且宣威肺癌以非小细胞肺癌为主，其发病机制远未阐明。前B细胞白血病同源盒基因（PBX1）是同源盒基因家族成员之一，其编码转录因子PBX1，在多种组织中广泛表达。既往研究表明，Pbx1 与器官生长发育、细胞分化相关；新近研究表明，Pbx1 在一些实体瘤的发生、发展中也发挥着重要作用。PBX1在肺癌、黑色素瘤、卵巢癌、乳腺癌等多种肿瘤中高表达，与肿瘤的转移和不良预后有关。PBX1可作为肺癌预后的独立因素也已被确认。我们研究发现：PBX1在非小细胞肺癌中高表达，与非小细胞肺癌患者的不良预后有关，提示PBX1与非小细胞肺癌的恶性进展密切相关。在宣威肺癌细胞系XWLC-05和国际通用非小细胞肺癌细胞系A549中，稳定沉默Pbx1,细胞增殖能力显著降低，细胞周期阻滞于G1期，进一步的体内实验也表明，稳定沉默Pbx1抑制了裸鼠皮下移植瘤的生长，提示Pbx1与肺癌恶性增殖相关。Notch信号通路是一条高度保守的、与细胞命运密切相关的信号通路，包括4个受体和5个配体，Notch3为其中一个受体。新近研究显示：Notch3 能够激活Pbx1 表达。我们的实验也发现，通过慢病毒或γ-酶抑制剂下调Notch3后，Pbx1的表达也下调了。进一步用生物信息学分析发现：Pbx1 在Cyclin D1 启动子区有五个可能的结合基序。我们通过ChIP实验也验证了这一结合。由此，我们提出肺癌细胞增殖可能存在的信号转导机制：Notch3 介导Pbx1 异常调控Cyclin D1 表达，导致细胞恶性增殖。本项目获得了Notch3 激活Pbx1 调控Cyclin D1表达的可靠证据；阐明了肺癌增殖相关信号转导通路的作用机制,为肺癌新靶点治疗提供科学依据。