PPP3CB下调Sp1转录活性抑制胰腺癌侵袭转移的机制研究

The high invasiveness and metastasis of pancreatic cancer is a major cause for its poor prognosis. Our previous research reported that the phosphorylated transcription factor Sp1 can bind to the promoter region of COX2 and enhance the invasion and metastasis of pancreatic cancer by promoting angiogenesis. In the pre-experiment, gene co-expression network analysis suggested that the protein phosphatase 3 catalytic subunit beta (PPP3CB) was associated with Sp1, and PPP3CB is a key factor involved in the dephosphorylation of substrate. Further studies revealed that pancreatic cancer cell lines with lower expression of PPP3CB demonstrated higher invasiveness and metastasis. After interfering with PPP3CB, the expression of p-Sp1 was increased in pancreatic cancer cell lines, and the invasiveness and metastasis of cells were enhanced. In addition, PPP3CB expression in pancreatic cancer tissues was lower than that in adjacent normal tissues, and the expression of PPP3CB and Sp1 in cancer tissues was correlated with lymph node metastasis. On the base of these findings, we will use bioinformatics and classical molecular biology methods to investigate the expression and function of PPP3CB in pancreatic cancer, as well as the dephosphorylation of Sp1 by PPP3CB. In vitro and in vivo studies will be conducted to investigate the biological function of PPP3CB in down-regulating the transcriptional activity of Sp1 by dephosphorylation to inhibit the invasion and metastasis of pancreatic cancer. The results will be verified in pancreatic cancer tissues and used in providing evidence for the new target and treatment strategy of pancreatic cancer.

胰腺癌的高侵袭转移性是其预后差的一大主因。课题组既往研究报道，转录因子Sp1可结合于COX2启动子区，并促进其介导的胰腺癌侵袭转移。预实验中，基因共表达网络分析提示钙调神经磷酸酶A亚基β异构体（PPP3CB）和Sp1相关，而PPP3CB是底物去磷酸化的关键因子。进一步研究发现低表达PPP3CB的胰腺癌细胞株具有更强侵袭转移性，干扰PPP3CB后，细胞中p-Sp1表达增高，细胞侵袭转移能力增强。此外，胰腺癌中PPP3CB表达低于癌旁组织，且PPP3CB和Sp1均与淋巴结转移相关。基于上述结果，我们将采用生信分析和分子生物学等方法，研究PPP3CB在胰腺癌中的表达和功能，探讨PPP3CB对Sp1的去磷酸化修饰，体内体外研究PPP3CB下调Sp1转录活性抑制胰腺癌侵袭转移的机制，并在临床样本中验证，从而阐明PPP3CB在胰腺癌侵袭转移中的生物学效应，最终为胰腺癌靶点的寻找和诊疗策略制定提供依据。

胰腺癌是全世界男女癌症相关死亡率的第七大主要原因，其5年总生存率不到10％。我国胰腺癌的发病率为90.1/100000，在所有肿瘤中排第9位，死亡率为79.4/100000，在所有肿瘤中排第6位。初诊时，约78％的胰腺癌患者失去手术切除机会，近50%的患者伴有远处转移。尽管在胰腺癌的治疗方面已取得一定进展，但化学疗法仍是目前转移性胰腺癌的主要治疗方法，仅有一小部分患者能从靶向治疗和免疫治疗获益。磷蛋白磷酸酶（phosphoprotein phosphatase，PPP）是丝/苏氨酸蛋白磷酸酶中一大类，包含PP1、PP2A、PP3（钙调神经磷酸酶）、PP4、PP5、PP6和PP7，既往研究发现其参与了多个生理病理过程，但在胰腺癌发生发展中的作用少有报导。本研究通过生物信息学挖掘公共数据库发现胰腺癌患者肿瘤组织中的PPP3CB表达水平显著低于癌旁组织。进一步通过组织芯片验证发现肿瘤组织中PPP3CB的表达与性别、核分级、肿瘤的N分期、癌旁组织中PPP3CB表达水平显著相关。此外，胰腺癌患者PPP3CB表达越低，预后越差。通过Wb和qPCR发现PPP3CB在胰腺癌细胞株中表达低于正常胰腺细胞。干扰PPP3CB表达后，胰腺癌细胞侵袭转移能力明显增强，反之则减弱。在胰腺癌细胞株中加入PPP3CB抑制剂CsA后，胰腺癌细胞侵袭转移能力有所增强。裸鼠成瘤实验也在体内对上述结果进一步加以证明。另外，本研究通过生信分析筛选出PPP3CB的下游分子ATOH8，并通过Co-IP、免疫荧光等实验进一步验证两者的相关性及相互作用，从而发现PPP3CB通过去磷酸化修饰来激活ATOH8，使其从细胞质转入细胞核内。通过Chip seq筛选出ATOH8下游的靶基因Sp1，进一步的功能实验发现细胞核内的ATOH8通过转录抑制Sp1来抑制胰腺癌的侵袭转移。本研究初步阐明了PPP3CB去磷酸化调控ATOH8/Sp1信号轴抑制胰腺癌侵袭转移的分子机制，为胰腺癌的精准治疗和新靶点的寻找提供理论依据，也为抗癌药物的研发提供新的思路。下一步我们将继续通过体内及体外实验深入研究调控PPP3CB的上游信号分子UBE4A，其如何通过泛素化下调PPP3CB表达来促进胰腺癌侵袭转移的分子机制。