趋化因子CXCL14异常表达在介导线粒体氧化损伤引起的结直肠癌发生发展中的生物学功能及作用机制研究

Colorectal cancer (CRC) is a common malignancy which causes global health burden. Recent studies showed that chemokines play important roles in the proliferation and metastasis of CRC. Our previous studies have screened chemokine CXCL14 that is closely related to the metastasis of CRC using cDNA microarray. Validation of the candidate protein in a cohort of clinical samples revealed that chemokine CXCL14 is closely associated with tumor stage, histological grade, the survival prognosis of patients with CRC. In vitro studies demonstrated that CXCL14 profoundly induced proliferation, migration and invasion of CRC cells: the suppression of CXCL14 reduced the proliferative, migratory and invasive capacity of CRC cells; the enforced expression of CXCL14 elevated the proliferative, migratory and invasive capacity of CRC cells (Zeng et al. J Transl Med 2013). Our further functional analysis revealed that ROS could up-regulate the expression of CXCL14 in the CRC cells, and induce the proliferation and migration of CRC cells. Mitochondrial dysfunction will lead to the production of ROS, which may in turn affect certain redox-sensitive molecules and further lead to cell proliferation and invasion. So far, the association between abnormal CXCL14 expression and mitochondrial dysfunction is unclear. Clarifying these mechanisms is of great significance for cancer therapy. Based on our previous work, in this project, we will apply the molecular and cell biology strategies combined with animal models to comprehensively interrogate the association between mitochondrial dysfunction and abnormal CXCL14 expression, explore the biological functions of mitochondrial dysfunction-induced ROS and abnormal CXCL14 level in the colorectal cancer promotion and progression, and clarify the mechanisms of the abnormal expression of CXCL14 on colorectal cancer growth and metastasis mediated mitochondria dysfunction and oxidative stress. Our study aims to evaluate the potential of CXCL14 as a drug target in CRC, which will pave the road for translational medicine.

结直肠癌是临床上常见的恶性肿瘤。近年来研究发现趋化因子在结直肠癌生长转移过程中有重要作用。前期我们通过cDNA microarray筛选到趋化因子CXCL14，经临床样本验证发现CXCL14表达与结直肠癌分期、病理分级和患者生存预后密切相关。体外实验发现CXCL14可显著影响结直肠癌的增殖、迁移和侵袭能力，结果发表于J Trans Med杂志。后续研究发现：ROS能上调结直肠癌细胞CXCL14的表达，促进结直肠癌细胞增殖和迁移。线粒体氧化损伤会诱导大量ROS的产生，促进肿瘤生长和转移。目前对于CXCL14异常表达与线粒体功能线粒体氧化损伤之间的关系尚不清楚。本项目拟在此基础上，综合采用多种分子肿瘤学研究技术，深入研究趋化因子CXCL14异常表达在介导线粒体氧化损伤引起的结直肠癌发生发展中的生物学功能及作用机制研究，探讨CXCL14作为结直肠癌药物靶标的潜在应用价值。

通过加入外源H2O2处理结直肠癌细胞系HCT116和SW620，我们发现胞内活性氧水平也显著提高，通过MTT实验、CKK8实验、细胞克隆形成实验、流式细胞术、划痕实验、Transwell小室等的实验结果说明胞内高ROS水平可导致结直肠癌细胞的增殖和迁移。通过Western blot实验探讨其机制发现H2O2可引起细胞周期相关蛋白、转移相关蛋白表达水平的变化，同时H2O2可引起趋化因子CXCL14表达的变化。为了进一步研究结直肠癌细胞内高ROS水平和CXCL14异常表达的关系，我们构建了CXCL14稳定敲除或稳定过表达细胞系，发现CXCL14可促进结直肠癌细胞的增殖和迁移，并引起增殖和迁移相关蛋白表达水平的变化。同时我们发现CXCL14可引起NF-κB和ERK表达的变化。对两种结直肠癌细胞系，我们进行了不同的研究：在SW620细胞系中，H2O2处理后能够上调CXCL14 的表达和AP-1磷酸化水平；加入ROS清除剂NAC进行预处理，H2O2对CXCL14和AP-1磷酸化水平表达的影响会被减弱；加入AP-1抑制剂即SP600125进行预处理，H2O2对CXCL14表达的促进作用同样也会得到消除。以上结果表明在SW620细胞中，ROS能够促进CXCL14的表达，并且这种促进作用是AP-1所依赖的。H2O2处理SW620细胞后能够促进NF-κB磷酸化蛋白的表达，加入NAC进行预处理后H2O2对NF-κB磷酸化水平表达的影响会被减弱。CXCL14过表达后能够提高NF-κB磷酸化蛋白的表达，并且促进NF-κB下游与抗凋亡相关的Bcl2、Survivin蛋白的表达，与EMT过程相关的Vimentin蛋白的表达、与细胞周期相关的CyclinA和CDK2蛋白的表达。以上结果表明CXCL14可能通过激活NF-κB信号通路，调节NF-κB下游蛋白的表达，进而促进SW620细胞的增殖和迁移。综合上述实验结果，ROS可通过ROS→AP-1→CXCL14→NF-κB信号通路调控SW620细胞的增殖和迁移。在HCT116细胞系中，H2O2处理后同样能够上调CXCL14 的表达，同时胞内ERK磷酸化水平显著升高。加入ROS清除剂NAC进行预处理，H2O2对CXCL14和ERK磷酸化水平表达的影响会被减弱。为了进一步验证CXCL14和ERK的关系，我们构建了CXCL14稳定表达或敲除的细胞系，结果显