Huang Ren-Hong, Quan Ying-Jun, Chen Jin-Hong, Wang Ting-Feng, Xu Ming, Ye Min, Yuan Hao, Zhang Chong-Jie, Liu Xiao-Jian, Min Zhi-Jun
Department of Gastrointestinal Surgery, Shanghai Pudong Hospital, Fudan University Pudong Medical Center, Shanghai, China.
Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, China.
Cell Physiol Biochem. 2017;41(5):1851-1864. doi: 10.1159/000471933. Epub 2017 Apr 3.
Osteopontin (OPN) is highly expressed in colorectal cancer (CRC) and is associated with disease progression in vivo. High levels of OPN have been demonstrated to predict low survival rates in CRC. Autophagy is a process of self-digestion, which is thought to play a significant role in carcinogenesis. However, the mechanisms of OPN's effects on CRC cell autophagy have not been elucidated. Therefore, we aimed to investigate possible mechanisms of OPN's effects on CRC autophagy.
HCT116 cell proliferation, apoptosis, and migration and invasion ability were identified by cell counting k¡t-8 assay, flow cytometry, wound healing assay, and transwell chamber invasion assay, respectively. The ratios of proteins LC3-II/LC3-I, P62, and Atg7 were analyzed by Western-blot. Expressions of Beclin-1, Atg4b, Bnip3, and Vps34, both in transcriptional and translational levels, were analyzed and compared by RT-PCR and Western blot. Immunofluorescence and co-focusing experiments were used to investigate the formation of autophagosomes.
The results showed that OPN can promote cell proliferation, migration, and invasion, as well as inhibit cell apoptosis. It was also demonstrated that OPN could inhibit cell autophagy. Further experiments revealed that the inhibitory effect of OPN on autophagy could be reversed by blocking the p38 MAPK pathway in HCT116 cells.
OPN is involved in HCT116 cell progression and is capable of inhibiting cell autophagy possibly by activating the p38 MAPK signaling pathway, implying that OPN could be a potential novel molecular therapeutic biomarker in patients with CRC.
骨桥蛋白（OPN）在结直肠癌（CRC）中高表达，且与体内疾病进展相关。高水平的OPN已被证明可预测CRC的低生存率。自噬是一种自我消化过程，被认为在致癌过程中起重要作用。然而，OPN对CRC细胞自噬的影响机制尚未阐明。因此，我们旨在研究OPN影响CRC自噬的可能机制。
分别通过细胞计数试剂盒-8法、流式细胞术、伤口愈合试验和Transwell小室侵袭试验鉴定HCT116细胞的增殖、凋亡以及迁移和侵袭能力。通过蛋白质免疫印迹法分析蛋白质LC3-II/LC3-I、P62和Atg7的比例。通过逆转录-聚合酶链反应（RT-PCR）和蛋白质免疫印迹法分析并比较Beclin-1、Atg4b、Bnip3和Vps34在转录和翻译水平的表达。采用免疫荧光和共聚焦实验研究自噬体的形成。
结果表明，OPN可促进细胞增殖、迁移和侵袭，同时抑制细胞凋亡。还证明OPN可抑制细胞自噬。进一步实验表明，在HCT116细胞中阻断p38丝裂原活化蛋白激酶（MAPK）途径可逆转OPN对自噬的抑制作用。
OPN参与HCT116细胞进展，可能通过激活p38 MAPK信号通路抑制细胞自噬，这意味着OPN可能是CRC患者潜在的新型分子治疗生物标志物。
