STOML2 通过激活 MAPK 信号通路与 PHB 相互作用，促进结直肠癌细胞增殖。

BACKGROUND: Highly expressed STOML2 has been reported in a variety of cancers, yet few have detailed its function and regulatory mechanism. This research aims to reveal regulatory mechanism of STOML2 and to provide evidence for clinical therapeutics, via exploration of its role in colorectal cancer, and identification of its interacting protein. METHODS: Expression level of STOML2 in normal colon and CRC tissue from biobank in Nanfang Hospital was detected by pathologic methods. The malignant proliferation of CRC induced by STOML2 was validated via gain-of-function and loss-of-function experiments, with novel techniques applied, such as organoid culture, orthotopic model and endoscopy monitoring. Yeast two-hybrid assay screened interacting proteins of STOML2, followed by bioinformatics analysis to predict biological function and signaling pathway of candidate proteins. Target protein with most functional similarity to STOML2 was validated with co-immunoprecipitation, and immunofluorescence were conducted to co-localize STOML2 and PHB. Pathway regulated by STOML2 was detected with immunoblotting, and subsequent experimental therapy was conducted with RAF inhibitor Sorafenib. RESULTS: STOML2 was significantly overexpressed in colorectal cancer and its elevation was associated with unfavorable prognosis. Knockdown of STOML2 suppressed proliferation of colorectal cancer, thus attenuated subcutaneous and orthotopic tumor growth, while overexpressed STOML2 promoted proliferation in cell lines and organoids. A list of 13 interacting proteins was screened out by yeast two-hybrid assay. DTYMK and PHB were identified to be most similar to STOML2 according to bioinformatics in terms of biological process and signaling pathways; however, co-immunoprecipitation confirmed interaction between STOML2 and PHB, rather than DTYMK, despite its highest rank in previous analysis. Co-localization between STOML2 and PHB was confirmed in cell lines and tissue level. Furthermore, knockdown of STOML2 downregulated phosphorylation of RAF1, MEK1/2, and ERK1/2 on the MAPK signaling pathway, indicating common pathway activated by STOML2 and PHB in colorectal cancer proliferation. CONCLUSIONS: This study demonstrated that in colorectal cancer, STOML2 expression is elevated and interacts with PHB through activating MAPK signaling pathway, to promote proliferation both in vitro and in vivo. In addition, combination of screening assay and bioinformatics marks great significance in methodology to explore regulatory mechanism of protein of interest.

背景：高度表达的 STOML2 已在多种癌症中被报道，但很少有研究详细阐明其功能和调控机制。本研究旨在通过探索 STOML2 在结直肠癌中的作用及其相互作用蛋白，揭示其调控机制，并为临床治疗提供依据。

方法：通过病理方法检测南方医院生物库中正常结肠和 CRC 组织中 STOML2 的表达水平。应用新型技术，如类器官培养、原位模型和内镜监测，通过 gain-of-function 和 loss-of-function 实验验证 STOML2 诱导 CRC 恶性增殖。应用酵母双杂交筛选 STOML2 的相互作用蛋白，通过生物信息学分析预测候选蛋白的生物学功能和信号通路。与 STOML2 功能最相似的靶蛋白通过 co-immunoprecipitation 验证，免疫荧光检测 STOML2 和 PHB 的共定位。通过免疫印迹检测 STOML2 调控的通路，随后用 RAF 抑制剂索拉非尼进行实验性治疗。

结果：STOML2 在结直肠癌中显著过表达，其升高与不良预后相关。敲低 STOML2 抑制结直肠癌细胞增殖，从而减弱皮下和原位肿瘤生长，而过表达 STOML2 促进细胞系和类器官的增殖。酵母双杂交筛选出 13 个相互作用蛋白。根据生物过程和信号通路的生物信息学分析，DTYMK 和 PHB 被鉴定为与 STOML2 最相似的蛋白；然而，尽管 DTYMK 在之前的分析中排名最高，但 co-immunoprecipitation 证实了 STOML2 与 PHB 而不是 DTYMK 相互作用。在细胞系和组织水平证实了 STOML2 与 PHB 的共定位。此外，敲低 STOML2 下调 MAPK 信号通路中 RAF1、MEK1/2 和 ERK1/2 的磷酸化，表明 STOML2 和 PHB 在结直肠癌细胞增殖中激活共同通路。

结论：本研究表明，在结直肠癌中，STOML2 表达上调，并通过激活 MAPK 信号通路与 PHB 相互作用，促进体外和体内的增殖。此外，筛选实验和生物信息学的结合在探索感兴趣蛋白质的调控机制的方法学上具有重要意义。