CLSTN3在结直肠癌中的促癌作用及其与淀粉样前体蛋白的相互作用研究

Oncogenic role of CLSTN3 in colorectal cancer and its interaction with amyloid precursor protein

摘要:

背景与目的 CLSTN3是近年来新发现的结直肠癌（CRC）候选生物标志物，但其在CRC中的表达特征、临床意义及作用机制尚不明确。本研究探讨CLSTN3在CRC中的表达、预后价值及其与淀粉样前体蛋白（APP）的相互作用关系。方法 收集313例CRC石蜡组织及17例新鲜组织标本，采用免疫组织化学及Western blot检测CLSTN3和APP表达，并分析其与临床病理特征及预后的关系。构建CLSTN3过表达HCT8细胞和CLSTN3敲减HCT116细胞模型，通过CCK-8实验及裸鼠皮下成瘤实验评估CLSTN3对CRC细胞增殖能力的影响。利用GeneMANIA数据库预测CLSTN3相关互作分子，并采用免疫共沉淀验证CLSTN3与APP的相互作用。结果 CLSTN3在CRC组织及细胞系中的表达均明显高于正常组织及正常结肠细胞（均P<0.05）。免疫组化结果显示，CLSTN3在CRC组织中的阳性表达率明显高于癌旁组织（68.7% vs. 27.2%）。CLSTN3高表达与TNM分期、肿瘤坏死及神经脉管侵犯密切相关（均P<0.05）。Kaplan-Meier生存分析显示，CLSTN3高表达患者的总体生存（OS）及无病生存（DFS）均明显降低（均P<0.001）。多因素Cox分析表明，CLSTN3高表达是CRC患者OS及DFS的独立危险因素。体外及体内实验结果显示，CLSTN3过表达可显著促进CRC细胞增殖及移植瘤生长，而CLSTN3敲减则明显抑制肿瘤生长。生物信息学分析提示APP可能是CLSTN3的重要互作分子。免疫共沉淀证实CLSTN3与APP在CRC细胞中存在直接相互作用。Western blot结果显示，CRC组织中CLSTN3与APP表达呈正相关（r=0.604，P=0.012），且二者在伴肿瘤坏死组织中的表达水平更高。结论 CLSTN3在CRC中异常高表达，并通过促进肿瘤细胞生长参与CRC进展。CLSTN3高表达提示不良预后，其可能通过与APP相互作用发挥促癌效应。CLSTN3有望成为CRC新的预后标志物及潜在治疗靶点。

Abstract:

Background and Aims CLSTN3 has recently been identified as a potential biomarker for colorectal cancer (CRC); however, its expression pattern, clinical significance, and underlying mechanisms in CRC remain unclear. This study aimed to investigate the biological role and prognostic value of CLSTN3 and its interaction with amyloid precursor protein (APP) in CRC.Methods A total of 313 paraffin-embedded CRC specimens and 17 fresh CRC tissue samples were collected. Immunohistochemistry and Western blot analyses were performed to evaluate the expression of CLSTN3 and APP and their associations with clinicopathological characteristics and prognosis. HCT8 cells with CLSTN3 overexpression and HCT116 cells with CLSTN3 knockdown were established. Cell proliferation was assessed using CCK-8 assays and nude mouse xenograft experiments. Potential interacting molecules of CLSTN3 were predicted using the GeneMANIA database, and the interaction between CLSTN3 and APP was validated by co-immunoprecipitation assays.Results CLSTN3 expression was significantly increased in CRC tissues and cell lines compared with normal controls (all P<0.05). Immunohistochemistry demonstrated that the positive expression rate of CLSTN3 was markedly higher in CRC tissues than in adjacent tissues (68.7% vs. 27.2%). High CLSTN3 expression was significantly associated with TNM stage, tumor necrosis, and neurovascular invasion (all P<0.05). Kaplan-Meier analysis showed that patients with high CLSTN3 expression had significantly worse overall survival (OS) and disease-free survival (DFS) (both P<0.001). Multivariate Cox regression analysis identified CLSTN3 overexpression as an independent prognostic factor for both OS and DFS in CRC patients. Functional experiments demonstrated that CLSTN3 overexpression significantly promoted CRC cell proliferation and tumor growth in vivo, whereas CLSTN3 knockdown inhibited tumor growth. Bioinformatics analysis suggested APP as a key interacting molecule of CLSTN3. Co-immunoprecipitation assays confirmed a direct interaction between CLSTN3 and APP in CRC cells. Western blot analysis further showed a positive correlation between CLSTN3 and APP expression in CRC tissues (r=0.604, P=0.012), and both proteins were more highly expressed in tumors with necrosis.Conclusion CLSTN3 is aberrantly upregulated in CRC and promotes tumor progression by enhancing cancer cell growth. High CLSTN3 expression predicts poor prognosis and may exert oncogenic effects through interaction with APP. CLSTN3 may serve as a novel prognostic biomarker and potential therapeutic target for CRC.