1.河南省漯河市中心医院 病理科，河南 漯河 462300;2.漯河医学高等专科学校第二附属医院 病理科;中南大学湘雅医院 3. 癌变机理与靶向治疗研究中心;3.肿瘤蛋白质转化医学湖南省高校重点实验室， 湖南 长沙 410008
杨娟，河南省漯河市中心医院副主任医师，主要从事妇乳及消化肿瘤方面的研究。
国家自然科学青年基金资助项目（82103638）；湖南省自然科学青年基金资助项目（2022JJ40805）。
1.Department of Pathology, Luohe Central Hospital, Luohe, Henan 462300, China;2.Luohe Medical College Second Affiliated Hospital, Luohe, Henan 462300, China;3.Research Center of Carcinogenesis and Targeted Therapy, 4. Higher Educational Key Laboratory for Cancer Proteomics and Translational Medicine of Hunan Province, Xiangya Hospital, Central South University, Changsha 410008, China
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背景与目的 F-BOX蛋白（FBP）家族成员F-box only protein 43（FBXO43）在肝癌和结直肠癌等消化系统肿瘤中高表达，促进肿瘤恶性进展，且研究显示，FBXO43促进p53降解，发挥促瘤功能。为此本研究进一步探讨FBXO43在胃癌中的表达及其在胃癌恶性进展中的功能与相关机制。方法 基于TCGA、GTEx和Kaplan-Meier Plotter等在线数据库，分析FBXO43在胃癌组织中的表达及其与胃癌患者预后的相关性。用Western blot和qPCR检测FBXO43在胃癌细胞与正常胃黏膜上皮细胞中的表达水平；用免疫组化检测在胃癌组织与癌旁组织中FBXO43的蛋白水平。利用脂质体转染特异性靶向FBXO43和p53的小分子干扰RNA分子（siFBXO43和sip53），分别或同时敲低HGC27和MGC803细胞中的FBXO43和p53的表达，利用CCK8、平板克隆形成、Transwell侵袭和迁移等实验，检测细胞生长、增殖、迁移和侵袭能力的影响；利用免疫共沉淀（Co-IP）检测FBXO43和p53的相互作用情况，以及敲低FBXO43后，p53的总泛素化水平。结果 TCGA和GTEx数据显示，FBXO43在胃癌中表达水平明显上调（均P<0.05）；Kaplan-Meier Plotter数据显示，FBXO43高表达的胃癌患者总生存期（HR=1.39，95% CI=1.09~1.78，P=0.007 6）、无进展生存期（HR=1.35，95% CI=1.04~1.76，P=0.023）、进展后生存期（HR=1.6，95% CI=1.18~2.17，P=0.002 1）均显著缩短。Western blot、qPCR和免疫组化结果显示，FBXO43在胃癌细胞和组织中上调，FBXO43蛋白水平与胃癌患者肿瘤大小、远处转移、TNM分期明显有关（均P<0.05）。CCK8、平板克隆形成、Transwell侵袭和迁移结果显示，敲低FBXO43表达，胃癌细胞的增殖、侵袭和迁移能力明显减弱（均P<0.05）。敲低FBXO43表达，上调p53蛋白的水平。Co-IP结果显示，FBXO43与p53可以相互共沉淀，敲低FBXO43，p53的总泛素化水平显著增加。功能实验结果显示，同时敲低p53，FBXO43敲低对胃癌细胞的增殖、侵袭和迁移能力的抑制作用被拮抗，回复胃癌细胞的体外恶性表型（均P<0.05）。结论 FBXO43在胃癌中高表达，与胃癌患者预后不良密切相关；FBXO43的作用机制可能是与p53相互作用，促进p53泛素化和降解，进而发挥促进胃癌细胞恶性进展，FBXO43有望成为胃癌治疗的靶点。
Background and Aims F-box protein (FBP) family member F-box only protein 43 (FBXO43) is highly expressed in digestive system tumors such as liver cancer and colorectal cancer, promoting malignant progression of tumors. Research has shown that FBXO43 promotes the degradation of p53, exerting oncogenic functions. Therefore, this study was conducted to further explore the expression of FBXO43 in gastric cancer and its role and related mechanisms in the malignant progression of gastric cancer.Methods Based on online databases such as TCGA, GTEx, and Kaplan-Meier Plotter, the expression of FBXO43 in gastric cancer tissues and its correlation with the prognosis of gastric cancer patients were analyzed. Western blot and qPCR were used to detect the expression levels of FBXO43 in gastric cancer cells and normal gastric mucosal epithelial cells. Immunohistochemical staining was performed to detect the protein levels of FBXO43 in gastric cancer and adjacent tissues. Specific small interfering RNA molecules targeting FBXO43 and p53 (siFBXO43 and sip53) were transfected into HGC27 and MGC803 cells to knock down the expression of FBXO43 and p53 alone or simultaneously. Cell Counting Kit-8 (CCK8) assay, colony formation assay, Transwell invasion and migration assays were used to detect the effects of FBXO43 knockdown on the proliferation, invasion, and migration abilities of gastric cancer cells. Co-immunoprecipitation (Co-IP) was used to detect the interaction between FBXO43 and p53, as well as the total ubiquitination level of p53 after FBXO43 knockdown.Results TCGA and GTEx data showed that the expression level of FBXO43 was significantly upregulated in gastric cancer (both P<0.05). Kaplan-Meier Plotter data showed that high expression of FBXO43 was significantly associated with shortened overall survival (HR=1.39, 95% CI=1.09-1.78, P=0.007 6), progression-free survival (HR=1.35, 95% CI=1.04-1.76, P=0.023), and post-progression survival (HR=1.6, 95% CI=1.18-2.17, P=0.002 1) of gastric cancer patients. Western blot, qPCR, and immunohistochemistry results showed that FBXO43 was upregulated in gastric cancer cells and tissues, and the protein level of FBXO43 was significantly associated with tumor size, distant metastasis, and TNM stage of gastric cancer patients (all P<0.05). CCK8 assay, colony formation assay, Transwell invasion, and migration assays showed that knockdown of FBXO43 expression significantly inhibited the proliferation, invasion, and migration abilities of gastric cancer cells (all P<0.05). Knockdown of FBXO43 expression upregulated the protein level of p53. Co-IP results showed that FBXO43 and p53 could co-immunoprecipitate with each other, and knockdown of FBXO43 significantly increased the total ubiquitination level of p53. Functional experiments showed that simultaneous knockdown of p53 antagonized the inhibitory effects of FBXO43 knockdown on the proliferation, invasion, and migration abilities of gastric cancer cells, restoring the malignant phenotype of gastric cancer cells in vitro (all P<0.05).Conclusion FBXO43 is highly expressed in gastric cancer and is closely associated with poor prognosis in gastric cancer patients. The mechanism of action of FBXO43 may involve interaction with p53, promoting p53 ubiquitination and degradation, thereby promoting the malignant progression of gastric cancer. FBXO43 is expected to become a therapeutic target for gastric cancer.
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杨娟,刘春梅,吴涵,赵路,卢珊珊. FBXO43在胃癌中的表达及其生物学功能与作用机制[J].中国普通外科杂志,2024,33(4):612-623.
DOI:10.7659/j. issn.1005-6947.2024.04.011
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- 收稿日期:2024-03-15
- 最后修改日期:2024-04-17
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- 在线发布日期: 2024-04-29