背景:乳腺癌是全世界女性最常见的肿瘤。糖尿病是一种全球性的慢性代谢性疾病,发病率逐年上升。据报道,糖尿病可以积极调节许多肿瘤的发展。然而,高血糖环境调节乳腺癌的具体机制尚不清楚。PFKFB3(6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶3)是糖尿病糖酵解过程的关键调节因子,以及乳腺癌的启动子.所以,我们希望在本研究中探讨PFKFB3与伴有高血糖的乳腺癌患者的不良预后之间的潜在联系.
方法:细胞培养物用于构建不同葡萄糖的乳腺癌细胞系。采用免疫组织化学方法分析良性乳腺组织中PFKFB3蛋白水平,浸润性导管癌伴糖尿病和浸润性导管癌不伴糖尿病。采用Kaplan-Meier绘图仪数据库和GEO数据库(GSE61304)分析不同PFKFB3表达的乳腺癌患者的生存期。采用Westernblot方法分析乳腺癌细胞中PFKFB3、上皮间质转化(EMT)相关蛋白和细胞外调节蛋白激酶(ERK)的蛋白水平。利用基因集癌症分析(GSCA)来研究PFKFB3的潜在下游信号传导途径。TargetScan和OncomiR用于探索高血糖导致PFKFB3过度表达的潜在机制。利用转染(包括siRNA和miRNA转染引物)来抑制或模拟相应RNA的表达。细胞功能测定(包括细胞计数,MTT,菌落形成,伤口愈合,和细胞迁移测定)用于探索乳腺癌细胞的增殖和迁移。
结果:在这项研究中,我们通过体外细胞实验和组织学实验证明,PFKFB3在合并高血糖的乳腺癌中的表达高于正常血糖的乳腺癌。PFKFB3的过表达降低了乳腺癌患者的生存期,并与乳腺癌合并糖尿病的许多临床病理参数相关。PFKFB3在高血糖环境中促进乳腺癌的增殖和迁移,可能受miR-26的调控。此外,PFKFB3在高血糖环境中刺激乳腺癌的上皮-间质转化。在下游机制探索方面,我们预测并验证了PFKFB3通过RAS/MAPK通路对乳腺癌合并高血糖的促癌作用。
结论:结论:PFKFB3可在高血糖状态下过度表达,可能成为乳腺癌合并糖尿病的潜在治疗靶点。
BACKGROUND: Breast cancer is the most common tumor in women worldwide. Diabetes mellitus is a global chronic metabolic disease with increasing incidence. Diabetes mellitus has been reported to positively regulate the development of many tumors. However, the specific mechanism of hyperglycemic environment regulating breast cancer remains unclear. PFKFB3 (6-phosphofructose-2-kinase/fructose-2, 6-bisphosphatase 3) is a key regulatory factor of the glycolysis process in diabetes mellitus, as well as a promoter of breast cancer. So, we want to explore the potential link between PFKFB3 and the poor prognosis of breast cancer patients with hyperglycemia in this study.
METHODS: Cell culture was utilized to construct different-glucose breast cancer cell lines. Immunohistochemistry was adopted to analyze the protein level of PFKFB3 in benign breast tissues, invasive ductal carcinoma with diabetes and invasive ductal carcinoma without diabetes. The Kaplan-Meier plotter database and GEO database (GSE61304) was adopted to analyze the survival of breast cancer patients with different PFKFB3 expression. Western blot was adopted to analyze the protein level of PFKFB3, epithelial-mesenchymal transition (EMT)-related protein and extracellular regulated protein kinases (ERK) in breast cancer cells. Gene Set Cancer Analysis (GSCA) was utilized to investigate the potential downstream signaling pathways of PFKFB3. TargetScan and OncomiR were utilized to explore the potential mechanism of PFKFB3 overexpression by hyperglycemia. Transfections (including siRNAs and miRNA transfection premiers) was utilized to restrain or mimic the expression of the corresponding RNA. Cell functional assays (including cell counting, MTT, colony formation, wound-healing, and cell migration assays) were utilized to explore the proliferation and migration of breast cancer cells.
RESULTS: In this study, we demonstrated that the expression of PFKFB3 in breast cancer complicated with hyperglycemia was higher than that in breast cancer with euglycemia through cell experiment in vitro and histological experiment. PFKFB3 overexpression decreased the survival period of breast cancer patients and was correlated with a number of clinicopathological parameters of breast cancer complicated with diabetes. PFKFB3 promoted the proliferation and migration of breast cancer in a hyperglycemic environment and might be regulated by miR-26. In addition, PFKFB3 stimulated epithelial-mesenchymal transition of breast cancer in a hyperglycemic environment. In terms of downstream mechanism exploration, we predicted and verified the cancer-promoting effect of PFKFB3 in breast cancer complicated with hyperglycemia through RAS/MAPK pathway.
CONCLUSIONS: In conclusion, PFKFB3 could be overexpressed by hyperglycemia and might be a potential therapeutic target for breast cancer complicated with diabetes.