甲状腺乳头状癌组织PFKFB3表达及其意义

Expression and effects of PFKFB3 in thyroid papillary carcinoma

目的肿瘤细胞产生能力的方式是糖酵解,且糖酵解与恶性肿瘤的发生、发展及转移关系非常密切。本研究的目的是评估甲状腺中糖酵解限速酶6磷酸果糖2激酶/果糖-2,6-二磷酸酶(6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3,PFKFB3)的表达水平,及其在甲状腺细胞生长、迁移、体内成瘤的影响,以期为甲状腺治疗提供新思路。方法收集2015-01-01-2016-12-31右江民族医学院附属医院病理科存档蜡块标本28例及11例癌周正常甲状腺组织标本。应用免疫组织化学技术检测甲状腺和正常甲状腺组织中PFKFB3的表达。采用PFKFB3的抑制剂PFK15抑制甲状腺细胞PFKFB3的表达,并观察PFKFB3抑制对甲状腺癌细胞生长、迁移、体内成瘤的影响。结果免疫组化结果显示,PFKFB3在甲状腺组织中表达阳性率达85.7%(24/28),明显高于正常甲状腺组织中的PFKFB3的18.2%(2/11),F=3.24,P<0.001。蛋白质印迹检测结果同样证实,甲状腺癌组织中PFKFB3蛋白表达水平明显高于正常甲状腺组织。MTT试验表明,与对照组(1.1±0.12)相比,0.25(0.82±0.08)、2.5(0.61±0.16)和5μmol/L(0.15±0.05)PFK15可明显抑制SW579细胞的增殖活性(F值分别为3.47、2.35和3.77,均P<0.05)和增殖相关分子的表达。细胞划痕实验结果显示,刮除细胞24h后,0.25、2.5和5μmol/L PFK15处理组的SW579细胞向划痕区域迁移,但其划痕区域的细胞数量分别为86±14(F=2.59,P<0.001)、57±12(F=3.52,P<0.001)和46±8(F=2.15,P<0.001),明显少于对照组的102±13。Transwell小室表明,0.25、2.5和5μmol/L PFK15处理组中,穿过基质胶的SW579细胞数量分别为71±6(F=2.35,P<0.001)、58±11(F=3.68,P<0.001)和41±7(F=2.38,P<0.001),明显少于对照组的91±8。裸鼠移植瘤实验结果显示,注射浓度为1[(661±168)mm^3,F=3.42,P<0.01]和5mg/kg[(514±147)mm^3,F=2.87,P<0.001)]组裸鼠的肿瘤体积明显小于对照组的(924±254)mm^3,且0.25、2.5和5μmol/L PFK15组肿瘤质量[(201±74)mg,F=1.27,P>0.05;(142±42)mg,F=3.56,P<0.01;(112±32)mg,F=3.57,P<0.001]明显小于对照组的(223±87)mg。裸鼠肺转移模型结果显示,0.25、2.5和5μmol/L PFK15组裸鼠肺部转移灶数目[(12±4)个,F=3.66,P<0.05;(4±3)个,F=2.86,P<0.01;(2±1.5)个,F=3.31,P<0.001]明显少于对照组的(16±5)个。结论 PFKFB3在甲状腺组织中高表达,且在甲状腺癌的增殖、迁移、侵袭和转移中起重要作用。抑制PFKFB3可作为甲状腺生物治疗的潜在分子靶点。

OBJECTIVE Tumor cells manage their energy production in a most peculiar way,glycolysis.Importantly,glycolysis is closely related to tumorigenesis,tumor development and tumor metastasis.The study aimed to investigate the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3（PFKFB3）in thyroid cancer,and to detect the role of PFKFB3 in the proliferation,migration,invasion and tumorigenesis of thyroid carcinoma cells.METHODS Totally 28 thyroid cancer paraffin block specimens and 11 cancer peripheral normal thyroid tissue were collected from a 1 st January 2015 to 31 st December 2016 in the the Affiliated Hospital,Youjiang Medical University for Nationalities.Immunohistochemistry staining was applied to detect the expression of PFKFB3 in thyroid carcinoma and normal thyroid tissue.The roles of PFKFB3 in the proliferation,migration,invasion and tumorigenesis of thyroid carcinoma cells were determined when PFKFB3 was blocked by PFK15.RESULTS The results of IHC staining showed that positive expression of PFKFB3（85.7%）were enhanced significantly in thyroid carcinoma when compared with normal thyroid（18.2%,F=3.24,P〈0.001）.The enhanced expression of PFKFB3 was confirmed by the results of western blot.The results of MTT assay showed that,compared with control group（1.1±0.12）,the proliferation activity of thyroid cancer cells was significantly suppressed by treatment with PFK15 [PFK15（0.25μmol/L）,0.82±0.08,F =3.47,P〈0.05;PFK15（2.5μmol/L）,0.61±0.16,F=2.35,P〈0.001;PFK15（5μmol/L）,0.15±0.05,F=3.77,P〈0.001].The expression of proliferative molecules were also inhibited by PFK15.The number of cells migrated to the scratch area in the PFK15 treated groups[PFK15（0.25μmol/L）,86±14,F=2.59,P〈0.001;PFK15（2.5μmol/L）,57±12,F=3.52,P〈0.001;PFK15（5μmol/L）,46±8,F=2.15,P〈0.001]were significantly less than control group（102±13）.Addition of PFK15 to the upper compartment transwell invasion model [PFK15（0.25μmol/L）,71±6,F =2.35,P〈0.001;PFK15（2.5μmol/L）,58±11,F=3.68,P〈0.001;PFK15（5μmol/L）,41±7,F=2.38,P〈0.001]resulted in the significant inhibition of the cancer cell invasion in reconstituted basement membrane when compared with control group（91±8）.In cancer bearing mice model,PFK15 significantly inhibited the development of cancer[（1 mg/kg,（661±168）mm^3,F=3.42,P〈0.01;5 mg/kg,（514±147）mm^3,F=2.87,P〈0.001 vs control,（924±254）mm^3].The tumor weight of PFK15 treated group[PFK15（0.25μmol/L）,（201±74）mg,F=1.27,P〉0.05;PFK15（2.5μmol/L）,（142±42）mg,F=3.56,P〈0.01;PFK15（5μmol/L）,（112±32）mg,F=3.57,P〈0.001]were significantly less than the control group（223±87）mg].In pulmonary metastasis model,PFK15 significantly reduced the lung metastasis nodule [PFK15（0.25μmol/L）,（12±4）mg,F=3.66,P〈0.05;PFK15（2.5μmol/L）,（4±3）mg,F=2.86,P〈0.01;PFK15（5μmol/L）,（2±1.5）mg,F=3.31,P〈0.001;vs control group（16±5）mg].CONCLUSIONS PFKFB3 was highly expressed in thyroid carcinoma and the enhanced PFKFB3 may play an important role in the development of thyroid carcinoma.Blockage of PFKFB3 offered a promising strategy for the treatment of thyroid carcinoma.