PFKFB3促进腺样囊性癌ACC-2细胞增殖、迁移、侵袭和转移

PFKFB3 promotes cell proliferation,migration,invasion and metastasis in adenoid cystic carcinoma ACC-2 cells

目的研究腺样囊性癌中糖酵解限速酶6-磷酸果糖2激酶/果糖-2,6-二磷酸酶3(PFKFB3)的表达水平,及其对腺样囊性癌细胞增殖、迁移、体内成瘤的影响。方法应用免疫组织化学技术和Western blotting检测29例腺样囊性癌和10例癌旁正常唾液腺组织中PFKFB3的表达水平。腺病毒载体沉默腺样囊性癌ACC-2细胞中PFKFB3,通过细胞增殖实验、细胞划痕实验、Transwell小室实验、裸鼠移植瘤模型和裸鼠肺转移模型观察PFKFB3沉默对细胞生长、迁移、侵袭、体内成瘤和远处转移的影响。结果免疫组织化学染色结果显示,PFKFB3在腺样囊性癌组织中阳性率为93.1%(27/29),在正常唾液腺中阳性率为20.0%(2/10),差异有统计学意义(χ^2=20.84,P<0.001)。四甲基偶氮唑蓝(MTT)增殖实验结果显示,72 h时,PFKFB3沉默后的腺样囊性癌ACC-2(PFKFB3--ACC-2)细胞组吸光度为1.8±0.2,明显低于ACC-2细胞组吸光度4.7±0.8,差异具有统计学意义(t=3.582,P=0.001)。细胞划痕实验结果表明,刮除细胞24 h后,PFKFB3--ACC-2细胞组划痕区细胞数量为(99.8±13.2)个,低于ACC-2组[(263.0±97.4)个],差异具有统计学意义(t=2.868,P=0.029)。Transwell小室检测结果发现,24 h时穿过基质胶的PFKFB3--ACC-2细胞数量[(17.6±2.1)个]明显少于ACC-2细胞[(28.6±3.8)个],差异有统计学意义(t=4.452,P=0.004)。PFKFB3--ACC-2细胞组肿瘤体积为(623.5±134.1)mm^3,明显小于ACC-2细胞组肿瘤体积[(1 621.0±278.1)mm^3],差异有统计学意义(t=4.213,P=0.001)。ACC-2细胞组裸鼠肺部转移灶数目为(18.1±3.2)个,明显多于PFKFB3--ACC-2细胞组[(4.1±2.2)个,t=6.322,P=0.001]。结论腺样囊性癌组织中PFKFB3表达水平明显高于正常唾液腺组织,且高表达的PFKFB3在腺样囊性癌细胞增殖、迁移、侵袭和转移等生物学行为中起促进作用。

Objective To evaluate the expression of 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) in adenoid cystic carcinoma and the effects of PFKFB3 on the proliferation, migration, invasion and tumorigenesis in vivo of adenoid cystic carcinoma cells.Methods Immunohistochemistry and Western blotting were applied to detect the expression of PFKFB3 in 29 cases of adenoid cystic carcinoma tissues and 10 cases of para-carcinoma normal salivary gland tissues. PFKFB3 in adenoid cystic carcinoma ACC-2 cells was silenced by adenovirus vector. The effects of PFKFB3 silence on cell proliferation, migration, invasion and tumorigenesis in vivo and distant metastasis of adenoid cystic carcinoma cells were evaluated by cell proliferation assay, wound healing assay, Transwell assay, xenografted mice model and lung metastasis mice model.Results The results of immunohistochemical staining showed that the positive expression rate of PFKFB3 in adenoid cystic carcinoma tissues was 93.1% (27/29), and the positive expression rate of PFKFB3 in normal salivary gland tissues was 20.0% (2/10), with a significant difference (χ^2=20.84, P<0.001). The results of methyl thiazolyl tetrazolium (MTT) assay showed that, compared with ACC-2 group, the proliferation activity of cancer cells with silence of PFKFB3 (PFKFB3--ACC-2) was significantly suppressed for 72 h (1.8±0.2 vs. 4.7±0.8, t=3.582, P=0.001). The results of wound healing assay showed that after scraping cells away for 24 h, the number of cells in the scratch area was 99.8±13.2 in the PFKFB3--ACC-2 group, which was significantly less than that in the ACC-2 group (263.0±97.4, t=2.868, P=0.029). Transwell results indicated that the number of cells passing through matrigel was 17.6±2.1 in the PFKFB3--ACC-2 group, which was less than that in the ACC-2 group (28.6±3.8, t=4.452, P=0.004). The tumor volume in the PFKFB3--ACC-2 [(623.5±134.1) mm3] was smaller than that in the ACC-2 group [(1621.0±278.1) mm^3,t=4.213, P=0.001]. More pulmonary metastases were found in the ACC-2 group than PFKFB3--ACC-2 group (18.1±3.2 vs. 4.1±2.2, t=6.322, P=0.001).Conclusion The expression of PFKFB3 is higher in adenoid cystic carcinoma tissue than normal salivary gland tissue, and the highly expressed PFKFB3 plays a driving role in the proliferation, migration, invasion and metastasis in adenoid cystic carcinoma.