巨噬细胞移动抑制因子促进有氧糖酵解与直肠癌细胞耐药性的关系

Relationship of macrophage migration inhibition factor enhancing aerobic glycolysis to drug resistance of colorectal cancer cells

目的:探讨巨噬细胞移动抑制因子(MIF)在结直肠癌细胞耐药中的作用和机制。方法:用5-氟尿嘧啶(5-FU)药物浓度持续递增法诱导人结直肠癌细胞株LoVo细胞构建5-FU耐药的人结肠癌LoVo/5-FU细胞。检测指标包括细胞对5-FU的敏感性(IC_(50))、细胞MIF蛋白表达、细胞葡萄糖摄取水平、细胞中乳酸脱氢酶(LDH)活性、细胞培养基上清中乳酸水平,分别用CCK-8法、Western blot、2-NBDG法、微孔法、试剂盒法检测。比较LoVo/5-FU细胞与亲本LoVo细胞间上述指标的差异,并检测用siRNA或慢病毒技术干扰或过表达MIF蛋白,或用PFKFB3抑制剂PFK-15抑制有氧糖酵解后,LoVo/5-FU细胞上述指标的变化。结果:成功构建LoVo/5-FU细胞,该细胞的MIF蛋白表达、对5-FU的IC_(50)、葡萄糖摄取、LDH活性和乳酸生成水平都较其亲本LoVo细胞明显升高(均P<0.05);在LoVo/5-FU细胞上,siRNA干扰MIF后表现为MIF蛋白表达、对5-FU的IC_(50)、葡萄糖摄取、LDH活性和乳酸生成水平均明显减少,而MIF蛋白过表达后表现为上述指标的明显升高(均P<0.05);用PFK-15抑制有氧糖酵解后,LoVo/5-FU细胞对5-FU的IC_(50)、葡萄糖摄取、乳酸水平均明显降低(均P<0.05),但LDH活性无明显变化(P>0.05),PFK-15对MIF过表达的LoVo/5-FU细胞也有同样作用(均P<0.05)。结论:MIF通过上调LoVo细胞有氧糖酵解,诱导其对5-FU耐药能力的增加。

Objective： To investigation the action and mechanism ofmacrophage migration inhibition factor （MIF） in drug resistance of colorectal cancer. Methods： The 5-fluorouracil （5-FU）-resistant human colon cancer LoVo/5-FU cells were established by stepwise exposure of human colon cancer LoVo cells to increasing concentrations of 5-FU. The studied parameters included the sensitivity of cells to 5-FU （ICS0）, MIF protein expression, glucose uptake ability of cells, lactic dehydrogenase （LDH） activity of cells and lactate production from cultured cells supernatant, which were detected by CCK-8 assa）5 Western blot analysis, 2-NBDG method, microporous assay and kit assay, respectively. q-he differences in above parameters between LoVo/5-FU cells and their parent LoVo cells were compared, and changes in these parameters in LoVo/5-FU cells before and after MIF interference and overexpression by siRNA and lentivirus transfection, or inhibition of aerobic glycolysis by PFKFB3 inhibitor PFK-15 were examined. Results： The LoVo/5-FU cells were successfully constructed, which showed significantly increased MIF protein expression, ICs0 to 5-FU, glucose uptake, LDH activity and lactate production level compared with their parent LoVo cells （all P〈0.05）. In LoVo/5-FU ceils, the ICs0 to 5-FU, glucose uptake, LDH activity and lactate production level were significantly decreased after MIF interference by siRNA, while those above parameters were significantly increased after MIF overexpression （all P〈0.05）. After inhibition of aerobic glycolysis by PFK-15, the ICs0 to 5-FU, glucose uptake and lactate production level in LoVo/5-FU cells were significantly decreased （all P〈0.05）, but the LDH activity showed no significant change （P〉0.05）, and the same effects were exerted by PFK- 15 in LoVo/5-FU cells with MIF overexpression （all P〈0.05）. Conclusion： MIF enhanced the resistance of LoVo cells to 5-FU by increasing the aerobic glycolysis.