丙酮酸激酶M2亚型在肾癌迁移侵袭中的作用及其机制  

Role of pyruvate kinase type M2 in the migration and invasion of renal cell carcinoma and possible mechanisms

作　　者：王新君[1] 白培明[1] 赵晓昆[2] 罗广承[1] 

机构地区：[1]厦门大学附属中山医院泌尿外科,361004 [2]中南大学湘雅二医院泌尿外科,长沙410011

出　　处：《中华实验外科杂志》2017年第12期2057-2059,共3页Chinese Journal of Experimental Surgery

基　　金：福建省卫生和计划生育委员会青年科研课题（2015-2-47）

摘　　要：目的探讨敲低丙酮酸激酶M2亚型（PKM2）对肾癌细胞株ACHN迁移侵袭的影响及其机制。方法构建靶向PKM2基因的pLV载体，筛选稳定转染的细胞株。采用细胞计数试剂盒（CCK-8）法检测敲低PKM2对ACHN细胞增殖的影响。在Transwell小室的上室和下室之间不放置基质胶测定细胞的迁移能力，在Transwell小室的上室和下室之放置基质胶测定细胞的侵袭能力。通过实时定量反转录聚合酶链反应（RT-qPCR）和Western blot的方法检测E-钙黏蛋白（E-cadherin）和波形蛋白（Vimentin）的变化。结果CCK-8实验结果显示敲低PKM2抑制了ACHN的增殖。对照组的葡萄糖消耗率和乳酸生成率分别为（3.493±0.179）和（3.553±0.185） nmol/106 cells/min，而shPKM2组的葡萄糖消耗率和乳酸生成率分别为（1.457±0.081）和（5.820±0.352） nmol/106 cells/min（P＝0.001，P＝0.005）。Transwell迁移实验结果显示shPKM2组和对照组到达下室的细胞数分别为（62±6）个和（39±5）个（P＝0.001）。Transwell侵袭实验结果显示shPKM2组和对照组到达下室的细胞数分别为（40±7）个和（26±6）个（P＝0.009）。通过Western blot和RT-qPCR检测shPKM2组中E-cadherin蛋白和mRNA的相对表达量分别为1.560±0.110和2.534±0.342，而对照组中E-cadherin蛋白和mRNA的相对表达量分别为0.833±0.070和1.312±0.284（P＝0.001，P＝0.009），shPKM2组中Vimentin蛋白和mRNA的相对表达量分别为0.340±0.110和1.631±0.123，而对照组中Vimentin蛋白和mRNA的相对表达量为0.653±0.100和3.530±0.511（P＝0.022，P＝0.003）。结论敲低PKM2表达显著抑制了肾癌细胞的有氧糖酵解、增殖、迁移和侵袭能力，并影响了上皮-间充质转化（EMT）过程。ObjectiveTo analyze the alterations of glucose metabolism upon the knockdown of pyruvate kinase type M2 （PKM2） in renal cell carcinoma cell line ACHN, and explore the potential role of PKM2 in migration and invasion of renal cell carcinoma and the possible mechnisms.MethodsThe pLV lentivirus vector with PKM2-short hairpin RNA （shRNA） was constructed and the stably expressed cell line was generated. The effects of PKM2 knockdown on proliferation of ACHN cells were detected by cell counting kit-8 （CCK-8） assay, migration and invasion ability detected by Transwell assay, and alteration of Warburg effect detected by measuring glucose uptake and lactate production. Western blotting and real-time reverse transcriptase-polymerase chain reaction （RT-qPCR） were performed to examine the expression of E-cadherin and Vimentin after the knockdown of PKM2.ResultsCCK-8 assay showed that cell viability of shPKM2 group was significantly lower than that in control group. The glucose uptake and lactate production in control group were （3.493±0.179） and （3.553±0.185） nmol/106 cells/min repectively, while those in shPKM2 group were （1.457±0.081） and （5.820±0.352） nmol/106 cells/min （P=0.001, P=0.005）. The number of migrating cells in control and shPKM2 groups was 62±6 and 39±5 （P=0.001）, and that of invasive cells in control and shPKM2 groups was 40±7 and 26±6 （P=0.009）, respectively. The relative expressions of E-cadherin protein and mRNA in shPKM2 group （1.560±0.110 and 2.534±0.342） were higher than that in control group （0.833±0.070 and 1.312±0.284） by Western blotting and RT-qPCR （P=0.001, P=0.009）. The relative expressions of Vimentin protein and mRNA in shPKM2 group （0.340±0.110 and 1.631±0.123） were lower than that in control group （0.653±0.100 and 3.530±0.511, P=0.022, P=0.003）.ConclusionKnockdown of PKM2 can impair the glucose metabolism and suppress cell proliferation, migration and invasion in vitro and in vivo. Knockdown of PKM2 decreases the

关 键 词：肾透明细胞癌 丙酮酸激酶M2亚型 迁移 侵袭 上皮一间充质转化 

分 类 号：R737.11[医药卫生—肿瘤]