摘要
目的探讨泛素蛋白连接酶L3(UBE2L3)对肺腺癌细胞有氧糖酵解的作用及其分子机制。方法通过生物信息学方法分析UBE2L3 mRNA表达水平与肺腺癌患者的总生存期的关系以及对肺腺癌组织中有氧糖酵解水平的影响。选择UBE2L3表达水平较低的HCC827细胞为研究对象,构建过表达质粒细胞HCC827/UBE2L3及HCC827/NC;选择UBE2L3表达水平较高的H1299细胞为研究对象,构建敲低细胞H1299/UBE2L3及H1299/NC,收集细胞用于后续实验。采用CCK-8法检测细胞增殖能力,采用葡萄糖测定试剂盒测定葡萄糖消耗量,采用乳酸检测试剂盒测定乳酸生成量,采用Western blot检测有氧糖酵解关键蛋白M2-型丙酮酸激酶(PKM2)、L-乳酸脱氢酶(LDHA)表达水平。另外再通过磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)抑制剂Y294002阻断PI3K/Akt信号通路后检测细胞有氧糖酵解的变化。结果肺腺癌组织中UBE2L3蛋白表达水平高于正常肺组织(P<0.05)。UBE2L3高表达组患者总生存期低于低表达组(P<0.05)。UBE2L3 mRNA表达水平与LDHA和PKM2 mRNA表达水平均呈正相关(r=0.258和0.404,均P<0.01)。HCC827/UBE2L3组细胞在48、72、96 h时增殖能力均高于HCC827/NC组(均P<0.05),H1299/si-NC组细胞在48、72、96 h时增殖能力均高于H1299/si-UBE2L3组(均P<0.05)。HCC827/UBE2L3组细胞葡萄糖消耗量、乳酸生成量均大于HCC827/NC组,PKM2和LDHA蛋白表达水平均高于HCC827/NC组;H1299/si-UBE2L3组细胞葡萄糖消耗量、乳酸生成量均小于H1299/si-NC组,PKM2、LDHA蛋白表达水平均低于H1299/si-NC组,差异均有统计学意义(均P<0.05)。与HCC827/UBE2L3+二甲基亚砜组相比,经过抑制剂Y294002处理的HCC827/UBE2L3细胞增殖能力下降,Akt活性被抑制,磷酸化Akt、LDHA和PKM2蛋白表达水平均下降,葡萄糖消耗量、乳酸生成量均下降。结论UBE2L3可以通过激活PI3K/Akt信号通路从而促进肺腺癌细胞的有氧糖酵解过程。
Objective To investigate the effect of UBE2L3 on aerobic glycolysis in lung adenocarcinoma cells and its molecular mechanism.Methods Bioinformatics was used to analyze the relationship between UBE2L3 mRNA expression level and overall survival of lung adenocarcinoma patients and the effect on aerobic glycolysis level in lung adenocarcinoma tissues.UBE2L3-overexpresssing HCC827 cell line(HCC827/UBE2L3)and UBE2L3-knockdown cell line(H1299/si-UBE2L3)were constructed;HCC827/NC and H1299/si-NC cells were used for controls,respectively.Cell proliferation capacity was determined by CCK-8 method,glucose consumption was determined by glucose assay kit,lactic acid production was determined by lactic acid assay kit,and expression levels of key proteins of aerobic glycolysis pyruvate kinase M2(PKM2)and lactate dehydrogenase A(LDHA)were detected by Western blot.In addition,phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt)signaling pathway was blocked by PI3K inhibitor Y294002,and the changes of aerobic glycolysis in cells were detected.Results Bioimformatics analysis showed that UBE2L3 protein expression level in lung adenocarcinoma tissue was higher than that in normal lung tissue(P<0.05);the overall survival of high expression group was lower than that of low expression group(P<0.05);the mRNA expression levels of UBE2L3 were positively correlated with those of LDHA and PKM2(r=0.258 and 0.404,both P<0.01).The proliferating ability of HCC827/UBE2L3 cells was higher than that in HCC827/NC cells at 48,72 and 96 h(all P<0.05),and proliferating ability of H1299/si-NC cells was higher than that in H1299/si-UBE2L3 cells at48,72 and 96 h(all P<0.05).The glucose consumption and lactic acid production in HCC827/UBE2L3 cells were higher than those in HCC827/NC cells,and the protein expression levels of PKM2 and LDHA in HCC827/NC cells were higher than those in HCC827/NC cells.The glucose consumption and lactic acid production in H1299/si-UBE2L3 cells were lower than those in H1299/si-NC cells,and the protein levels of PKM2 and LDHA in H1299/si-NC cells were lower than those in H1299/Si-NC cells(all P<0.05).Compared with the HCC827/UBE2L3+DMSO group,the proliferation ability of HCC827/UBE2L3 cells treated with inhibitor Y294002 was decreased,the activity of Akt was inhibited,the protein expression levels of p-Akt,LDHA and PKM2were decreased,glucose consumption and lactic acid production were also decreased.Conclusion UBE2L3 can promote aerobic glycolysis of lung adenocarcinoma cells by activating PI3K/Akt signaling pathway.
作者
潘欢
杨帆
戚维波
马兴杰
PAN Huan;YANG Fan;QI Weibo;MA Xingjie(Central Laboratory,the First Hospital of Jiaxing,Jiaxing 314000,China;不详)
出处
《浙江医学》
CAS
2023年第1期14-20,共7页
Zhejiang Medical Journal
基金
浙江省自然科学基金项目(LQ20H160058)
嘉兴市科技计划项目(2019AD32250)。
