沉默干扰素调控因子2降低胰腺癌细胞有氧糖酵解水平的实验研究
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摘要
目的:研究干扰素调控因子2(interferon regulatory factor 2,IRF-2)对胰腺癌细胞有氧糖酵解水平的影响。方法:胰腺癌细胞感染含有IRF-2-shRNA的慢病毒及对照空载体病毒记为干扰组和阴性组,以不做处理的细胞作为对照组,用Realtime PCR和Western blot方法测定细胞中IRF-2水平,MTT方法测定胰腺癌细胞的增殖情况,Western blot方法测定细胞内糖酵解关键酶己糖激酶2(HK2)、丙酮酸激酶M2亚型(PKM2)蛋白、NF-κBp65(NF-κBp65亚型)、细胞核增殖抗原(Ki-67)表达水平,用试剂盒检测细胞乳酸生成及葡萄糖消耗水平,同时检测细胞中三磷酸腺苷(ATP)合成水平。结果:干扰组细胞中的IRF-2 mRNA和蛋白水平均明显低于对照组,而阴性组细胞中IRF-2 mRNA和蛋白水平与对照组比较没有明显差异。干扰组细胞的OD值明显降低,细胞中HK2、PKM2、NF-κBp65、Ki-67蛋白水平降低,同时细胞乳酸生成及葡萄糖消耗量均降低,细胞合成的ATP减少,与对照组相比,差异有统计学意义(P<0.05)。阴性组细胞OD值、HK2蛋白水平、PKM2蛋白水平、Ki-67蛋白水平、NF-κBp65蛋白水平、乳酸生成、葡萄糖消耗量、ATP水平与对照组相比,差异均没有统计学意义(P> 0. 05)。结论:沉默IRF-2抑制胰腺癌细胞中糖酵解关键酶表达,降低细胞糖酵解水平,抑制胰腺癌细胞增殖。
Objective:To study the effect of IRF-2 on aerobic glycolysis in pancreatic cancer cells.Methods:Pancreatic cancer cells infected with lentivirus containing IRF-2-shRNA and control empty virus were classified as interference group and negative group.The cells not treated were used as the control group.The IRF-2 levels in cells were measured by Realtime PCR and Western blot.MTT assay was used to determine the proliferation of pancreatic cancer cells.Western blot method was used to detect the expression levels of HK2,PKM2,NF-κBp65,Ki-67 proteins in glycolysis.The cell lactate production and glucose consumption were detected by kit.At the same time,the level of ATP synthesis in the cells was also detected.Results:The transcriptional and protein levels of IRF-2 in the interference group were significantly lower than those in the control group.The IRF-2 transcription and protein levels in the negative group were not significantly different from those in the control group.The OD values of the cells in the interference group decreased significantly,the level of HK2,PKM2,NF-κBp65,Ki-67 protein in the cells decreased,at the same time,lactic acid generation and glucose consumption decreased,ATP decreased in cell synthesis.Compared with the control group,the difference was statistically significant(P<0.05).The OD value,HK2 protein level,PKM2 protein level,NF-κBp65 protein level,Ki-67 protein level,lactate production,glucose consumption and ATP level in the negative group were compared with those in the control group,and the difference was not statistically significant(P>0.05).Conclusion:Silence IRF-2 inhibits the expression of key glycolysis enzymes in pancreatic cancer cells,reduces the level of glycolysis and inhibits the proliferation of pancreatic cancer cells.
Objective:To study the effect of IRF-2 on aerobic glycolysis in pancreatic cancer cells.Methods:Pancreatic cancer cells infected with lentivirus containing IRF-2-shRNA and control empty virus were classified as interference group and negative group.The cells not treated were used as the control group.The IRF-2 levels in cells were measured by Realtime PCR and Western blot.MTT assay was used to determine the proliferation of pancreatic cancer cells.Western blot method was used to detect the expression levels of HK2,PKM2,NF-κBp65,Ki-67 proteins in glycolysis.The cell lactate production and glucose consumption were detected by kit.At the same time,the level of ATP synthesis in the cells was also detected.Results:The transcriptional and protein levels of IRF-2 in the interference group were significantly lower than those in the control group.The IRF-2 transcription and protein levels in the negative group were not significantly different from those in the control group.The OD values of the cells in the interference group decreased significantly,the level of HK2,PKM2,NF-κBp65,Ki-67 protein in the cells decreased,at the same time,lactic acid generation and glucose consumption decreased,ATP decreased in cell synthesis.Compared with the control group,the difference was statistically significant(P<0.05).The OD value,HK2 protein level,PKM2 protein level,NF-κBp65 protein level,Ki-67 protein level,lactate production,glucose consumption and ATP level in the negative group were compared with those in the control group,and the difference was not statistically significant(P>0.05).Conclusion:Silence IRF-2 inhibits the expression of key glycolysis enzymes in pancreatic cancer cells,reduces the level of glycolysis and inhibits the proliferation of pancreatic cancer cells.
引文
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[23] Xu RH,Pelicano H,Zhou Y,et al.Inhibition of glycolysis in cancer cells:A novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia[J].Cancer Research,2005,65(2):613-621.
[24] Kaplan O,Firon M,Vivi A,et al.HGF/SF activates glycolysis and oxidative phosphorylation in DA3 murine mammary cancer cells[J].Neoplasia,2000,2(4):365-377.
[25] Qin Y,Cheng C,Lu H,et al.miR-4458 suppresses glycolysis and lactate production by directly targeting hexokinase2 in colon cancer cells[J].Biochem Biophys Res Commun,2016,469(1):37-43.
[26] Han DF.p65-PKM2 mediated fenofibrate-induced inhibition of glucose metabolism in human glioblastoma cells[D].Nanjing:Nanjing Medical University,2015.[韩东风.非诺贝特通过抑制p65-PKM2信号通路调控人脑胶质瘤细胞糖酵解[D].南京:南京医科大学,2015.]
[27] Xu Q,Liu LZ,Yin Y,et al.Regulatory circuit of PKM2/NF-κB/miR-148a/152-modulated tumor angiogenesis and cancer progression[J].Oncogene,2015,34(43):5482-5493.
[2] Court CM,Ankeny JS,Sho S,et al.Reality of single circulating tumor cell sequencing for molecular diagnostics in pancreatic cancer[J].Journal of Molecular Diagnostics Jmd,2016,18(5):688-696.
[3] Wai WVK,Hang D,Xu L,et al.Rh2E2,a novel metabolic suppressor,specifically inhibits energy-based metabolism of tumor cells[J].Oncotarget,2016,7(9):9907-9924.
[4] Huang CJ,Chou CM,Lien HW,et al.IRF9-Stat2 fusion protein as an innate immune inducer to activate Mx and interferon-stimulated gene expression in zebrafish larvae[J].Marine Biotechnology,2017,19(3):310-319.
[5] Minamino K,Takahara K,Adachi T,et al.IRF-2 regulates B-cell proliferation and antibody production through distinct mechanisms[J].International Immunology,2012,24(9):573-581.
[6] Connett JM,Badri L,Giordano TJ,et al.Interferon regulatory factor 1 (IRF-1) and IRF-2 expression in breast cancer tissue microarrays[J].Journal of Interferon & Cytokine Research the Official Journal of the International Society for Interferon & Cytokine Research,2005,25(10):587-594.
[7] Wang Y,Liu DP,Chen PP,et al.Involvement of IFN regulatory factor (IRF)-1 and IRF-2 in the formation and progression of human esophageal cancers[J].Cancer Research,2007,67(6):2535-2543.
[8] Cui L,Deng Y,Rong Y,et al.IRF-2 is over-expressed in pancreatic cancer and promotes the growth of pancreatic cancer cells[J].Tumour Biology the Journal of the International Society for Oncodevelopmental Biology & Medicine,2012,33(1):247-255.
[9] Chattopadhyay S,Kuzmanovic T,Ying Z,et al.Ubiquitination of the transcription factor IRF-3 activates RIPA,the apoptotic pathway that protects mice from viral pathogenesis[J].Immunity,2016,44(5):1151-1161.
[10] Schwartz JA,Clayton KL,Mujib S,et al.Tim-3 is a marker of plasmacytoid dendritic cell dysfunction during HIV infection and is associated with the recruitment of IRF7 and p85 into lysosomes and with the submembrane displacement of TLR9[J].Journal of Immunology,2017,198(8):3181-3194.
[11] Li Y,Huang R,Wang L,et al.MicroRNA-762 promotes breast cancer cell proliferation and invasion by targeting IRF7 expression[J].Cell Proliferation,2016,48(6):643-649.
[12] Wang Y.The role of IRF and CCN in the development of esophageal and lung cancer [D].Shanghai:Institute of Nutrition Science,Shanghai Academy of Life Sciences,Chinese Academy of Sciences,and the Shanghai Institute of Life Sciences,Chinese Academy of Sciences,2008.[王岩.IRF及CCN在食管癌和肺癌发生发展中的作用[D].上海:中国科学院上海生命科学研究院营养科学研究所,中国科学院上海生命科学研究院,2008.]
[13] Zhang M,Zhang L,Cui M,et al.miR-302b inhibits cancer-related inflammation by targeting ERBB4,IRF2 and CXCR4 in esophageal cancer[J].Oncotarget,2017,8(30):49053-49063.
[14] Sakai T,Mashima H,Yamada Y,et al.The roles of interferon regulatory factors 1 and 2 in the progression of human pancreatic cancer[J].Pancreas,2014,43(6):909-916.
[15] Qian XL.Study on the mechanism of enhancing the drug resistance of gastric cancer cells by enhancing aerobic glycolysis [D].Zhejiang:Zhejiang University,2017.[钱晓玲.胃癌细胞通过提高有氧糖酵解增强耐药性的机制研究[D].浙江:浙江大学,2017.]
[16] Pusapati RV,Daemen A,Wilson C,et al.mTORC1-dependent metabolic reprogramming underlies escape from glycolysis addiction in cancer cells[J].Cancer Cell,2016,29(4):548-562.
[17] Gang L,Li YI,Gao X.Overexpression of microRNA-133b sensitizes non-small cell lung cancer cells to irradiation through the inhibition of glycolysis[J].Oncology Letters,2016,11(4):2903-2908.
[18] Wu H,Ying M,Hu X.Lactic acidosis switches cancer cells from aerobic glycolysis back to dominant oxidative phosphorylation[J].Oncotarget,2016,7(26):40621-40629.
[19] Pavlides S,Whitaker-Menezes D,Castello-Cros R,et al.The reverse Warburg effect:Aerobic glycolysis in cancer associated fibroblasts and the tumor stroma[J].Cell Cycle,2009,8(23):3984-4001.
[20] Heminger K,Jain V,Kadakia M,et al.Altered gene expression induced by ionizing radiation and glycolytic inhibitor 2-deoxy-glucose in a human glioma cell line:Implications for radio sensitization[J].Cancer Biology & Therapy,2006,5(7):815-823.
[21] Jiang Y,Wu GH,He GD,et al.The effect of silencing HIF-1α gene in BxPC-3 cell line on glycolysis-related gene expression,cell growth,invasion,and apoptosis[J].Nutrition & Cancer,2015,67(8):1316-1325.
[22] Yeh CS,Wang JY,Chung FY,et al.Significance of the glycolytic pathway and glycolysis related-genes in tumorigenesis of human colorectal cancers[J].Oncology Reports,2008,19(1):81-91.
[23] Xu RH,Pelicano H,Zhou Y,et al.Inhibition of glycolysis in cancer cells:A novel strategy to overcome drug resistance associated with mitochondrial respiratory defect and hypoxia[J].Cancer Research,2005,65(2):613-621.
[24] Kaplan O,Firon M,Vivi A,et al.HGF/SF activates glycolysis and oxidative phosphorylation in DA3 murine mammary cancer cells[J].Neoplasia,2000,2(4):365-377.
[25] Qin Y,Cheng C,Lu H,et al.miR-4458 suppresses glycolysis and lactate production by directly targeting hexokinase2 in colon cancer cells[J].Biochem Biophys Res Commun,2016,469(1):37-43.
[26] Han DF.p65-PKM2 mediated fenofibrate-induced inhibition of glucose metabolism in human glioblastoma cells[D].Nanjing:Nanjing Medical University,2015.[韩东风.非诺贝特通过抑制p65-PKM2信号通路调控人脑胶质瘤细胞糖酵解[D].南京:南京医科大学,2015.]
[27] Xu Q,Liu LZ,Yin Y,et al.Regulatory circuit of PKM2/NF-κB/miR-148a/152-modulated tumor angiogenesis and cancer progression[J].Oncogene,2015,34(43):5482-5493.