Axin2基因对肝癌细胞中Wnt/β-catenin信号通路相关分子表达的调控及机制
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摘要
目的探讨Axin2基因对肝癌细胞中Wnt/β-catenin信号通路相关分子表达的调控及其机制。方法通过TOPflash试验检测Axin2基因对Wnt/β-catenin信号通路的影响;荧光定量PCR(Quantitative Real-time PCR,QPCR)法检测Axin2基因在正常肝细胞LO2及3种肝癌细胞HepG2、HHCC、HB611中的表达水平;对肝癌细胞HepG2中Axin2的表达进行干扰,并检测Wnt/β-catenin信号通路相关基因(β-catenin、Cyclin A、CDK2、Wnt5a、STAT3、EGFR、APC)mRNA转录水平及相关蛋白(β-catenin、Cyclin A、CDK2、APC)的表达量。结果 Axin2对Wnt/β-catenin信号通路有显著抑制作用,且呈剂量依赖性(P <0. 05);3种肝癌细胞Axin2的表达水平显著低于正常肝细胞LO2(P <0. 05);干扰HepG2细胞中Axin2基因表达后,Axin2基因m RNA转录及蛋白表达水平降低,Wnt信号通路下游基因β-catenin、Cyclin A、CDK2、Wnt5a、STAT3和EGFR基因mRNA转录水平及β-catenin、Cyclin A、CDK2蛋白表达量均上升,而APC基因mRNA转录水平及蛋白表达量降低。结论 Axin2基因通过调控Wnt/β-catenin信号通路相关基因的表达抑制肝癌细胞的生成,本研究为寻找新的肝癌治疗靶点及防治药物提供了实验依据。
Objective To investigate the mechanism of Axin2 gene in regulation of expression of Wnt/β-catenin signaling pathway associated molecules in liver cancer cells. Methods The effect of Axin2 gene on Wnt/β-catenin signaling pathway was evaluated by TOPflash test. The expression levels of Axin2 gene in hepatoma cell lines HepG2,HHCC and HB611 and normal liver cell line LO2 were determined by Q-PCR. HepG2 cells were transfected with constructed RNAi plasmid for Axin2 gene,and determined for the transcription levels of m RNAs of Wnt/β-catenin signaling pathway associated genes(β-catenin,Cyclin A,CDK2,Wnt5 a,STAT3,EGFR and APC)and the expressions of Wnt/β-catenin signaling pathway associated proteins(β-catenin,Cyclin A,CDK2 and APC). Results Axin2 gene showed significantly dose-dependent inhibitory effect on Wnt/β-catenin signaling pathway(P < 0. 05). The expression levels of Axin2 in three kinds of hepatoma cells were significantly lower than that in normal liver cells(P < 0. 05). After transfection of HepG2 cells with RNAi plasmid,both the mRNA transcription and protein expression levels of Axin2 gene decreased significantly,while mRNA transcription levels of β-catenin,Cyclin A,CDK2,Wnt5 a,STAT3 and EGFR and protein expression levels of β-catenin,Cyclin A and CDK2 increased significantly. However,both the mRNA transcription and protein expression levels of APC decreased significantly. Conclusion Axin2 gene inhibited the generation of liver cancer cells by regulating the expression of Wnt/β-catenin signaling pathway associated genes,which provided an experimental basis for searching the new therapeutic targets and drugs for treatment of liver cancer.
Objective To investigate the mechanism of Axin2 gene in regulation of expression of Wnt/β-catenin signaling pathway associated molecules in liver cancer cells. Methods The effect of Axin2 gene on Wnt/β-catenin signaling pathway was evaluated by TOPflash test. The expression levels of Axin2 gene in hepatoma cell lines HepG2,HHCC and HB611 and normal liver cell line LO2 were determined by Q-PCR. HepG2 cells were transfected with constructed RNAi plasmid for Axin2 gene,and determined for the transcription levels of m RNAs of Wnt/β-catenin signaling pathway associated genes(β-catenin,Cyclin A,CDK2,Wnt5 a,STAT3,EGFR and APC)and the expressions of Wnt/β-catenin signaling pathway associated proteins(β-catenin,Cyclin A,CDK2 and APC). Results Axin2 gene showed significantly dose-dependent inhibitory effect on Wnt/β-catenin signaling pathway(P < 0. 05). The expression levels of Axin2 in three kinds of hepatoma cells were significantly lower than that in normal liver cells(P < 0. 05). After transfection of HepG2 cells with RNAi plasmid,both the mRNA transcription and protein expression levels of Axin2 gene decreased significantly,while mRNA transcription levels of β-catenin,Cyclin A,CDK2,Wnt5 a,STAT3 and EGFR and protein expression levels of β-catenin,Cyclin A and CDK2 increased significantly. However,both the mRNA transcription and protein expression levels of APC decreased significantly. Conclusion Axin2 gene inhibited the generation of liver cancer cells by regulating the expression of Wnt/β-catenin signaling pathway associated genes,which provided an experimental basis for searching the new therapeutic targets and drugs for treatment of liver cancer.
引文
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[16]KIM W K,BYUN W S,CHUNG H J,et al.Esculetin suppresses tumor growth and metastasis by targeting Axin2/E-cadherin axis in colorectal cancer[J].Biochem Pharmacol,2018,152:71-83.
[17]ABSHAGEN K,SENNE M,GENZ B,et al.Differential effects of Axin2 deficiency on the fibrogenic and regenerative response in livers of bile duct-ligated mice[J].Eur Surg Res,2015,55(4):328-340.
[18]DING Z,SHI C,JIANG L,et al.Oncogenic dependency onβ-catenin in liver cancer cell lines correlates with pathway activation[J].Oncotarget,2017,8(70):114526-114539.
[19]CHAI S,NG K Y,TONG M,et al.Octamer 4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells[J].Hepatology,2016,64(6):2062-2076.
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[3]BAN D,OGURA T,AKAHOSHI K,et al.Current topics in the surgical treatments for hepatocellular carcinoma[J].Ann Gastroenterol Surg,2018,2(2):137-146.
[4]GOESSLING W,NORTH T E,LOEWER S,et al.Genetic interaction of PGE2 and Wnt signaling regulates developmental specification of stem cells and regeneration[J].Cell,2009,136(6):1136-1147.
[5]GILES R H,VAN ES J H,CLEVERS H,et al.Caught up in a Wnt storm:Wnt signaling in cancer[J].Biochim Biophys Acta,2003,1653(1):1-24.
[6]YU S,WANG Z,SU Z,et al.Gigantol inhibits Wnt/β-catenin signaling and exhibits anticancer activity in breast cancer cells[J].BMC Complement Altern Med,2018,18(1):59-67.doi:10.1186/s12906-018-2108-x.
[7]MINDE D P,ANVARIAN Z,RUDIGER S G,et al.Messing up disorder:how do missense mutations in the tumor suppressor protein APC lead to cancer[J].Mol Cancer,2011(10):101-110.doi:10.1186/1476-4598-10-101.
[8]GORDON M D,NUSSE R.Wnt Signaling:multiple pathways,multiple receptors,and multiple transcription factors[J].J Biol Chem,2006,281(32):22429-22433.
[9]MA L,WANG X,JIA T,et al.Tankyrase inhibitors attenuate Wnt/β-catenin signaling and inhibit growth of hepatocellular carcinoma cells[J].Oncotarget,2015,6(28):25390-25401.
[10]BHANVADIA R R,VANOPSTALL C,BRECHKA H,et al.MEIS1 and MEIS2 expression and prostate cancer progression:a role for HOXB13 binding partners in metastatic disease[J].Clin Cancer Res,2018,34(15):3668-3680.doi:10.1158/1078-0432.CCR-17-3673.
[11]YANG J,ZENG Y,KANG J,et al.Effect of DR5-targeted docetaxel-loaded lipid microbubble combined with ultrasound targeted microbubble destruction on proliferation and apoptosis of human liver cancer Hep G2 cells[J].Chin J Biologicals,2016,29(4):389-392.(in Chinese)杨健,曾妍,康娟,等.载多烯紫杉醇的靶向脂质超声微泡联合超声靶向微泡破裂技术对人肝癌HepG2细胞增殖及凋亡的影响[J].中国生物制品学杂志,2016,29(4):389-392.
[12]XING L,ANBARCHIAN T,TSAI J M,et al.Wnt/β-catenin signaling regulates ependymal cell development and adult homeostasis[J].PNAS,2018,5:doi:10.1073/pnas.18032-97115.
[13]JARVINEN E,SHIMOMURA-KUROKI J,BALIC,et al.Mesenchymal Wnt/β-catenin signaling limits tooth number[J].Development,2018,145(4).doi:10.1242/dev.158048.
[14]MURALIMANOHARAN S,KWAK Y T,MENDELSON C R.Redox-sensitive transcription factor NRF2 enhances trophoblast differentiation via induction of miR-1246 and aromatase[J].Endocrinology,2018,159(5):2022-2033.
[15]BAHL C,SINGH N,BEHERA D,et al.High-order gene interactions between the genetic polymorphisms in Wnt and AhRpathway in modulating lung cancer susceptibility[J].Personalized Med,2017,14(6):487-502.
[16]KIM W K,BYUN W S,CHUNG H J,et al.Esculetin suppresses tumor growth and metastasis by targeting Axin2/E-cadherin axis in colorectal cancer[J].Biochem Pharmacol,2018,152:71-83.
[17]ABSHAGEN K,SENNE M,GENZ B,et al.Differential effects of Axin2 deficiency on the fibrogenic and regenerative response in livers of bile duct-ligated mice[J].Eur Surg Res,2015,55(4):328-340.
[18]DING Z,SHI C,JIANG L,et al.Oncogenic dependency onβ-catenin in liver cancer cell lines correlates with pathway activation[J].Oncotarget,2017,8(70):114526-114539.
[19]CHAI S,NG K Y,TONG M,et al.Octamer 4/microRNA-1246 signaling axis drives Wnt/β-catenin activation in liver cancer stem cells[J].Hepatology,2016,64(6):2062-2076.