[1]赵金金,崔非非,莫清江,等.缺氧诱导因子-1α通过调控蛋白激酶B/核因子-κB信号通路促进肝癌细胞干细胞标志物CD133的表达[J].新乡医学院学报,2022,39(4):301-307.[doi:10.7683/xxyxyxb.2022.04.001]
　ZHAO Jinjin,CUI Feifei,MO Qingjiang,et al.Hypoxia inducible factor-1α promotes the expression of stem cell marker CD133 in hepatoma cells by regulating protein kinase B/nuclear factor-κB signaling pathway[J].Journal of Xinxiang Medical University,2022,39(4):301-307.[doi:10.7683/xxyxyxb.2022.04.001]
点击复制

缺氧诱导因子-1α通过调控蛋白激酶B/核因子-κB信号通路促进肝癌细胞干细胞标志物CD133的表达
分享到：

《新乡医学院学报》[ISSN:1004-7239/CN:41-1186/R]

卷:
39
期数:
2022年4
页码:
301-307
栏目:
基础研究
出版日期:
2022-04-05

文章信息/Info

Title:
Hypoxia inducible factor-1α promotes the expression of stem cell marker CD133 in hepatoma cells by regulating protein kinase B/nuclear factor-κB signaling pathway
作者:
赵金金1; 崔非非1; 莫清江1; 汪　磊1; 林志强1; 张海光2; 焦路阳1
（1.新乡医学院第一附属医院检验科，河南　卫辉　453100；2.新乡医学院第一附属医院妇产科，河南　卫辉　453100）
Author(s):
ZHAO Jinjin1; CUI Feifei1; MO Qingjiang1; WANG Lei1; LIN Zhiqiang1; ZHANG Haiguang2; JIAO Luyang1
(1.Department of Clinical Laboratory,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China;2.Department of Obsterics and Gynecology,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China)
关键词:
肝癌; 缺氧诱导因子-1α; 蛋白激酶B; 核因子-κB; 干细胞标志物; CD133
Keywords:
hepatoma; hypoxia inducible factor-1α; protein kinase B; nuclear factor-κB; stem cell marker; CD133
分类号:
R735.7
DOI:
10.7683/xxyxyxb.2022.04.001
文献标志码:
A
摘要:
目的　探讨缺氧诱导因子-1α（HIF-1α）诱导肝癌细胞中干细胞标志物CD133表达的分子机制及意义。方法　将HepG2细胞随机分为空白对照组、对照组和实验组，空白对照组HepG2细胞不做转染，对照组和实验组HepG2细胞分别转染空载体慢病毒pHBLV-ZsGreen-Puro和HIF-1α高表达慢病毒pHBLV-HIF1A-3flag-ZsGreen-Puro。使用荧光显微镜观察对照组和实验组细胞中绿色荧光蛋白(GFP)的表达，并计算转染效率；采用Western blot法检测3组细胞中HIF-1α蛋白及蛋白激酶B（Akt）/核因子-κB（NF-κB）信号通路蛋白磷酸化蛋白激酶B（p-Akt）、Akt、p-p65、p65蛋白的相对表达量，流式细胞术检测对照组和实验组CD133阳性细胞率。另取实验组细胞随机分为HepG2-HIF-1α组、Akt激酶抑制剂组和NF-κB信号通路抑制剂组，Akt激酶抑制剂组和NF-κB信号通路抑制剂组细胞分别给予终浓度为0.4、0.8、1.6、3.2、6.3、12.5、25.0 μmol·L-1 Akt激酶抑制剂及终浓度为3.0、6.0、12.5、25.0、50.0、100.0、200.0、400.0 μmol·L-1 NF-κB信号通路抑制剂干预；采用四甲基偶氮唑盐法检测Akt激酶抑制剂组和NF-κB信号通路抑制剂组细胞活力，流式细胞术检测HepG2-HIF-1α组、Akt激酶抑制剂组和NF-κB信号通路抑制剂组CD133阳性细胞率的变化。结果　对照组和实验组GFP 阳性细胞比例显著高于空白对照组（P<0.05）；对照组与实验组GFP阳性细胞比例比较差异无统计学意义（P>0.05）。实验组细胞中HIF-1α相对表达量显著高于空白对照组及对照组（P<0.05）；空白对照组与对照组细胞中HIF-1α相对表达量比较差异无统计学意义（P>0.05）。对照组和实验组CD133阳性细胞率分别为（4.42±0.29）%、（15.43±0.41）%，实验组CD133阳性细胞率显著高于对照组（t=22.160，P<0.05）。实验组细胞中p-Akt/Akt、p-p65/p65显著高于对照组（P<0.05）。0.4、0.8、1.6、3.2、6.3、12.5、25.0 μmol·L-1Akt激酶抑制剂组细胞活力比较差异无统计学意义（F=1.301，P>0.05）；3.0、6.0、12.5、25.0、50.0、100.0、200.0、400.0 μmol·L-1 NF-κB信号通路抑制剂组细胞活力比较差异无统计学意义（F=2.300，P>0.05）。Akt激酶抑制剂组和NF-κB信号通路抑制剂组CD133阳性细胞率低于HepG2-HIF-1α组（t=10.17 0、8.932,P<0.05）；Akt激酶抑制剂组与NF-κB信号通路抑制剂组CD133阳性细胞率比较差异无统计学意义（t=1.745，P>0.05）。结论　HIF-1α可通过Akt/NF-κB信号通路促进肝癌细胞中干细胞标志物CD133的表达，增加肝癌细胞的干细胞特性。
Abstract:
Objective　To investigate the mechanism and significance of the expression of stem cell marker CD133 in hepatoma cells induced by hypoxia inducible factor-1α(HIF-1α).Methods　HepG2 cells were randomly divided into blank control group,control group and experimental group;HepG2 cells in the blank control group were not transfected,the cells in the control group and experimental group were transfected with empty vector lentivirus pHBLV-ZsGreen-Puro or HIF-1a high expression lentivirus pHBLV-HIF1A-3flag-ZsGreen-Puro,respectively.The transfection efficiency of cells in the control group and experimental group was confirmed by the expression of green fluorescent protein (GFP) through fluorescence microscope;the relative expressions of HIF-1a and phosphorylated protein kinase B(p-Akt),protein kinase B(Akt),p-p65 and p65 protein in Akt/nuclear factor-κB(NF-κB) pathway in the three groups were detected by Western blot;the rarte of CD133 positive cells in the control group and experimental group were detected by flow cytometry.In addition,the cells in the experimental group were randomly divided into HepG2-HIF-1α group,Akt kinase inhibitor group and NF-κB signal pathway inhibitor group;the cells in the Akt kinase inhibitor group and NF-κB signal pathway inhibitor group were treated with the final concentration of 0.4,0.8,1.6,3.2,6.3,12.5,25.0 μmol·L-1 Akt kinase inhibitor or 3.0,6.0,12.5,25.0,50.0,100.0,200.0,400.0 μmol·L-1 NF-κB signal pathway inhibitor;the cell viability in the Akt kinase inhibitor group and NF-κB signal pathway inhibitor group was detected by tetramethylazozole salt assay.The rate of CD133 positive cells in the three groups was detected by flow cytometry.Results　The proportion of GFP positive cells in the control group and experiment group was significantly higher than that in the blank control group (P<0.05);there was no significant difference in the proportion of GFP positive cells between the control group and the experimental group (P>0.05).The relative expression of HIF-1a in the experimental group was significantly higher than that in blank control group and control group (P<0.05),there was no significant difference in the relative expression of HIF-1a between the blank control group and control group (P>0.05).The rate of CD133 positive cells in the control group and the experimental group was (4.42±0.29)% and (15.43±0.41)%，respectively.The rate of CD133 positive cells in the experimental group was significantly higher than that in the control group (t=22.160,P<0.05).The p-Akt/Akt and p-p65/p65 in the experimental group were significantly higher than those in the control group (P<0.05).There was no significant difference in the cell viability among the,0.4,0.8,1.6,3.2,6.3,12.5 and 25.0 μmol·L-1 Akt kinase inhibitor groups (F=1.301,P>0.05);there was no significant difference in the cell viability among the 3.0,6.0,12.5,25.0,50.0,100.0,200.0,400.0 μmol·L-1 NF-κB signal pathway inhibitor groups (F=2.300,P>0.05).The rate of CD133 positive cells in the Akt kinase inhibitor group and NF-κB signal pathway inhibitor group was significantly lower than that in the HepG2-HIF-1α group(t=10.170,8.932;P<0.05).There was no significant difference in the rate of CD133 positive cells between the Akt kinase inhibitor group and NF-κB signal pathway inhibitor group (t=1.745,P>0.05).Conclusion　HIF-1α can promote the expression of stem cell marker CD133 in hepatoma cells through AKT/NF-κB signaling pathway,enhance the characteristics of hepatoma stem cells.

参考文献/References:

［1］　ZHANG J,QI Y P,MA N,et al.Overexpression of Epcam and CD133 correlates with poor prognosis in dual-phenotype hepatocellular carcinoma［J］.J Cancer,2020,11(11):3400-3406.
［2］　CAO X,WU W,WANG D,et al.Glycogen synthase kinase GSK3α promotes tumorigenesis by activating HIF1/VEGFA signaling pathway in NSCLC tumor［J］.Cell Commun Signal,2022,20(1):32.
［3］　KAPPLER M,PABST U,WEINHOLD C,et al.Causes and consequences of a glutamine induced normoxic HIF1 activity for the tumor metabolism［J］.Int J Mol Sci,2019,20(19):4742.
［4］　AZORN E P,LETECHPIA DE LEN C,GARCA-REYNA M G,et al.Mathematical description of the effect of HIF inhibition on the radiobiological response of LNCaP cells［J］.Appl Radiat Isot,2022,184:110157.
［5］　SEMENZA G L.Breakthrough science:hypoxia-inducible factors,oxygen sensing,and disorders of hematopoiesis［J］.Blood,2021,Epub ahead of print.
［6］　MUOZ-GALVN S,FELIPE-ABRIO B,VERDUGO E M,et al.Downregulation of MYPT1 increases tumor resistance in ovarian cancer by targeting the Hippo pathway and increasing the stemness［J］.Mol Cancer,2020,19(1):7.
［7］　SAMANTA D,PARK Y,NI X,et al.Chemotherapy induces enrichment of CD47+/CD73+/ PDL1+ immune evasive triple-negative breast cancer cells［J］.Proc Natl Acad Sci U S A.,2018,115(6):E1239-E1248.
［8］　赵金金,张海光,崔非非,等.缺氧诱导因子1α对肝癌细胞HepG2干细胞特性及表阿霉素敏感性的影响［J］.临床肝胆病杂志,2021,37(2):354-357.
ZHAO J J,ZHANG H G,CUI F F,et al.Effect of hypoxia-inducible factor-1α on stemness and epirubicin sensitivity of HepG2 hepatoma cells［J］.J Clin Hepatol,2021,37(2):354-357.
［9］　SONG M,BODE A M,DONG Z,et al.AKT as a therapeutic target for cancer［J］.Cancer Res,2019,79(6):1019-1031.
［10］　ZINATIZADEH M R,SCHOCK B,CHALBATANI G M,et al.The nuclear factor kappa B (NF-κB) signaling in cancer development and immune diseases［J］.Genes Dis,2021,8(3):287-297.
［11］　王玉君,林秀艳,王涛.miR-582-5p靶向调控AKT3对甲状腺乳头状癌细胞增殖和凋亡的影响 ［J］.新乡医学院学报,2018,35(11):954-960.
WANG Y J,LIN X Y,WANG T.Effect of miR-582-5p targeting AKT3 on proliferation and apoptosis of papillary thyroid carcinoma cells［J］.J Xinxiang Med Univ,2018,35(11):954-960.
［12］　XIANG L,SEMENZA G L.Hypoxia-inducible factors promote breast cancer stem cell specification and maintenance in response to hypoxia or cytotoxic chemotherapy［J］.Adv Cancer Res,2019,141:175-212.
［13］　CASADO-MEDRANO V,BARRIO-REAL L,WANG A,et al.Distinctive requirement of PKCε in the control of Rho GTPases in epithelial and mesenchymally transformed lung cancer cells［J］.Oncogene,2019,38(27):5396-5412.
［14］　LAN J,LU H,SAMANTA D,et al.Hypoxia-inducible factor 1-dependent expression of adenosine receptor 2B promotes breast cancer stem cell enrichment［J］.Proc Natl Acad Sci U S A,2018,115(41):E9640-E9648.
［15］　PARK J H,SEO J H,JEON H Y,et al.Lentivirus-mediated VEGF knockdown suppresses gastric cancer cell proliferation and tumor growth in vitro and in vivo［J］.Onco Targets Ther,2020,13:1331-1341.
［16］　WANG R,LI Y,TSUNG A,et al.iNOS promotes CD24+CD133+ liver cancer stem cell phenotype through a TACE/ADAM17-dependent notch signaling pathway［J］.Proc Natl Acad Sci U S A,2018,115(43):E10127-E10136.
［17］　GALASSI C,VITALE I,GALLUZZI L.Using epigenetic modifiers to target cancer stem cell immunoevasion［J］.Cancer Cell,2021,39(12):1573-1575.
［18］　WAKIZAKA K,YOKOO H,KAMIYAMA T,et al.CD133 and epithelial cell adhesion molecule expressions in the cholangiocarcinoma component are prognostic factors for combined hepatocellular cholangiocarcinoma［J］.Hepatol Res,2020,50(2):258-267.
［19］　WU J,ZHU P,LU T,et al.The long non-coding RNA LncHDAC2 drives the self-renewal of liver cancer stem cells via activation of Hedgehog signaling［J］.J.Hepatol,2019,70(5):918-929.
［20］　VORA P,VENUGOPAL C,SALIM S K,et al.The rational development of CD133-targeting immunotherapies for glioblastoma［J］.Cell Stem Cell,2020,26(6):832-844.
［21］　LIU F,QIAN Y.The role of CD133 in hepatocellular carcinoma［J］.Cancer Biol Ther,2021,22(4):291-300.
［22］　LIU C,CHEN Y J,FAN M H,et al.Characteristics of CD133-sustained chemoresistant cancer stem-like cells in human ovarian carcinoma［J］.Int J Mol Sci,2020,21(18):6467.
［23］　FU X,ZHU X,QIN F,et al.Linc00210 drives Wnt/β-catenin signaling activation and liver tumor progression through CTNNBIP1-dependent manner［J］.Mol Cancer,2018,17(1):73.
［24］　CHEN Z Z,HUANG L,WU Y H,et al.LncSox4 promotes the self-renewal of liver tumour-initiating cells through Stat3-mediated Sox4 expression［J］.Nat Commun,2016,7:12598.
［25］　JEON T,KO M J,SEO Y R,et al.Silencing CDCA8 suppresses hepatocellular carcinoma growth and stemness via restoration of ATF3 tumor suppressor and inactivation of AKT/β-catenin signaling［J］.Cancers (Basel),2021,13(5):1055.
［26］　TIAN W,LI J,WANG Z,et al.HYD-PEP06 suppresses hepatocellular carcinoma metastasis,epithelial-mesenchymal transition and cancer stem cell-like properties by inhibiting PI3K/Akt and WNT/β-catenin signaling activation［J］.Acta Pharm Sin B,2021,11(6):1592-1606.
［27］　KAHRAMAN D C,KAHRAMAN T,CETIN-ATALAY R.Targeting PI3K/Akt/mTOR pathway identifies differential expression and functional role of IL8 in liver cancer stem cell enrichment［J］.Mol Cancer Ther,2019,18(11):2146-2157.
［28］　HUAN H B,YANG D P,WEN X J,et al.HOXB7 accelerates the malignant progression of hepatocellular carcinoma by promoting stemness and epithelial-mesenchymal transition［J］.J Exp Clin Cancer Res,2017,36(1):86.
［29］　WEI X,YOU X,ZHANG J,et al.MicroRNA-1305 inhibits the stemness of LCSCs and tumorigenesis by repressing the UBE2T-dependent Akt-signaling pathway［J］.Mol Ther Nuc Acids,2019,16:721-732.
［30］　HE J,GERSTENLAUER M,CHAN L K,et al.Block of NF-κB signaling accelerates MYC-driven hepatocellular carcinogenesis and modifies the tumor phenotype towards combined hepatocellular cholangiocarcinoma［J］.Cancer Lett,2019,458:113-122.
［31］　HU B,XU Y,LI Y C,et al.CD13 promotes hepatocellular carcinogenesis and sorafenib resistance by activating HDAC5-LSD1-NF-κB oncogenic signaling［J］.Clin Transl Med,2020,10(8):e233.

相似文献/References:

[1]王克霞,张荣波,唐小龙,等.肝细胞性肝癌与乙型肝炎病毒感染相关性的研究[J].新乡医学院学报,1997,14(03):281.
[2]杨瑞民,张凯,李玉侠,等.巨块型肝癌肝动脉栓塞后无水酒精注射疗效观察[J].新乡医学院学报,1997,14(04):382.
[3]甄艳华,杨利霞.肝癌患者肝动脉血流动力学的彩色多普勒研究[J].新乡医学院学报,2008,25(01):062.
[4]沈洁,阮健,郑航,等.ZD6474对肝癌HepG2细胞的抑制作用[J].新乡医学院学报,2010,27(03):234.
[5]黄长山,余伟,王云检,等.外科Apgar评分在肝癌术后并发症预测中的应用 TitleFilter('chTitle');[J].新乡医学院学报,2010,27(04):365.
[6]孙春伟,吴广银.哺乳动物雷帕霉素靶蛋白对肝癌细胞系Hep3B中侧群细胞的影响[J].新乡医学院学报,2011,28(03):307.
[7]王　雷１,王煜霞２,薛会朝１. 肝癌患者肿瘤坏死因子α和白细胞介素８水平变化及其与临床病理特征的关系[J].新乡医学院学报,2015,32(08):735.
[8]罗羽田,李世朋,徐红伟,等.铁死亡在肝癌中的调控机制及作用研究进展[J].新乡医学院学报,2021,38(1):091.[doi:10.7683/xxyxyxb.2021.01.020]
[9]王政强,韩锋博.肝癌切除术联合肝动脉化疗栓塞术治疗中晚期肝癌疗效观察[J].新乡医学院学报,2021,38(10):957.[doi:10.7683/xxyxyxb.2021.10.012]
　WANG Zhengqiang,HAN Fengbo.Effect of hepatectomy combined with transcatheter hepatic arterial chemoembolization in the treatment of middle-advanced liver cancer[J].Journal of Xinxiang Medical University,2021,38(4):957.[doi:10.7683/xxyxyxb.2021.10.012]
[10]李　妍,朱绍辉,张　彬.肝癌患者肝切除术后感染性并发症的相关危险因素分析[J].新乡医学院学报,2016,33(9):767.[doi:10.7683/xxyxyxb.2016.09.007]
　LI Yan,ZHU Shao-hui,ZHANG Bin.Analysis of the risk factors for infectious complications in patients with hepatocellular carcinoma after hepatectomy[J].Journal of Xinxiang Medical University,2016,33(4):767.[doi:10.7683/xxyxyxb.2016.09.007]

更新日期/Last Update: 2022-04-05