Kruppel样转录因子6介导Bel7404、Huh7细胞中谷氨酰胺酶2通路的肿瘤增殖调控

Krueppel-like factor 6 regulates tumor proliferation in bel7404,huh7 cells by mediating glutaminase2 pathway

目的探讨Kruppel样转录因子6(KLF6)基因调控谷氨酰胺酶2(GLS2)对人肝癌细胞系Bel7404、Huh7增殖的影响。方法采用定量聚合酶链反应(qPCR)和蛋白印迹法(Western blot)检测正常肝细胞和肝癌细胞中KLF6的mRNA和蛋白表达(上海细胞库)。沉默KLF6基因, 细胞计数试剂盒(CCK-8)和细胞增殖实验(EdU)检测细胞增殖;结合前期实验基础、转录因子结合位点数据库(JASPAR)辅助筛选KLF6结合GLS2结合序列;染色质免疫共沉淀(CHIP)验证转录因子KLF6结合GLS2的序列;qPCR、Western blot检测沉默KLF6, GLS2的mRNA和蛋白的表达量。结果 KLF6在人肝癌细胞系HepG2(0.12±0.01)、HepG3B(0.11±0.02)、BEL-7404(0.44±0.02)、Huh7(0.34±0.03)中mRNA表达低于正常细胞HL7702(1.00±0.01), 具有统计学意义(t=172.80、113.90、61.64、44.41, P<0.01);Western blot检测KLF6在HepG2(0.57±0.02)、HepG3B(0.53±0.01)、BEL-7404(0.73±0.01)、Huh7(0.66±0.01)中蛋白表达水平低于HL7702, 差异有统计学意义(t=37.79、42.17、31.40、40.13, P<0.01);沉默KLF6基因, CCK-8检测Huh7在0、24、48、72 h增殖能力(0.20±0.01、0.30±0.01、0.45±0.04、0.63±0.02)高于对照组(0.20±0.01、0.29±0.01、0.35±0.03、0.47±0.01), 差异有统计学意义(F=43.46, P<0.01);CCK-8检测Bel7404在0、24、48、72 h增殖能力(0.20±0.01、0.27±0.01、0.42±0.02、0.62±0.04)高于对照组(0.20±0.01、0.26±0.01、0.36±0.01、0.44±0.02), 差异有统计学意义(F=141.40, P<0.01);EdU检测Huh7中3个不同抑制剂sh-KLF6-1、sh-KLF6-2、sh-KLF6-3增殖率(56.83%、52.68%、45.43%)高于对照组(35.32%), 差异有统计学意义(t=6.68、4.10、3.86, P<0.01);同理检测Bel7404中3个不同抑制剂增殖率(53.61%、49.32%、42.16%)高于对照组(32.75%), 差异有统计学意义(t=3.48、2.89、2.31, P<0.01);沉默KLF6基因, Huh7细胞在3种不同抑制剂sh-KLF6-1、sh-KLF6-2、sh-KLF6-3中GLS2的mRNA表达(0.01±0.00、0.13±0.01、0.44±0.02)低于对照组(1.01±0.01), 差异有统计学意义(t=94.63、23.54、27.04, P<0.01);实验组蛋白表达(0.41±0.01、0.64±0.10、0.87±0.02)低于对照组(1.00±0.01), 差异有统计学意义(t=15.11、9.57、3.88, P<0.01);Bel7404细胞在sh-KLF6-1、sh-KLF6-2、sh-KLF6-3中GLS2的mRNA表达(0.04±0.01、0.29±0.10、0.33±0.02)低于对照组(1.00±0.01), 差异有统计学意义(t=30.21、21.82、20.92, P<0.01);实验组蛋白表达(0.36±0.01、0.66±0.10、0.72±0.02)低于对照组(1.00±0.01), 差异有统计学意义(t=59.99、36.50、21.80, P<0.01)。结论 KLF6介导的GLS2调控途径显著抑制肝癌细胞的增殖。

Objective To investigate the effect of kruppel-like transcription factor 6(KLF6)gene regulating glutaminase 2(GLS2)on the proliferation of human hepatoma cell lines Bel7404 and Huh7.Methods The mRNA and protein expressions of KLF6 in normal and hepatocellular carcinoma cells were detected by quantitative polymerase chain reaction(qPCR)and Western blotting(Shanghai Cell Bank).KLF6 gene was silenced,cell counting kit(CCK-8)and cell proliferation assay(EdU)were used to detect cell proliferation.Combined with previous experimental basis and transcription factor binding site database(JASPAR),KLF6 binding GLS2 binding sequence was screened.Chromatin immunoprecipitation(CHIP)verifies the binding sequence of transcription factor KLF6 to GLS2.The mRNA and protein expression levels of silenced KLF6 and GLS2 were detected by qPCR and Western blotting.Results KLF6 mRNA expression in HepG2(0.12±0.01),HepG3B(0.11±0.02),BEL-7404(0.44±0.02),Huh7(0.34±0.03)was lower than that in HL7702(1.00±0.01).There was statistical significance(t=172.8,113.9,61.64,44.41,P<0.01).The protein expression level of KLF6 in HepG2(0.57±0.02),HepG3B(0.53±0.01),BEL-7404(0.73±0.01),Huh7(0.66±0.01)was lower than that of HL7702 by Western blotting.The difference was statistically significant(t=37.79,42.17,31.40,40.13,P<0.01).Silencing the KLF6 gene,CCK-8 detected the proliferation of Huh7 cells at 0,24,48 and 72 h(0.20±0.01,0.30±0.01,0.45±0.04,0.63±0.02)compared with the control group(0.20±0.001,0.29±0.01,0.35±0.03,0.47±0.01)Increased and had statistical significance(F=43.46,P<0.01).CCK-8 detected the proliferation of Bel7404 cells at 0,24,48,72 h(0.20±0.002,0.27±0.01,0.42±0.02,0.62±0.04)compared with the control group(0.20±0.01,0.26±0.01,0.36±0.01,0.44±0.02)significantly increased,and had statistical significance(F=141.4,P<0.01);The proliferation rates of three different inhibitors sh-KLF6-1,sh-KLF6-2 and sh-KLF6-3 in Huh7 were significantly increased by EdU(56.83%,52.68%and 45.43%)compared with the control group(35.32%),with statistical significance(t=6.68,4.10,3.86).P<0.01);The same method detected the proliferation rates of three different inhibitors in liver cancer cells Bel7404(53.61%,49.32%and 42.16%)were significantly higher than those in the control group(32.75%).There was statistical significance(t=3.48,2.89,2.31,P<0.01).By silencing KLF6 gene,GLS2 mRNA expression of Huh7 cells in three different inhibitors sh-KLF6-1,sh-KLF6-2 and sh-KLF6-3(0.01±0.00,0.13±0.01,0.44±0.02)was down-regulated compared with control group(1.01±0.01).There was statistical significance(t=94.63,23.54,27.04,P<0.01).The protein expression in experimental group(0.41±0.01,0.64±0.10,0.87±0.02)was significantly lower than that in control group(1.00±0.01,t=15.11,9.57,3.88,P<0.01).GLS2 mRNA expression of Bel7404 cells in sh-KLF6-1,sh-KLF6-2,sh-KLF6-3(0.04±0.01,0.29±0.10,0.33±0.02)was down-regulated compared with control group(1.00±0.01).There was statistical significance(t=30.21,21.82,20.92,P<0.01).Protein expression in experimental group(0.36±0.01,0.66±0.10,0.72±0.02)was down-regulated compared with control group(1.00±0.01),with statistical significance(t=59.99,36.50,21.80,P<0.01).Conclusion KLF6-mediated GLS2 regulation can significantly inhibit the proliferation of HCC cells.